TUMOR CELL VACCINES

Abstract

The present disclosure provides an allogeneic whole cell cancer vaccine platform that includes compositions and methods for treating and preventing cancer. Provided herein are compositions containing a therapeutically effective amount of cells from one or more cancer cell lines, some or all of which are modified to (i) inhibit or reduce expression of one or more immunosuppressive factors by the cells, and/or (ii) express or increase expression of one or more immunostimulatory factors by the cells, and/or (iii) express or increase expression of one or more tumor-associated antigens (TAAs), including TAAs that have been mutated, and which comprise cancer cell lines that natively express a heterogeneity of tumor associated antigens and/or neoantigens, and/or (iv) express one or more tumor fitness advantage mutations, including but not limited to acquired tyrosine kinase inhibitor (TKI) resistance mutations, EGFR activating mutations, and/or (v) express modified ALK intracellular domain(s), and/or express one or more driver mutations. Also provided herein are methods of making and preparing the vaccine compositions and methods of use thereof.

Description

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a text file. The name of the text file containing the Sequence Listing is “56087_Seqlisting.txt”, which was created on Oct. 28, 2021 and is 379,266 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.

BACKGROUND

Cancer is a leading cause of death. Recent breakthroughs in immunotherapy approaches, including checkpoint inhibitors, have significantly advanced the treatment of cancer, but these approaches are neither customizable nor broadly applicable across indications or to all patients within an indication. Furthermore, only a subset of patients are eligible for and respond to these immunotherapy approaches. Therapeutic cancer vaccines have the potential to generate anti-tumor immune responses capable of eliciting clinical responses in cancer patients, but many of these therapies have a single target or are otherwise limited in scope of immunomodulatory targets and/or breadth of antigen specificity. The development of a therapeutic vaccine customized for an indication that targets the heterogeneity of the cells within an individual tumor remains a challenge.

A vast majority of therapeutic cancer vaccine platforms are inherently limited in the number of antigens that can be targeted in a single formulation. The lack of breadth in these vaccines adversely impacts efficacy and can lead to clinical relapse through a phenomenon called antigen escape, with the appearance of antigen-negative tumor cells. While these approaches may somewhat reduce tumor burden, they do not eliminate antigen-negative tumor cells or cancer stem cells. Harnessing a patient's own immune system to target a wide breadth of antigens could reduce tumor burden as well as prevent recurrence through the antigenic heterogeneity of the immune response. Thus, a need exists for improved whole cell cancer vaccines. Provided herein are methods and compositions that address this need.

SUMMARY

In various embodiments, the present disclosure provides an allogeneic whole cell cancer vaccine platform that includes compositions and methods for treating and preventing cancer. The present disclosure provides compositions and methods that are customizable for the treatment of various solid tumor indications and target the heterogeneity of the cells within an individual tumor. The compositions and methods of embodiments of the present disclosure are broadly applicable across solid tumor indications and to patients afflicted with such indications. In some embodiments, the present disclosure provides compositions of cancer cell lines that (i) are modified as described herein and (ii) express a sufficient number and amount of tumor associated antigens (TAAs) such that, when administered to a subject afflicted with a cancer, cancers, or cancerous tumor(s), a TAA-specific immune response is generated.

In one embodiment, the present disclosure provides a composition comprising a therapeutically effective amount of at least 1 modified cancer cell line, wherein the cell line or a combination of the cell lines comprises cells that express at least 5 tumor associated antigens (TAAs) associated with a cancer of a subject intended to receive said composition, and wherein said composition is capable of eliciting an immune response specific to the at least 5 TAAs, and wherein the cell line or combination of the cell lines have been modified to express at least 1 peptide comprising at least 1 oncogene driver mutation. In one embodiment, the cell line or combination of the cell lines have been modified to express at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more peptides, wherein each peptide comprises at least 1 oncogene driver mutation.

In other embodiments, an aforementioned composition is provided wherein the cell line or a combination of the cell lines are modified to express or increase expression of at least 1 immunostimulatory factor. In other embodiments, an aforementioned composition is provided wherein the cell line or a combination of the cell lines are modified to inhibit or decrease expression of at least 1 immunosuppressive factor. In other embodiments, an aforementioned composition is provided wherein the cell line or a combination of the cell lines are modified to (i) express or increase expression of at least 1 immunostimulatory factor, and (ii) inhibit or decrease expression of at least 1 immunosuppressive factor. In other embodiments, an aforementioned composition is provided wherein the cell line or a combination of the cell lines are modified to express or increase expression of at least 1 TAA that is either not expressed or minimally expressed by one or all of the cell lines. In one embodiment, the cell line or a combination of the cell lines are further modified to express or increase expression of at least 1 peptide comprising at least 1 tumor fitness advantage mutation selected from the group consisting of an acquired tyrosine kinase inhibitor (TKI) resistance mutation, an EGFR activating mutation, and/or a modified ALK intracellular domain (modALK-IC). In another embodiment, the composition comprises at least 2 modified cancer lines, wherein one modified cell line comprises cells that have been modified to express at least 1 peptide comprising at least 1 acquired tyrosine kinase inhibitor (TKI) resistance mutation, and at least 1 peptide comprising at least 1 EGFR activating mutation, and a different modified cell line comprises cells that have been modified to express a modified ALK intracellular domain (modALK-IC). In still another embodiment, the cell line or combination of the cell lines have been modified to express at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more peptides, wherein each peptide comprises at least 1 acquired tyrosine kinase inhibitor (TKI) resistance mutation.

In other embodiments, an aforementioned composition is provided wherein the at least 1 acquired tyrosine kinase inhibitor (TKI) resistance mutation is selected from the group consisting of at least 1 EGFR acquired tyrosine kinase inhibitor (TKI) resistance mutation and at least 1 ALK acquired tyrosine kinase inhibitor (TKI) resistance mutation. In another embodiment, the cell line or combination of the cell lines have been modified to express at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more peptides, wherein each peptide comprises at least 1 EGFR activating mutation.

In other embodiments, an aforementioned composition is provided wherein the composition is capable of stimulating an immune response in a subject receiving the composition. In still another embodiment, the cell line or a combination of the cell lines are modified to (i) express at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more peptides, wherein each peptide comprises at least 1 oncogene driver mutation, (ii) express or increase expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunostimulatory factors, (iii) inhibit or decrease expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunosuppressive factors, and/or (iv) express or increase expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 TAAs that are either not expressed or minimally expressed by one or all of the cell lines, and wherein at least one of the cell lines is a cancer stem cell line. In yet another embodiment, the cancer stem line is selected from the group consisting of JHOM-2B, OVCAR-3, OV56, JHOS-4, JHOC-5, OVCAR-4, JHOS-2, EFO-21, CFPAC-1, Capan-1, Panc 02.13, SUIT-2, Panc 03.27, SK-MEL-28, RVH-421, Hs 895.T, Hs 940.T, SK-MEL-1, Hs 936.T, SH-4, COLO 800, UACC-62, NCI-H2066, NCI-H1963, NCI-H209, NCI-H889, COR-L47, NCI-H1092, NCI-H1436, COR-L95, COR-L279, NCI-H1048, NCI-H69, DMS 53, HuH-6, Li7, SNU-182, JHH-7, SK-HEP-1, Hep 382.1-7, SNU-1066, SNU-1041, SNU-1076, BICR 18, CAL-33, YD-8, CAL-29, KMBC-2, 253J, 253J-BV, SW780, SW1710, VM-CUB-1, BC-3C, KNS-81, TM-31, NMC-G1, GB-1, SNU-201, DBTRG-05MG, YKG-1, ECC10, RERF-GC-1B, TGBC-11-TKB, SNU-620, GSU, KE-39, HuG1-N, NUGC-4, SNU-16, OCUM-1, C2BBe1, Caco-2, SNU-1033, SW1463, COLO 201, GP2d, LoVo, SW403, CL-14, HCC2157, HCC38, HCC1954, HCC1143, HCC1806, HCC1599, MDA-MB-415, CAL-51, K052, SKNO-1, Kasumi-1, Kasumi-6, MHH-CALL-3, MHH-CALL-2, JVM-2, HNT-34, HOS, OUMS-27, T1-73, Hs 870.T, Hs 706.T, SJSA-1, RD-ES, U2OS, SaOS-2, and SK-ES-1. In still another embodiment, the cell line or cell lines are: (a) non-small cell lung cancer cell lines and/or small cell lung cancer cell lines selected from the group consisting of NCI-H460, NCIH520, A549, DMS 53, LK-2, and NCI-H23; (b) small cell lung cancer cell lines selected from the group consisting of DMS 114, NCI-H196, NCI-H1092, SBC-5, NCI-H510A, NCI-H889, NCI-H1341, NCIH-1876, NCI-H2029, NCI-H841, DMS 53, and NCI-H1694; (c) prostate cancer cell lines and/or testicular cancer cell lines selected from the group consisting of PC3, DU-145, LNCAP, NEC8, and NTERA-2cl-D1; (d) colorectal cancer cell lines selected from the group consisting of HCT-15, RKO, HuTu-80, HCT-116, and LS411N; (e) breast and/or triple negative breast cancer cell lines selected from the group consisting of Hs-578T, AU565, CAMA-1, MCF-7, and T-47D; (f) bladder and/or urinary tract cancer cell lines selected from the group consisting of UM-UC-3, J82, TCCSUP, HT-1376, and SCaBER; (g) head and/or neck cancer cell lines selected from the group consisting of HSC-4, Detroit 562, KON, HO-1-N-1, and OSC-20; (h) gastric and/or stomach cancer cell lines selected from the group consisting of Fu97, MKN74, MKN45, OCUM-1, and MKN1; (i) liver cancer and/or hepatocellular cancer (HCC) cell lines selected from the group consisting of Hep-G2, JHH-2, JHH-4, JHH-5, JHH-6, Li7, HLF, HuH-1, HuH-6, and HuH-7; (j) glioblastoma cancer cell lines selected from the group consisting of DBTRG-05MG, LN-229, SF-126, GB-1, and KNS-60; (k) ovarian cancer cell lines selected from the group consisting of TOV-112D, ES-2, TOV-21G, OVTOKO, and MCAS; (l) esophageal cancer cell lines selected from the group consisting of TE-10, TE-6, TE-4, EC-GI-10, OE33, TE-9, TT, TE-11, OE19, and OE21; (m) kidney and/or renal cell carcinoma cancer cell lines selected from the group consisting of A-498, A-704, 769-P, 786-O, ACHN, KMRC-1, KMRC-2, VMRC-RCZ, and VMRC-RCW; (n) pancreatic cancer cell lines selected from the group consisting of PANC-1, KP-3, KP-4, SUIT-2, and PSN11; (o) endometrial cancer cell lines selected from the group consisting of SNG-M, HEC-1-B, JHUEM-3, RL95-2, MFE-280, MFE-296, TEN, JHUEM-2, AN3-CA, and Ishikawa; (p) skin and/or melanoma cancer cell lines selected from the group consisting of RPMI-7951, MeWo, Hs 688(A).T, COLO 829, C32, A-375, Hs 294T, Hs 695T, Hs 852T, and A2058; or (q) mesothelioma cancer cell lines selected from the group consisting of NCI-H28, MSTO-211H, IST-Mes1, ACC-MESO-1, NCI-H2052, NCI-H2452, MPP 89, and IST-Mes2.

In other embodiments, an aforementioned composition is provided wherein the oncogene driver mutation is in one or more oncogenes selected from the group consisting of ACVR2A, AFDN, ALK, AMER1, ANKRD11, APC, AR, ARID1A, ARID1B, ARID2, ASXL1, ATM, ATR, ATRX, AXIN2, B2M, BCL9, BCL9L, BCOR, BCORL1, BRAF, BRCA2, CACNA1D, CAD, CAMTA1, CARD11, CASP8, CDH1, CDH11, CDKN1A, CDKN2A, CHD4, CIC, COL1A1, CPS1, CREBBP, CTNNB1, CUX1, DICER1, EGFR, ELF3, EP300, EP400, EPHA3, EPHA5, EPHB1, ERBB2, ERBB3, ERBB4, ERCC2, FAT1, FAT4, FBXW7, FGFR3, FLT4, FOXA1, GATA3, GNAS, GRIN2A, HGF, HRAS, IDH1, IRS1, IRS4, KAT6A, KDM2B, KDM6A, KDR, KEAP1, KMT2A, KMT2B, KMT2C, KMT2D, KRAS, LARP4B, LRP1B, LRP5, LRRK2, MAP3K1, MDC1, MEN1, MGA, MGAM, MKI67, MTOR, MYH11, MYH9, MYO18A, MYO5A, NCOA2, NCOR1, NCOR2, NF1, NFATC2, NFE2L2, NOTCH1, NOTCH2, NOTCH3, NSD1, NTRK3, NUMA1, PBRM1, PCLO, PDE4DIP, PDGFRA, PDS5B, PIK3CA, PIK3CG, PIK3R1, PLCG2, POLE, POLQ, PREX2, PRKDC, PTCH1, PTEN, PTPN13, PTPRB, PTPRC, PTPRD, PTPRK, PTPRS, PTPRT, RANBP2, RB1, RELN, RICTOR, RNF213, RNF43, ROBO1, ROS1, RPL22, RUNX1T1, SETBP1, SETD1A, SLX4, SMAD2, SMAD4, SMARCA4, SOX9, SPEN, SPOP, STAG2, STK11, TCF7L2, TET1, TGFBR2, TP53, TP53BP1, TPR, TRRAP, TSC1, UBR5, ZBTB20, ZFHX3, ZFP36L1, or ZNF521.

In other embodiments, an aforementioned composition is provided wherein the one or more oncogenes comprise PTEN (SEQ ID NO: 39), TP53 (SEQ ID NO:41), EGFR (SEQ ID NO: 43), PIK3CA (SEQ ID NO: 47), and/or PIK3R1 (SEQ ID NO: 45). In one embodiment, PTEN (SEQ ID NO: 39) comprises driver mutations selected from the group consisting of R130Q, G132D, and R173H; TP53 (SEQ ID NO: 41) comprises driver mutations selected from the group consisting of R158H, R175H, H179R, V216M, G245S, R248W, R273H, and C275Y; EGFR (SEQ ID NO: 43) comprises driver mutations selected from the group consisting of G63R, R108K, R252C, A289D, H304Y, G598V, S645C, and V774M; PIK3CA (SEQ ID NO: 47) comprises driver mutations selected from the group consisting of M1043V and H1047R; and PIK3R1 (SEQ ID NO: 45) comprises the driver mutation G376R.

In other embodiments, an aforementioned composition is provided wherein the one or more oncogenes comprise TP53 (SEQ ID NO: 41), SPOP (SEQ ID NO: 57), and/or AR (SEQ ID NO: 59). In one embodiment, TP53 (SEQ ID NO: 41) comprises driver mutations selected from the group consisting of R175H, Y220C, and R273C; SPOP (SEQ ID NO: 57) comprises driver mutations selected from the group consisting of Y87C, F102V, and F133L; and AR (SEQ ID NO: 59) comprises driver mutations selected from the group consisting of L702H, W742C, and H875Y.

In still other embodiments, an aforementioned composition is provided wherein the one or more oncogenes comprise TP53 (SEQ ID NO: 41), PIK3CA (SEQ ID NO: 47), and KRAS (SEQ ID NO: 77). In another embodiment, TP53 (SEQ ID NO: 41) comprises driver mutations selected from the group consisting of R110L, C141Y, G154V, V157F, R158L, R175H, C176F, H214R, Y220C, Y234C, M237I, G245V, R249M, I251F, R273L, and R337L; PIK3CA (SEQ ID NO: 47) comprises driver mutations selected from the group consisting of E542K and H1047R; and KRAS (SEQ ID NO: 77) comprises driver mutations selected from the group consisting of G12A and G13C.

In yet other embodiments, an aforementioned composition is provided wherein the one or more oncogenes comprise TP53 (SEQ ID NO: 41), PIK3CA (SEQ ID NO: 47), FBXW7 (SEQ ID NO: 104), SMAD4 (SEQ ID NO: 106), GNAS (SEQ ID NO: 114), ATM (SEQ ID NO: 108), KRAS (SEQ ID NO: 77), CTNNB1 (SEQ ID NO: 110), and ERBB3 (SEQ ID NO: 112). In one embodiment, TP53 (SEQ ID NO: 41) comprises driver mutations selected from the group consisting of R273C, G245S, and R248W; PIK3CA (SEQ ID NO: 47) comprises driver mutations selected from the group consisting of E542K, R88Q, M1043I, and H1047Y; FBXW7 (SEQ ID NO: 104) comprises driver mutations selected from the group consisting of R505C, S582L and R465H; SMAD4 (SEQ ID NO: 106) comprises driver mutations selected from the group consisting of R361H, GNAS (SEQ ID NO: 114) comprises driver mutations selected from the group consisting of R201H, ATM (SEQ ID NO: 108) comprises driver mutations selected from the group consisting of R337C; KRAS (SEQ ID NO: 77) comprises driver mutations selected from the group consisting of G12D, G12C and G12V; CTNNB1 (SEQ ID NO: 110) comprises driver mutations selected from the group consisting of S45F; and ERBB3 (SEQ ID NO: 112) comprises drive mutation V104M.

In other embodiments, an aforementioned composition is provided wherein the one or more oncogenes comprise TP53 (SEQ ID NO: 41) and PIK3CA (SEQ ID NO: 47). In another embodiment, TP53 (SEQ ID NO: 41) comprises driver mutations selected from the group consisting of Y220C, R248W and R273H; and PIK3CA (SEQ ID NO: 47) comprises driver mutations selected from the group consisting of N345K, E542K, E726K and H1047R.

In other embodiments, an aforementioned composition is provided wherein (a) the at least one immunostimulatory factor is selected from the group consisting of GM-CSF, membrane-bound CD40L, GITR, IL-15, IL-23, and IL-12, and (b) wherein the at least one immunosuppressive factors are selected from the group consisting of CD276, CD47, CTLA4, HLA-E, HLA-G, IDO1, IL-10, TGFβ1, TGFβ2, and TGFβ3.

The present disclosure provides compositions comprising cell lines. In embodiment, a composition is provided comprising cancer cell line LN-229, wherein the LN-229 cell line is modified in vitro to (i) express at least one immunostimulatory factor, at least one TAA that is either not expressed or minimally expressed by LN-229, and at least 1 peptide comprising at least 1 oncogene driver mutation; and (ii) decrease expression of at least one immunosuppressive factor. In another embodiment, the LN-229 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), modPSMA (SEQ ID NO: 30), and peptides comprising one or more driver mutation sequences selected from the group consisting of G63R, R108K, R252C, A289D, H304Y, S645C, and V774M of oncogene EGFR (SEQ ID NO: 51); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, a composition is provided comprising cancer cell line GB-1, wherein the GB-1 cell line is modified in vitro to (i) express at least one immunostimulatory factor, and at least 1 peptide comprising at least 1 oncogene driver mutation; and (ii) decrease expression of at least one immunosuppressive factor. In another embodiment, the GB-1 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), peptides comprising one or more driver mutation sequences selected from the group consisting of R130Q, G132D, and R173H of oncogene PTEN, R158H, R175H, H179R, V216M, G245S, R248W, R273H, and C275Y of oncogene TP53, G598V of oncogene EGFR, M1043V and H1047R of oncogene PIK3CA, and G376R of oncogene PIK3R1 (SEQ ID NO: 49); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, a composition is provided comprising cancer cell line SF-126, wherein the SF-126 cell line is modified in vitro to (i) express at least one immunostimulatory factor, at least one TAA that is either not expressed or minimally expressed by SF-126; and (ii) decrease expression of at least one immunosuppressive factor. In another embodiment, the SF-126 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTERT (SEQ ID NO: 28); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, a composition is provided comprising cancer cell line DBTRG-05MG, wherein the DBTRG-05MG cell line is modified in vitro to (i) express at least one immunostimulatory factor; and (ii) decrease expression of at least one immunosuppressive factor. In another embodiment, the DBTRG-05MG cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and CD276 shRNA (SEQ ID NO: 53).

In still another embodiment, a composition is provided comprising cancer cell line KNS-60, wherein the KNS-60 cell line is modified in vitro to (i) express at least one immunostimulatory factor, at least one TAA that is either not expressed or minimally expressed by KNS-60; and (ii) decrease expression of at least one immunosuppressive factor. In one embodiment, the KNS-60 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modMAGEA1 (SEQ ID NO: 32), EGFRvIII (SEQ ID NO: 32), hCMV-pp65 (SEQ ID NO: 32); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In yet another embodiment, a composition is provided comprising cancer cell line PC3, wherein the PC3 cell line is modified in vitro to (i) express at least one immunostimulatory factor, at least one TAA that is either not expressed or minimally expressed by PC3, and at least 1 peptide comprising at least 1 oncogene driver mutation; and (ii) decrease expression of at least one immunosuppressive factor. In another embodiment, the PC3 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTBXT (SEQ ID NO: 36), modMAGEC2 (SEQ ID NO: 36), and peptides comprising one or more driver mutation sequences selected from the group consisting of R175H, Y220C, and R273C of oncogene TP53, Y87C, F102V, and F133L of oncogene SPOP, and L702H, W742C, and H875Y of oncogene AR (SEQ ID NO: 61); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, a composition is provided comprising cancer cell line NEC8, wherein the NEC8 cell line is modified in vitro to (i) express at least one immunostimulatory factor; and (ii) decrease expression of at least one immunosuppressive factor. In one embodiment, the NEC8 cell line is modified in vitro to i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO: 3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In still another embodiment, a composition is provided comprising cancer cell line NTERA-2cl-D1, wherein the NTERA-2cl-D1 cell line is modified in vitro to (i) express at least one immunostimulatory factor; and (ii) decrease expression of at least one immunosuppressive factor. In another embodiment, the NTERA-2cl-D1 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO: 3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In yet another embodiment, a composition is provided comprising cancer cell line DU-145, wherein the DU-145 cell line is modified in vitro to (i) express at least one immunostimulatory factor, at least one TAA that is either not expressed or minimally expressed by DU-145; and (ii) decrease expression of at least one immunosuppressive factor. In one embodiment, the DU-145 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), and modPSMA (SEQ ID NO: 30); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In yet another embodiment, a composition is provided comprising cancer cell line LNCAP, wherein the LNCAP cell line is modified in vitro to (i) express at least one immunostimulatory factor; and (ii) decrease expression of at least one immunosuppressive factor. In one embodiment, the LNCAP cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO:3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, a composition is provided comprising cancer cell line NCI-H460, wherein the NCI-H460 cell line is modified in vitro to (i) express at least one immunostimulatory factor, at least one TAA that is either not expressed or minimally expressed by NCI-H460, and at least 1 peptide comprising at least 1 oncogene driver mutation; and (ii) decrease expression of at least one immunosuppressive factor. In another embodiment, the NCI-H460 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modBORIS (SEQ ID NO: 20), peptides comprising one or more TP53 driver mutations selected from the group consisting of R110L, C141Y, G154V, V157F, R158L, R175H, C176F, H214R, Y220C, Y234C, M237I, G245V, R249M, I251F, R273L, R337L, one or more PIK3CA driver mutations selected from the group consisting of E542K and H1047R, one or more KRAS driver mutations selected from the group consisting of G12A and G13C (SEQ ID NO: 79); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In still another embodiment, a composition is provided comprising cancer cell line A549, wherein the A549 cell line is modified in vitro to (i) express at least one immunostimulatory factor, at least one TAA that is either not expressed or minimally expressed by A549, at least 1 peptide comprising at least 1 oncogene driver mutation, and at least 1 EGFR activating mutation; and (ii) decrease expression of at least one immunosuppressive factor. In another embodiment, the A549 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTBXT (SEQ ID NO: 18), modWT1 (SEQ ID NO: 18), peptides comprising one or more KRAS driver mutations selected from the group consisting of G12D and G12 (SEQ ID NO: 18), peptides comprising one or more EGFR activating mutations selected from the group consisting of D761 E762insEAFQ, A763 Y764insFQEA, A767 S768insSVA, S768 V769insVAS, V769 D770insASV, D770 N771insSVD, N771repGF, P772 H773insPR, H773 V774insH, V774 C775insHV, G719A, L858R and L861Q (SEQ ID NO: 82); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, a composition is provided comprising cancer cell line NCI-H520, wherein the NCI-H520 cell line is modified in vitro to (i) express at least one immunostimulatory factor; and (ii) decrease expression of at least one immunosuppressive factor. In one embodiment, the NCI-H520 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In still another embodiment, a composition is provided comprising cancer cell line NCI-H23, wherein the NCI-H23 cell line is modified in vitro to (i) express at least one immunostimulatory factor, at least one TAA that is either not expressed or minimally expressed by NCI-H23, at least 1 EGFR acquired mutation, at least 1 ALK acquired resistance mutation, and ALK-IC; and (ii) decrease expression of at least one immunosuppressive factor. In one embodiment, the NCI-H23 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modMSLN (SEQ ID NO: 22), peptides comprising one or more EGFR tyrosine kinase inhibitor acquired resistance mutations selected from the group consisting of L692V, E709K, L718Q, G724S, T790M, C797S, L798I and L844V, one or more ALK tyrosine kinase inhibitor acquired resistance mutations selected from the group consisting of 1151Tins, C1156Y, I1171N, F1174L, V1180L, L1196M, G1202R, D1203N, S1206Y, F1245C, G1269A and R1275Q and modALK-IC (SEQ ID NO:94); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, a composition is provided comprising cancer cell line LK-2, wherein the LK-2 cell line is modified in vitro to (i) express at least one immunostimulatory factor; and (ii) decrease expression of at least one immunosuppressive factor. In another embodiment, the LK-2 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In yet another embodiment, a composition is provided comprising cancer cell line DMS 53, wherein the DMS 53 cell line is modified in vitro to (i) express at least one immunostimulatory factor; and (ii) decrease expression of at least one immunosuppressive factor. In another embodiment, a composition is provided comprising cancer cell line DMS 53, wherein the DMS 53 cell line is modified in vitro to (i) express at least one immunostimulatory factor; and (ii) decrease expression of at least one immunosuppressive factor, and wherein the modified DMS 53 cell line is adapted to serum-free media, wherein the adapted DMS 53 cell line has a doubling time less than or equal to approximately 200 hours, and wherein the adapted DMS 53 cell line expresses at least one immunostimulatory factor at a level approximately 1.2-fold to 1.6-fold greater than a modified DMS 53 cell line that is not adapted to serum-free media.

In one embodiment, the DMS 53 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 57). In still another embodiment, the DMS 53 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 57); wherein the modified DMS 53 cell line is adapted to serum-free media, wherein the adapted DMS 53 cell line has a doubling time less than or equal to approximately 200 hours, and wherein the adapted DMS 53 cell line expresses GM-CSF and/or IL-12 at a level approximately 1.2-fold or 1.5-fold greater, respectively, than a modified DMS 53 cell line that is not adapted to serum-free media.

In another embodiment, a composition is provided comprising a therapeutically effective amount of small cell lung cancer cell line DMS 53, wherein said cell line DMS 53 is modified to (i) knockdown TGFβ2, (ii) knockout CD276, and (iii) upregulate expression of GM-CSF, membrane bound CD40L, and IL-12. In yet another embodiment, a composition is provided comprising a therapeutically effective amount of small cell lung cancer cell line DMS 53, wherein said cell line DMS 53 is modified to (i) knockdown TGFβ2, (ii) knockout CD276, and (iii) upregulate expression of GM-CSF and membrane bound CD40L.

In still another embodiment, a composition is provided comprising cancer cell line HCT15, wherein the HCT15 cell line is modified in vitro to (i) express at least one immunostimulatory factor, and (ii) decrease expression of at least one immunosuppressive factor. In one embodiment, the HCT15 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), and TGFβ1 shRNA (SEQ ID NO: 54); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, a composition is provided comprising cancer cell line HUTU80, wherein the HUTU80 cell line is modified in vitro to (i) express at least one immunostimulatory factor, at least one TAA that is either not expressed or minimally expressed by HUTU80, and at least 1 peptide comprising at least 1 oncogene driver mutation; and (ii) decrease expression of at least one immunosuppressive factor. In one embodiment, the HUTU80 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modPSMA (SEQ ID NO: 30), and peptides comprising one or more driver mutation sequences selected from the group consisting of R273C of oncogene TP53, E542K of oncogene PIK3CA, R361H of oncogene SMAD4, R201H of oncogene GNAS, R505C of oncogene FBXW7, and R337C of oncogene ATM (SEQ ID NO: 116); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In yet another embodiment, a composition is provided comprising cancer cell line LS411N, wherein the LS411N cell line is modified in vitro to (i) express at least one immunostimulatory factor, and (ii) decrease expression of at least one immunosuppressive factor. In one embodiment, the L5411N cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, a composition is provided comprising cancer cell line HCT116, wherein the HCT116 cell line is modified in vitro to (i) express at least one immunostimulatory factor, at least one TAA that is either not expressed or minimally expressed by HCT116, and at least 1 peptide comprising at least 1 oncogene driver mutation; and (ii) decrease expression of at least one immunosuppressive factor. In another embodiment, the HCT116 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), modTBXT (SEQ ID NO: 18), modWT1 (SEQ ID NO: 18), and peptides comprising one or more driver mutation sequences selected from the group consisting of G12D and G12V of oncogene KRAS (SEQ ID NO: 77); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In still another embodiment, a composition is provided comprising cancer cell line RKO, wherein the RKO cell line is modified in vitro to (i) express at least one immunostimulatory factor, and at least 1 peptide comprising at least 1 oncogene driver mutation; and (ii) decrease expression of at least one immunosuppressive factor. In one embodiment, the RKO cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and peptides comprising one or more driver mutations sequences selected from the group consisting of R175H, G245S, and R248W of oncogene TP53, G12C of oncogene KRAS, R88Q, M1043I, and H1047Y of oncogene PIK3CA, S582L and R465H of oncogene FBXW7, S45F of oncogene CTNNB1), and V104M of oncogene ERBB3 (SEQ ID NO: 118); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, a composition is provided comprising cancer cell line CAMA-1, wherein the CAMA-1 cell line is modified in vitro to (i) express at least one immunostimulatory factor, and at least one TAA that is either not expressed or minimally expressed by CAMA-1; and (ii) decrease expression of at least one immunosuppressive factor. In another embodiment, the CAMA-1 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55), and modPSMA (SEQ ID NO: 30); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In still another embodiment, a composition is provided comprising cancer cell line AU565, wherein the AU565 cell line is modified in vitro to (i) express at least one immunostimulatory factor, at least one TAA that is either not expressed or minimally expressed by AU565, and at least 1 peptide comprising at least 1 oncogene driver mutation; and (ii) decrease expression of at least one immunosuppressive factor. In one embodiment, the AU565 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55), modTERT (SEQ ID NO: 28), and peptides comprising one or more driver mutation sequences selected from the group consisting of Y220C, R248W and R273H of oncogene TP53, and N345K, E542K, E726K and H1047L of oncogene PIK3CA (SEQ ID NO: 122); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In yet another embodiment, a composition is provided comprising cancer cell line HS-578T, wherein the HS-578T cell line is modified in vitro to (i) express at least one immunostimulatory factor, and (ii) decrease expression of at least one immunosuppressive factor. In one embodiment, the HS-578T cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, a composition is provided comprising cancer cell line MCF-7, wherein the MCF-7 cell line is modified in vitro to (i) express at least one immunostimulatory factor, and (ii) decrease expression of at least one immunosuppressive factor. In another embodiment, the MCF-7 cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, a composition is provided comprising cancer cell line T47D, wherein the T47D cell line is modified in vitro to (i) express at least one immunostimulatory factor, and at least one TAA that is either not expressed or minimally expressed by T47D; and (ii) decrease expression of at least one immunosuppressive factor. In one embodiment, the T47D cell line is modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), modTBXT (SEQ ID NO: 34) and modBORIS (SEQ ID NO: 34); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In some embodiment, an aforementioned composition is provided wherein the composition comprises approximately 1.0×10⁶-6.0×10⁷ cells of each cell line.

The present disclosure also provides kits according to some embodiments. In one embodiment, a kit is provided comprising one or more of the aforementioned compositions. In other embodiments, a kit is provided comprising at least one vial, said vial containing an aforementioned composition. In one embodiment, a kit is provided comprising 6 vials, wherein the vials each contain a composition comprising a cancer cell line, and wherein at least 2 of the 6 vials comprise a cancer cell line that is modified to (i) express or increase expression of at least 2 immunostimulatory factors, (ii) inhibit or decrease expression of at least 2 immunosuppressive factors, and (iii) express at least 1 peptide comprising at least 1 oncogene driver mutation. In another embodiment, at least 1 of the 6 vials comprises a cell line that is modified to express or increase expression of at least 1 peptide comprising at least 1 tumor fitness advantage mutation selected from the group consisting of an acquired tyrosine kinase inhibitor (TKI) resistance mutation, an EGFR activating mutation, and/or a modified ALK intracellular domain.

The present disclosure also provides unit doses as described herein. In one embodiment, a unit dose of a medicament for treating cancer is provided comprising at least 4 compositions of different cancer cell lines, wherein the cell lines comprise cells that collectively express at least 15 tumor associated antigens (TAAs) associated with the cancer. In another embodiment, a unit dose of a medicament for treating cancer is provided comprising at least 5 compositions of different cancer cell lines, wherein at least 2 compositions comprise a cell line that is modified to (i) express or increase expression of at least 2 immunostimulatory factors, (ii) inhibit or decrease expression of at least 2 immunosuppressive factors, and (iii) express at least 1 peptide comprising at least 1 oncogene driver mutation. In still another embodiment, a unit dose of a medicament for treating cancer is provided comprising at least 5 compositions of different cancer cell lines, wherein each cell line is modified to (i) express or increase expression of at least 2 immunostimulatory factors, (ii) inhibit or decrease expression of at least 2 immunosuppressive factors, and/or (iii) increase expression of at least 1 TAA that are either not expressed or minimally expressed by the cancer cell lines, and/or (iv) express at least 1 peptide comprising at least 1 oncogene driver mutation.

In some embodiments, an aforementioend kit is provided wherein at least 2 compositions comprise a cell line that is modified to express or increase expression of at least 1 peptide comprising at least 1 tumor fitness advantage mutation selected from the group consisting of an acquired tyrosine kinase inhibitor (TKI) resistance mutation, an EGFR activating mutation, and/or a modified ALK intracellular domain. In some embodiments, an aforementioend kit is provided wherein the unit dose comprises 6 compositions and wherein each composition comprises a different modified cell line. In one embodiment, prior to administration to a subject, 2 compositions are prepared, wherein the 2 compositions each comprises 3 different modified cell lines.

In one embodiment, a unit dose of a glioblastoma cancer vaccine is provided comprising 6 compositions, wherein each composition comprises one cancer cell line selected from the group consisting of LN-229, GB-1, SF-126, DBTRG-05MG, KNS-60 and DMS 53; wherein: (a) LN-229 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), modPSMA (SEQ ID NO: 30), and peptides comprising one or more driver mutation sequences selected from the group consisting of G63R, R108K, R252C, A289D, H304Y, S645C, and V774M of oncogene EGFR (SEQ ID NO: 51); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) GB-1 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), peptides comprising one or more driver mutation sequences selected from the group consisting of R130Q, G132D, and R173H of oncogene PTEN, R158H, R175H, H179R, V216M, G245S, R248W, R273H, and C275Y of oncogene TP53, G598V of oncogene EGFR, M1043V and H1047R of oncogene PIK3CA, and G376R of oncogene PIK3R1 (SEQ ID NO: 49); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) SF-126 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTERT (SEQ ID NO: 28); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) DBTRG-05MG is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and CD276 shRNA (SEQ ID NO: 53); (e) KNS-60 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modMAGEA1 (SEQ ID NO: 32), EGFRvIII (SEQ ID NO: 32), hCMV-pp65 (SEQ ID NO: 32); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52). In one embodiment, modified cell lines LN-229, GB-1 and SF-126 are combined into a first vaccine composition, and modified cell lines DBTRG-05MG, KNS-60 and DMS 53 are combined into a second vaccine composition.

In another embodiment, the present disclosure provides a unit dose of a prostate cancer vaccine comprising 6 compositions, wherein each composition comprises a cancer cell line selected from the group consisting of PC3, NEC8, NTERA-2cl-D1, DU145, LNCaP and DMS 53; wherein: (a) PC3 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTBXT (SEQ ID NO: 36), modMAGEC2 (SEQ ID NO: 36), and peptides comprising one or more driver mutation sequences selected from the group consisting of R175H, Y220C, and R273C of oncogene TP53, Y87C, F102V, and F133L of oncogene SPOP, and L702H, W742C, and H875Y of oncogene AR (SEQ ID NO: 61); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) NEC8 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO: 3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) NTERA-2cl-D1 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO: 3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) DU-145 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), and modPSMA (SEQ ID NO: 30); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) LNCAP is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO: 3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52). In another embodiment, modified cell lines PC3, NEC8 and NTERA-2cl-D1 are combined into a first vaccine composition, and modified cell lines DU145, LNCaP and DMS 53 are combined into a second vaccine composition.

In still another embodiment, the present disclosure provides a unit dose of a lung cancer vaccine comprising 6 compositions, wherein each composition comprises a cancer cell line selected from the group consisting of NCI-H460, A549, NCI-H520, NCI-H23, LK-2 and DMS 53; wherein: (a) NCI-H460 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modBORIS (SEQ ID NO: 20), peptides comprising one or more TP53 driver mutations selected from the group consisting of R110L, C141Y, G154V, V157F, R158L, R175H, C176F, H214R, Y220C, Y234C, M237I, G245V, R249M, I251F, R273L, R337L, one or more PIK3CA driver mutations selected from the group consisting of E542K and H1047R, one or more KRAS driver mutations selected from the group consisting of G12A and G13C (SEQ ID NO: 79); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) A549 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTBXT (SEQ ID NO: 18), modWT1 (SEQ ID NO: 18), peptides comprising one or more KRAS driver mutations selected from the group consisting of G12D and G12 (SEQ ID NO: 18), peptides comprising one or more EGFR activating mutations selected from the group consisting of D761 E762insEAFQ, A763 Y764insFQEA, A767 S768insSVA, S768 V769insVAS, V769 D770insASV, D770 N771insSVD, N771repGF, P772 H773insPR, H773 V774insH, V774 C775insHV, G719A, L858R and L861Q (SEQ ID NO: 82); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) NCI-H520 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) NCI-H23 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modMSLN (SEQ ID NO: 22), peptides comprising one or more EGFR tyrosine kinase inhibitor acquired resistance mutations selected from the group consisting of L692V, E709K, L718Q, G724S, T790M, C797S, L798I and L844V, one or more ALK tyrosine kinase inhibitor acquired resistance mutations selected from the group consisting of 1151Tins, C1156Y, I1171N, F1174L, V1180L, L1196M, G1202R, D1203N, S1206Y, F1245C, G1269A and R1275Q and modALK-IC (SEQ ID NO:94); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) LK-2 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52). In one embodiment, modified cell lines NCI-H460, A549 and NCI-H520 are combined into a first vaccine composition, and modified cell lines NCI-H23, LK-2 and DMS 53 are combined into a second vaccine composition.

In another embodiment, the present disclosure provides a unit dose of a colorectal vaccine comprising 6 compositions, wherein each composition comprises a cancer cell line selected from the group consisting of HCT15, HUTU80, LS411N, HCT116, RKO and DMS 53; wherein: (a) HCT15 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), and TGFβ1 shRNA (SEQ ID NO: 54); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) HUTU80 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modPSMA (SEQ ID NO: 30), and peptides comprising one or more driver mutation sequences selected from the group consisting of R273C of oncogene TP53, E542K of oncogene PIK3CA, R361H of oncogene SMAD4, R201H of oncogene GNAS, R505C of oncogene FBXW7, and R337C of oncogene ATM (SEQ ID NO: 116); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) LS411N is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) HCT116 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), modTBXT (SEQ ID NO: 18), modWT1 (SEQ ID NO: 18), and peptides comprising one or more driver mutation sequences selected from the group consisting of G12D and G12V of oncogene KRAS (SEQ ID NO: 77); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) RKO is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and peptides comprising one or more driver mutations sequences selected from the group consisting of R175H, G245S, and R248W of oncogene TP53, G12C of oncogene KRAS, R88Q, M1043I, and H1047Y of oncogene PIK3CA, S582L and R465H of oncogene FBXW7, S45F of oncogene CTNNB1), and V104M of oncogene ERBB3 (SEQ ID NO: 118); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52). In one embodiment, modified cell lines HCT15, HUTU80 and LS411N are combined into a first vaccine composition, and modified cell lines HCT116, RKO and DMS 53 are combined into a second vaccine composition.

In another embodiment, the present disclosure provides a unit dose of a breast cancer vaccine comprising 6 compositions, wherein each composition comprises a cancer cell line selected from the group consisting of CAMA-1, AU565, HS-578T, MCF-7, T47D and DMS 53; wherein: (a) CAMA-1 is modified to (i) express GM-CSF (SEQ ID NO: 52), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55), and modPSMA (SEQ ID NO: 30); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) AU565 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55), modTERT (SEQ ID NO: 28), and peptides comprising one or more driver mutation sequences selected from the group consisting of Y220C, R248W and R273H of oncogene TP53, and N345K, E542K, E726K and H1047L of oncogene PIK3CA (SEQ ID NO: 122); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) HS-578T is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) MCF-7 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) T47D is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), modTBXT (SEQ ID NO: 34), and modBORIS (SEQ ID NO: 34); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52). In one embodiment, modified cell lines CAMA-1, AU565, HS-578T are combined into a first vaccine composition, and modified cell lines MCF-7, T47D and DMS 53 are combined into a second vaccine composition.

The present disclosure provides methods of preparing the aforementioned compositions, as described herein. In one embodiment, the present disclosure provides a method of preparing a composition comprising a modified cancer cell line, said method comprising the steps of: (a) identifying one or more mutated oncogenes with >5% mutation frequency in a cancer; (b) identifying one or more driver mutations occurring in ≥0.5% of profiled patient samples in the mutated oncogenes identified in (a); (c) determining whether a peptide sequence comprising non-mutated oncogene amino acids and the driver mutation identified in (b) comprises a CD4 epitope, a CD8 epitope, or both CD4 and CD8 epitopes; (d) inserting a nucleic acid sequence encoding the peptide sequence comprising the driver mutation of (c) into a lentiviral vector; and (e) introducing the lentiviral vector into a cancer cell line, thereby producing a composition comprising a modified cancer cell line. In another embodiment, the method further comprises the steps of: (a) identifying one or more acquired resistance mutations and/or EGFR activating mutations in a cancer; (b) determining whether a peptide sequence comprising the one or more mutations identified in (a) comprises a CD4 epitope, a CD8 epitope, or both CD4 and CD8 epitopes; (c) inserting (i) a nucleic acid encoding the peptide sequence comprising the one or more mutations of (b) into a vector; and (d) introducing the vector into the cancer cell line, optionally wherein the cell line is further modified to express a modified ALK intracellular domain (modALK-IC). In another embodiment, the present disclosure provides an aforementioned method wherein said composition is capable of stimulating an immune response in a subject receiving the composition.

In still another embodiment, a method of stimulating an immune response in a subject is provided, the method comprising the steps of preparing a composition comprising a modified cancer cell line comprising the steps of: (a) identifying one or more mutated oncogenes with >5% mutation frequency in a cancer; (b) identifying one or more driver mutations occurring ≥0.5% of profiled patient samples in the mutated oncogenes identified in (a); (c) determining whether a peptide sequence comprising non-mutated oncogene amino acids and the driver mutation identified in (b) comprises a CD4 epitope, a CD8 epitope, or both CD4 and CD8 epitopes; (d) inserting a nucleic acid sequence encoding the peptide sequence comprising the driver mutation of (c) into a lentiviral vector; (e) introducing the lentiviral vector into a cancer cell line, thereby producing a composition comprising a modified cancer cell line; and (f) administering a therapeutically effective dose of the composition to the subject.

In yet another embodiment, a method of treating cancer in a subject is provided, the method comprising the steps of preparing a composition comprising a modified cancer cell line comprising the steps of: (a) identifying one or more mutated oncogenes with >5% mutation frequency in a cancer; (b) identifying one or more driver mutations occurring in ≥0.5% of profiled patient samples in the mutated oncogenes identified in (a); (c) determining whether a peptide sequence comprising non-mutated oncogene amino acids and the driver mutation identified in (b) comprises a CD4 epitope, a CD8 epitope, or both CD4 and CD8 epitopes; (d) inserting a nucleic acid sequence encoding the peptide sequence comprising the driver mutation of (c) into a lentiviral vector; (e) introducing the lentiviral vector into a cancer cell line, thereby producing a composition comprising a modified cancer cell line; and (f) administering a therapeutically effective dose of the composition to the subject.

In another embodiment, the present disclosure provides an aforementioned method wherein said method further comprises the steps of: (a) identifying one or more acquired resistance mutations and/or EGFR activating mutations in a cancer; (b) determining whether a peptide sequence comprising the one or more mutations identified in (a) comprises a CD4 epitope, a CD8 epitope, or both CD4 and CD8 epitopes; (c) inserting a nucleic acid encoding the peptide sequence comprising the one or more mutations of (b) into a vector; and (d) introducing the vector into the cancer cell line, optionally wherein the cell line is further modified to express a modified ALK intracellular domain (modALK-IC). In another embodiment, the present disclosure provides an aforementioned method wherein the cell line is further modified to express or increase expression of at least 1 immunostimulatory factor. In another embodiment, the present disclosure provides an aforementioned method wherein the cell line is further modified to inhibit or decrease expression of at least 1 immunosuppressive factor. In another embodiment, the present disclosure provides an aforementioned method wherein the cell line is further modified to (i) express or increase expression of at least 1 immunostimulatory factor, and (ii) inhibit or decrease expression of at least 1 immunosuppressive factor. In another embodiment, the present disclosure provides an aforementioned method wherein the cell line is further modified to express increase expression of at least 1 TAA that is either not expressed or minimally expressed by one or all of the cell lines. In one embodiment, (a) the at least one immunostimulatory factor is selected from the group consisting of GM-CSF, membrane-bound CD40L, GITR, IL-15, IL-23, and IL-12, and (b) wherein the at least one immunosuppressive factor is selected from the group consisting of CD276, CD47, CTLA4, HLA-E, HLA-G, IDO1, IL-10, TGFβ1, TGFβ2, and TGFβ3.

In still another embodiment, the present disclosure provides an aforementioned method wherein the cell line is a cancer stem cell line. In another embodiment, the present disclosure provides an aforementioned method wherein the composition comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified cancer cell lines. In another embodiment, the present disclosure provides an aforementioned method wherein two compositions, each comprising at least 2 modified cancer cell lines, are administered to the patient. In another embodiment, the present disclosure provides an aforementioned method wherein the two compositions in combination comprise at least 4 different modified cancer cell lines and wherein one composition comprises a cancer stem cell or wherein both compositions comprise a cancer stem cell. In another embodiment, the present disclosure provides an aforementioned method wherein the one or more mutated oncogenes has a mutation frequency of at least 5% in the cancer. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more mutated oncogenes are identified. In another embodiment, the present disclosure provides an aforementioned method wherein the one or more driver mutations identified in step (b) comprise missense mutations. In one embodiment, missense mutations in the same amino acid position occurring in ≥0.5% of profiled patient samples in each mutated oncogene of the cancer are identified in step (b) and selected for steps (c)-(f). In still another embodiment, the present disclosure provides an aforementioned method wherein the peptide sequence comprises a driver mutation flanked by approximately 15 non-mutated oncogene amino acids. In one embodiment, the driver mutation sequence is inserted approximately in the middle of the peptide sequence and wherein the peptide sequence is approximately 28-35 amino acids in length. In yet another embodiment, the present disclosure provides an aforementioned method wherein the peptide sequence comprises 2 driver mutations are flanked by approximately 8 non-mutated oncogene amino acids. In another embodiment, the present disclosure provides an aforementioned method wherein the vector is a lentivector. In one embodiment, the lentivector comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more peptide sequences, each comprising one or more driver mutations and/or acquired resistance mutations, and/or EGFR activating mutations, wherein each peptide sequence is optionally separated by a cleavage site. In another embodiment, the cleavage site comprises a furin cleavage site. In another embodiment, the present disclosure provides an aforementioned method wherein the vector is introduced into the at least one cancer cell line by transduction.

In still another embodiment, the present disclosure provides an aforementioned method wherein the subject is human. In another embodiment, the present disclosure provides an aforementioned method wherein the subject is afflicted with one or more cancers selected from the group consisting of lung cancer, prostate cancer, breast cancer, esophageal cancer, colorectal cancer, bladder cancer, gastric cancer, head and neck cancer, liver cancer, renal cancer, glioma, endometrial or uterine cancer, cervical cancer, ovarian cancer, pancreatic cancer, melanoma, and mesothelioma. In another embodiment, the present disclosure provides an aforementioned method wherein the cancer comprises a solid tumor. In yet another embodiment, the present disclosure provides an aforementioned method further comprising administering to the subject a therapeutically effective dose of one or more additional therapeutics selected from the group consisting of: a chemotherapeutic agent, cyclophosphamide, a checkpoint inhibitor, and all-trans retinoic acid (ATRA).

In yet another embodiment, the present disclosure provides an aforementioned method wherein the one or more mutated oncogenes is selected from the group consisting of ACVR2A, AFDN, ALK, AMER1, ANKRD11, APC, AR, ARID1A, ARID1B, ARID2, ASXL1, ATM, ATR, ATRX, AXIN2, B2M, BCL9, BCL9L, BCOR, BCORL1, BRAF, BRCA2, CACNA1D, CAD, CAMTA1, CARD11, CASP8, CDH1, CDH11, CDKN1A, CDKN2A, CHD4, CIC, COL1A1, CPS1, CREBBP, CTNNB1, CUX1, DICER1, EGFR, ELF3, EP300, EP400, EPHA3, EPHA5, EPHB1, ERBB2, ERBB3, ERBB4, ERCC2, FAT1, FAT4, FBXW7, FGFR3, FLT4, FOXA1, GATA3, GNAS, GRIN2A, HGF, HRAS, IDH1, IRS1, IRS4, KAT6A, KDM2B, KDM6A, KDR, KEAP1, KMT2A, KMT2B, KMT2C, KMT2D, KRAS, LARP4B, LRP1B, LRP5, LRRK2, MAP3K1, MDC1, MEN1, MGA, MGAM, MKI67, MTOR, MYH11, MYH9, MYO18A, MYO5A, NCOA2, NCOR1, NCOR2, NF1, NFATC2, NFE2L2, NOTCH1, NOTCH2, NOTCH3, NSD1, NTRK3, NUMA1, PBRM1, PCLO, PDE4DIP, PDGFRA, PDS5B, PIK3CA, PIK3CG, PIK3R1, PLCG2, POLE, POLQ, PREX2, PRKDC, PTCH1, PTEN, PTPN13, PTPRB, PTPRC, PTPRD, PTPRK, PTPRS, PTPRT, RANBP2, RB1, RELN, RICTOR, RNF213, RNF43, ROBO1, ROS1, RPL22, RUNX1T1, SETBP1, SETD1A, SLX4, SMAD2, SMAD4, SMARCA4, SOX9, SPEN, SPOP, STAG2, STK11, TCF7L2, TET1, TGFBR2, TP53, TP53BP1, TPR, TRRAP, TSC1, UBR5, ZBTB20, ZFHX3, ZFP36L1, or ZNF521.

In another embodiment, the present disclosure provides an aforementioned method wherein the one or more oncogenes comprise PTEN (SEQ ID NO: 39), TP53 (SEQ ID NO:41), EGFR (SEQ ID NO: 43), PIK3CA (SEQ ID NO: 47), and/or PIK3R1 (SEQ ID NO: 45) and the patient is afflicted with glioma. In one embodiment, PTEN (SEQ ID NO: 39) comprises driver mutations selected from the group consisting of R130Q, G132D, and R173H; TP53 (SEQ ID NO: 41) comprises driver mutations selected from the group consisting of R158H, R175H, H179R, V216M, G245S, R248W, R273H, and C275Y; EGFR (SEQ ID NO: 43) comprises driver mutations selected from the group consisting of G63R, R108K, R252C, A289D, H304Y, G598V, S645C, and V774M; PIK3CA (SEQ ID NO: 47) comprises driver mutations selected from the group consisting of M1043V and H1047R; and PIK3R1 (SEQ ID NO: 45) comprises the driver mutation G376R.

In another embodiment, the present disclosure provides an aforementioned method wherein peptide sequences comprising the driver mutations G598V of EGFR (SEQ ID NO: 43), R158H, R175H, H179R, V216M, G245S, R248W, R273H, and C275Y of TP53 (SEQ ID NO: 41), R130Q, G132D, and R173H of PTEN (SEQ ID NO: 39), G376R of PIK3CA (SEQ ID NO: 47), and M1043V and H1047R of PIK3R1 (SEQ ID NO: 45) are inserted into a first vector, and peptide sequences comprising the driver mutations G63R, R108K, R252C, A289D, H304Y, S645C, and V774M of EFGR (SEQ ID NO: 43) are inserted into a second vector. In another embodiment, wherein six compositions are prepared, wherein each composition comprises a cancer cell line selected from the group consisting of LN-229, GB-1, SF-126, DBTRG-05MG, KNS-60 and DMS 53; wherein: (a) LN-229 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), modPSMA (SEQ ID NO: 30), and peptides comprising one or more driver mutation sequences selected from the group consisting of G63R, R108K, R252C, A289D, H304Y, S645C, and V774M of oncogene EGFR (SEQ ID NO: 51); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) GB-1 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), peptides comprising one or more driver mutation sequences selected from the group consisting of R130Q, G132D, and R173H of oncogene PTEN, R158H, R175H, H179R, V216M, G245S, R248W, R273H, and C275Y of oncogene TP53, G598V of oncogene EGFR, M1043V and H1047R of oncogene PIK3CA, and G376R of oncogene PIK3R1 (SEQ ID NO: 49); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) SF-126 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTERT (SEQ ID NO: 28); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) DBTRG-05MG is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and CD276 shRNA (SEQ ID NO: 53); (e) KNS-60 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modMAGEA1 (SEQ ID NO: 32), EGFRvIII (SEQ ID NO: 32), hCMV-pp65 (SEQ ID NO: 32); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, the present disclosure provides an aforementioned method wherein the one or more oncogenes comprise TP53 (SEQ ID NO: 41), SPOP (SEQ ID NO: 57), and/or AR (SEQ ID NO: 59), and the patient is afflicted with prostate cancer. In another embodiment, TP53 (SEQ ID NO: 41) comprises driver mutations selected from the group consisting of R175H, Y220C, and R273C; SPOP (SEQ ID NO: 57) comprises driver mutations selected from the group consisting of Y87C, F102V, and F133L; and AR (SEQ ID NO: 59) comprises driver mutations selected from the group consisting of L702H, W742C, and H875Y. In another embodiment, peptide sequences comprising the driver mutations R175H, Y220, and R273C of TP53 (SEQ ID NO:41); Y87C, F102V, and F133L of SPOP (SEQ ID NO: 57); and L702H, W742C, and H875Y of AR (SEQ ID NO: 59) are inserted into a single vector. In another embodiment, six compositions are prepared, wherein each composition comprises a cancer cell line selected from the group consisting of PC3, NEC8, NTERA-2cl-D1, DU145, LNCaP and DMS 53; wherein: (a) PC3 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTBXT (SEQ ID NO: 36), modMAGEC2 (SEQ ID NO: 36), and peptides comprising one or more driver mutation sequences selected from the group consisting of R175H, Y220C, and R273C of oncogene TP53, Y87C, F102V, and F133L of oncogene SPOP, and L702H, W742C, and H875Y of oncogene AR (SEQ ID NO: 61); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) NEC8 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO: 3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) NTERA-2cl-D1 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO: 3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) DU-145 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), and modPSMA (SEQ ID NO: 30); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) LNCAP is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO: 3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In yet another embodiment, the present disclosure provides an aforementioned method wherein the one or more oncogenes comprise TP53 (SEQ ID NO: 41), PIK3CA (SEQ ID NO: 47), KRAS (SEQ ID NO: 77), and the patient is afflicted with lung cancer. In one embodiment, TP53 (SEQ ID NO: 41) comprises driver mutations selected from the group consisting of R110L, C141Y, G154V, V157F, R158L, R175H, C176F, H214R, Y220C, Y234C, M237I, G245V, R249M, I251F, R273L, and R337L; PIK3CA (SEQ ID NO: 47) comprises driver mutations selected from the group consisting of E542K and H1047R; and KRAS (SEQ ID NO: 77) comprises driver mutations selected from the group consisting of G12A and G13C. In another embodiment, peptide sequences comprising the driver mutations R110L, C141Y, G154V, V157F, R158L, R175H, C176F, H214R, Y220C, Y234, M237I, G245V, R249M, I251F, R273L, and R337L of TP53 (SEQ ID NO: 41); E542K and H1047R of PIK3CA (SEQ ID NO: 47); and G12A and G13C of KRAS (SEQ ID NO: 77) are inserted into a single lentiviral vector. In another embodiment, six compositions are prepared, wherein each composition comprises a cancer cell line selected from the group consisting of NCI-H460, A549, NCI-H520, NCI-H23, LK-2 and DMS 53; wherein: (a) NCI-H460 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modBORIS (SEQ ID NO: 20), peptides comprising one or more TP53 driver mutations selected from the group consisting of R110L, C141Y, G154V, V157F, R158L, R175H, C176F, H214R, Y220C, Y234C, M237I, G245V, R249M, I251F, R273L, R337L, one or more PIK3CA driver mutations selected from the group consisting of E542K and H1047R, one or more KRAS driver mutations selected from the group consisting of G12A and G13C (SEQ ID NO: 79); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) A549 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTBXT (SEQ ID NO: 18), modWT1 (SEQ ID NO: 18), peptides comprising one or more KRAS driver mutations selected from the group consisting of G12D and G12 (SEQ ID NO: 18), peptides comprising one or more EGFR activating mutations selected from the group consisting of D761 E762insEAFQ, A763 Y764insFQEA, A767 S768insSVA, S768 V769insVAS, V769 D770insASV, D770 N771insSVD, N771repGF, P772 H773insPR, H773 V774insH, V774 C775insHV, G719A, L858R and L861Q (SEQ ID NO: 82); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) NCI-H520 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) NCI-H23 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modMSLN (SEQ ID NO: 22), peptides comprising one or more EGFR tyrosine kinase inhibitor acquired resistance mutations selected from the group consisting of L692V, E709K, L718Q, G724S, T790M, C797S, L798I and L844V, one or more ALK tyrosine kinase inhibitor acquired resistance mutations selected from the group consisting of 1151Tins, C1156Y, I1171N, F1174L, V1180L, L1196M, G1202R, D1203N, S1206Y, F1245C, G1269A and R1275Q and modALK-IC (SEQ ID NO:94); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) LK-2 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, the present disclosure provides an aforementioned method wherein the one or more oncogenes comprise TP53 (SEQ ID NO: 41), PIK3CA (SEQ ID NO: 47), FBXW7 (SEQ ID NO: 104), SMAD4 (SEQ ID NO: 106), GNAS (SEQ ID NO: 114), ATM (SEQ ID NO: 108), KRAS (SEQ ID NO: 77), CTNNB1 (SEQ ID NO: 110), and ERBB3 (SEQ ID NO: 112). In one embodiment, TP53 (SEQ ID NO: 41) comprises driver mutations selected from the group consisting of R273C, G245S, and R248W; PIK3CA (SEQ ID NO: 47) comprises driver mutations selected from the group consisting of E542K, R88Q, M1043I, and H1047Y; FBXW7 (SEQ ID NO: 104) comprises driver mutations selected from the group consisting of R505C, S582L and R465H; SMAD4 (SEQ ID NO: 106) comprises driver mutations selected from the group consisting of R361H, GNAS (SEQ ID NO: 114) comprises driver mutations selected from the group consisting of R201H, ATM (SEQ ID NO: 108) comprises driver mutations selected from the group consisting of R337C; KRAS (SEQ ID NO: 77) comprises driver mutations selected from the group consisting of G12D, G12C and G12V; CTNNB1 (SEQ ID NO: 110) comprises driver mutations selected from the group consisting of S45F; and ERBB3 (SEQ ID NO: 112) comprises drive mutation V104M. In one embodiment, peptide sequences comprising the driver mutations R273C of oncogene TP53 (SEQ ID NO: 41), E542K of oncogene PIK3CA (SEQ ID NO: 47), R361H of oncogene SMAD4 (SEQ ID NO: 106), R201H of oncogene GNAS (SEQ ID NO: 114), R505C of oncogene FBXW7 (SEQ ID NO: 104), and R337C of oncogene ATM (SEQ ID NO: 108) are inserted into a first lentiviral vector, and peptide sequences comprising the driver mutations R175H, G245S, and R248W of oncogene TP53 (SEQ ID NO: 41), G12C of oncogene KRAS (SEQ ID NO: 77), R88Q, M1043I, and H1047Y of oncogene PIK3CA (SEQ ID NO: 47), S582L and R465H of oncogene FBXW7 (SEQ ID NO: 104), S45F of oncogene CTNNB1 (SEQ ID NO: 110), and V104M of oncogene ERBB3 (SEQ ID NO: 112) are inserted into a second lentiviral vector. In one embodiment, six compositions are prepared, wherein each composition comprises a cancer cell line selected from the group consisting of HCT15, HUTU80, LS411N, DMS 53, HCT116 and RKO; wherein: (a) HCT15 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), and TGFβ1 shRNA (SEQ ID NO: 54); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) HUTU80 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modPSMA (SEQ ID NO: 30), and peptides comprising one or more driver mutation sequences selected from the group consisting of R273C of oncogene TP53, E542K of oncogene PIK3CA, R361H of oncogene SMAD4, R201H of oncogene GNAS, R505C of oncogene FBXW7, and R337C of oncogene ATM (SEQ ID NO: 116); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) LS411N is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) HCT116 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), modTBXT (SEQ ID NO: 18), modWT1 (SEQ ID NO: 18), and peptides comprising one or more driver mutation sequences selected from the group consisting of G12D and G12V of oncogene KRAS (SEQ ID NO: 77); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) RKO is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and peptides comprising one or more driver mutations sequences selected from the group consisting of R175H, G245S, and R248W of oncogene TP53, G12C of oncogene KRAS, R88Q, M1043I, and H1047Y of oncogene PIK3CA, S582L and R465H of oncogene FBXW7, S45F of oncogene CTNNB1), and V104M of oncogene ERBB3 (SEQ ID NO: 118); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, the present disclosure provides an aforementioned method wherein the one or more oncogenes comprise TP53 (SEQ ID NO: 41) and PIK3CA (SEQ ID NO: 47). In another embodiment, TP53 (SEQ ID NO: 41) comprises driver mutations selected from the group consisting of Y220C, R248W and R273H; and PIK3CA (SEQ ID NO: 47) comprises driver mutations selected from the group consisting of N345K, E542K, E726K and H1047R. In another embodiment, six compositions are prepared, wherein each composition comprises a cancer cell line selected from the group consisting of CAMA-1, AU565, HS-578T, MCF-7, T47D and DMS 53 wherein: (a) CAMA-1 is modified to (i) express GM-CSF (SEQ ID NO: 52), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55), and modPSMA (SEQ ID NO: 30); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) AU565 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55), modTERT (SEQ ID NO: 28), and peptides comprising one or more driver mutation sequences selected from the group consisting of Y220C, R248W and R273H of oncogene TP53, and N345K, E542K, E726K and H1047L of oncogene PIK3CA (SEQ ID NO: 122); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) HS-578T is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) MCF-7 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) T47D is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), modTBXT (SEQ ID NO: 34), and modBORIS (SEQ ID NO: 34); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

The present disclosure, in one embodiment, provides a method of stimulating an immune response in a patient comprising administering to said patient a therapeutically effective amount of a unit dose of a cancer vaccine, wherein said unit dose comprises a composition comprising a cancer stem cell line and at least 3 compositions each comprising a different modified cancer cell line; wherein the cell lines are optionally modified to (i) express at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more peptides, wherein each peptide comprises at least 1 oncogene driver mutation, and/or (ii) express or increase expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunostimulatory factors, and/or (iii) inhibit or decrease expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunosuppressive factors, and/or (iv) express or increase expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 TAAs that are either not expressed or minimally expressed by one or all of the cell lines. In another embodiment, a method of treating cancer in a patient is provided comprising administering to said patient a therapeutically effective amount of a unit dose of a cancer vaccine, wherein said unit dose comprises a composition comprising a cancer stem cell line and at least 3 compositions each comprising a different modified cancer cell line; wherein the cell lines are optionally modified to (i) express at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more peptides, wherein each peptide comprises at least 1 oncogene driver mutation, and/or (ii) express or increase expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunostimulatory factors, and/or (iii) inhibit or decrease expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunosuppressive factors, and/or (iv) express or increase expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 TAAs that are either not expressed or minimally expressed by one or all of the cell lines.

In another embodiment, the present disclosure provides an aforementioned method wherein the unit dose comprises a composition comprising a cancer stem cell line and 5 compositions comprising the cell lines of (a) DBTRG-05MG, LN-229, SF-126, GB-1, and KNS-60; (b) PC3, DU-145, LNCAP, NEC8, and NTERA-2cl-D1; (c) NCI-H460, NCIH520, A549, DMS 53, LK-2, and NCI-H23; (d) HCT15, RKO, HUTU80, HCT116, and LS411N; or (e) Hs 578T, AU565, CAMA-1, MCF-7, and T-47D.

In another embodiment, the present disclosure provides a method of stimulating an immune response in a patient comprising administering to said patient a therapeutically effective amount of a unit dose of a glioblastoma cancer vaccine, wherein said unit dose comprises 6 compositions, wherein each composition comprises one cancer cell line selected from the group consisting of LN-229, GB-1, SF-126, DBTRG-05MG, KNS-60 and DMS 53; wherein: (a) LN-229 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), modPSMA (SEQ ID NO: 30), and peptides comprising one or more driver mutation sequences selected from the group consisting of G63R, R108K, R252C, A289D, H304Y, S645C, and V774M of oncogene EGFR (SEQ ID NO: 51); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) GB-1 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), peptides comprising one or more driver mutation sequences selected from the group consisting of R130Q, G132D, and R173H of oncogene PTEN, R158H, R175H, H179R, V216M, G245S, R248W, R273H, and C275Y of oncogene TP53, G598V of oncogene EGFR, M1043V and H1047R of oncogene PIK3CA, and G376R of oncogene PIK3R1 (SEQ ID NO: 49); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) SF-126 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTERT (SEQ ID NO: 28); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) DBTRG-05MG is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and CD276 shRNA (SEQ ID NO: 53); (e) KNS-60 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modMAGEA1 (SEQ ID NO: 32), EGFRvIII (SEQ ID NO: 32), hCMV-pp65 (SEQ ID NO: 32); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In still another embodiment, provided herein is a method of treating glioblastoma in a patient comprising administering to said patient a therapeutically effective amount of a unit dose of a glioblastoma cancer vaccine, wherein said unit dose comprises 6 compositions, wherein each composition comprises one cancer cell line selected from the group consisting of LN-229, GB-1, SF-126, DBTRG-05MG, KNS-60 and DMS 53; wherein: (a) LN-229 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), modPSMA (SEQ ID NO: 30), and peptides comprising one or more driver mutation sequences selected from the group consisting of G63R, R108K, R252C, A289D, H304Y, S645C, and V774M of oncogene EGFR (SEQ ID NO: 51); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) GB-1 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), peptides comprising one or more driver mutation sequences selected from the group consisting of R130Q, G132D, and R173H of oncogene PTEN, R158H, R175H, H179R, V216M, G245S, R248W, R273H, and C275Y of oncogene TP53, G598V of oncogene EGFR, M1043V and H1047R of oncogene PIK3CA, and G376R of oncogene PIK3R1 (SEQ ID NO: 49); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) SF-126 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTERT (SEQ ID NO: 28); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) DBTRG-05MG is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and CD276 shRNA (SEQ ID NO: 53); (e) KNS-60 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modMAGEA1 (SEQ ID NO: 32), EGFRvIII (SEQ ID NO: 32), hCMV-pp65 (SEQ ID NO: 32); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In still another embodiment, provided herein is a method of stimulating an immune response in a patient comprising administering to said patient a therapeutically effective amount of a unit dose of a prostate cancer vaccine, wherein said unit dose comprises 6 compositions, wherein each composition comprises a cancer cell line selected from the group consisting of PC3, NEC8, NTERA-2cl-D1, DU145, LNCaP and DMS 53; wherein: (a) PC3 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTBXT (SEQ ID NO: 36), modMAGEC2 (SEQ ID NO: 36), and peptides comprising one or more driver mutation sequences selected from the group consisting of R175H, Y220C, and R273C of oncogene TP53, Y87C, F102V, and F133L of oncogene SPOP, and L702H, W742C, and H875Y of oncogene AR (SEQ ID NO: 61); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) NEC8 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO: 3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) NTERA-2cl-D1 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO: 3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) DU-145 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), and modPSMA (SEQ ID NO: 30); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) LNCAP is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO: 3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In still another embodiment, provided herein is a method of treating glioblastoma in a patient comprising administering to said patient a therapeutically effective amount of a unit dose of a prostate cancer vaccine, wherein said unit dose comprises 6 compositions, wherein each composition comprises a cancer cell line selected from the group consisting of PC3, NEC8, NTERA-2cl-D1, DU145, LNCaP and DMS 53; wherein: (a) PC3 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTBXT (SEQ ID NO: 36), modMAGEC2 (SEQ ID NO: 36), and peptides comprising one or more driver mutation sequences selected from the group consisting of R175H, Y220C, and R273C of oncogene TP53, Y87C, F102V, and F133L of oncogene SPOP, and L702H, W742C, and H875Y of oncogene AR (SEQ ID NO: 61); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) NEC8 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO: 3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) NTERA-2cl-D1 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO: 3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) DU-145 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), and modPSMA (SEQ ID NO: 30); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) LNCAP is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), and membrane-bound CD40L (SEQ ID NO: 3); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In yet another embodiment, provided herein is a method of stimulating an immune response in a patient comprising administering to said patient a therapeutically effective amount of a unit dose of a NSCLC vaccine, wherein said unit dose comprises 6 compositions, wherein each composition comprises a cancer cell line selected from the group consisting of NCI-H460, A549, NCI-H520, NCI-H23, LK-2 and DMS 53; wherein: (a) NCI-H460 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modBORIS (SEQ ID NO: 20), peptides comprising one or more TP53 driver mutations selected from the group consisting of R110L, C141Y, G154V, V157F, R158L, R175H, C176F, H214R, Y220C, Y234C, M237I, G245V, R249M, I251F, R273L, R337L, one or more PIK3CA driver mutations selected from the group consisting of E542K and H1047R, one or more KRAS driver mutations selected from the group consisting of G12A and G13C (SEQ ID NO: 79); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) A549 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTBXT (SEQ ID NO: 18), modWT1 (SEQ ID NO: 18), peptides comprising one or more KRAS driver mutations selected from the group consisting of G12D and G12 (SEQ ID NO: 18), peptides comprising one or more EGFR activating mutations selected from the group consisting of D761 E762insEAFQ, A763 Y764insFQEA, A767 S768insSVA, S768 V769insVAS, V769 D770insASV, D770 N771insSVD, N771repGF, P772 H773insPR, H773 V774insH, V774 C775insHV, G719A, L858R and L861Q (SEQ ID NO: 82); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) NCI-H520 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) NCI-H23 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modMSLN (SEQ ID NO: 22), peptides comprising one or more EGFR tyrosine kinase inhibitor acquired resistance mutations selected from the group consisting of L692V, E709K, L718Q, G724S, T790M, C797S, L798I and L844V, one or more ALK tyrosine kinase inhibitor acquired resistance mutations selected from the group consisting of 1151Tins, C1156Y, I1171N, F1174L, V1180L, L1196M, G1202R, D1203N, S1206Y, F1245C, G1269A and R1275Q and modALK-IC (SEQ ID NO:94); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) LK-2 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In still another embodiment, provided herein is a method of treating NSCLC in a patient comprising administering to said patient a therapeutically effective amount of a unit dose of a NSCLC vaccine, wherein said unit dose comprises 6 compositions, wherein each composition comprises a cancer cell line selected from the group consisting of NCI-H460, A549, NCI-H520, NCI-H23, LK-2 and DMS 53; wherein: (a) NCI-H460 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modBORIS (SEQ ID NO: 20), peptides comprising one or more TP53 driver mutations selected from the group consisting of R110L, C141Y, G154V, V157F, R158L, R175H, C176F, H214R, Y220C, Y234C, M237I, G245V, R249M, I251F, R273L, R337L, one or more PIK3CA driver mutations selected from the group consisting of E542K and H1047R, one or more KRAS driver mutations selected from the group consisting of G12A and G13C (SEQ ID NO: 79); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) A549 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTBXT (SEQ ID NO: 18), modWT1 (SEQ ID NO: 18), peptides comprising one or more KRAS driver mutations selected from the group consisting of G12D and G12 (SEQ ID NO: 18), peptides comprising one or more EGFR activating mutations selected from the group consisting of D761 E762insEAFQ, A763 Y764insFQEA, A767 S768insSVA, S768 V769insVAS, V769 D770insASV, D770 N771insSVD, N771repGF, P772 H773insPR, H773 V774insH, V774 C775insHV, G719A, L858R and L861Q (SEQ ID NO: 82); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) NCI-H520 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) NCI-H23 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modMSLN (SEQ ID NO: 22), peptides comprising one or more EGFR tyrosine kinase inhibitor acquired resistance mutations selected from the group consisting of L692V, E709K, L718Q, G724S, T790M, C797S, L798I and L844V, one or more ALK tyrosine kinase inhibitor acquired resistance mutations selected from the group consisting of 1151Tins, C1156Y, I1171N, F1174L, V1180L, L1196M, G1202R, D1203N, S1206Y, F1245C, G1269A and R1275Q and modALK-IC (SEQ ID NO:94); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) LK-2 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In another embodiment, provided herein is a method of stimulating an immune response in a patient comprising administering to said patient a therapeutically effective amount of a unit dose of a colorectal cancer vaccine, wherein said unit dose comprises a first composition comprising cancer cell lines HCT15, HUTU80 and LS411N, and a second composition comprising cancer cell lines DMS 53, HCT116 and RKO wherein: (a) HCT15 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), and TGFβ1 shRNA (SEQ ID NO: 54); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) HUTU80 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modPSMA (SEQ ID NO: 30), and peptides comprising one or more driver mutation sequences selected from the group consisting of R273C of oncogene TP53, E542K of oncogene PIK3CA, R361H of oncogene SMAD4, R201H of oncogene GNAS, R505C of oncogene FBXW7, and R337C of oncogene ATM (SEQ ID NO: 116); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) LS411N is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) HCT116 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), modTBXT (SEQ ID NO: 18), modWT1 (SEQ ID NO: 18), and peptides comprising one or more driver mutation sequences selected from the group consisting of G12D and G12V of oncogene KRAS (SEQ ID NO: 77); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) RKO is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and peptides comprising one or more driver mutations sequences selected from the group consisting of R175H, G245S, and R248W of oncogene TP53, G12C of oncogene KRAS, R88Q, M1043I, and H1047Y of oncogene PIK3CA, S582L and R465H of oncogene FBXW7, S45F of oncogene CTNNB1), and V104M of oncogene ERBB3 (SEQ ID NO: 118); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In still another embodiment, provided herein is a method of treating colorectal cancer in a patient comprising administering to said patient a therapeutically effective amount of a unit dose of a colorectal cancer vaccine, wherein said unit dose comprises a first composition comprising cancer cell lines HCT15, HUTU80 and LS411N, and a second composition comprising cancer cell lines DMS 53, HCT116 and RKO wherein: (a) HCT15 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), and TGFβ1 shRNA (SEQ ID NO: 54); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) HUTU80 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modPSMA (SEQ ID NO: 30), and peptides comprising one or more driver mutation sequences selected from the group consisting of R273C of oncogene TP53, E542K of oncogene PIK3CA, R361H of oncogene SMAD4, R201H of oncogene GNAS, R505C of oncogene FBXW7, and R337C of oncogene ATM (SEQ ID NO: 116); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) LS411N is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) HCT116 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), modTBXT (SEQ ID NO: 18), modWT1 (SEQ ID NO: 18), and peptides comprising one or more driver mutation sequences selected from the group consisting of G12D and G12V of oncogene KRAS (SEQ ID NO: 77); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) RKO is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and peptides comprising one or more driver mutations sequences selected from the group consisting of R175H, G245S, and R248W of oncogene TP53, G12C of oncogene KRAS, R88Q, M1043I, and H1047Y of oncogene PIK3CA, S582L and R465H of oncogene FBXW7, S45F of oncogene CTNNB1), and V104M of oncogene ERBB3 (SEQ ID NO: 118); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In still another embodiment, provided herein is a method of stimulating an immune response in a patient comprising administering to said patient a therapeutically effective amount of a unit dose of a breast cancer vaccine, wherein said unit dose comprises 6 compositions, wherein each composition comprises a cancer cell line selected from the group consisting of CAMA-1, AU565, HS-578T, MCF-7, T47D and DMS 53; wherein: (a) CAMA-1 is modified to (i) express GM-CSF (SEQ ID NO: 52), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55), and modPSMA (SEQ ID NO: 30); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) AU565 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55), modTERT (SEQ ID NO: 28), and peptides comprising one or more driver mutation sequences selected from the group consisting of Y220C, R248W and R273H of oncogene TP53, and N345K, E542K, E726K and H1047L of oncogene PIK3CA (SEQ ID NO: 122); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) HS-578T is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) MCF-7 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) T47D is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), modTBXT (SEQ ID NO: 34), and modBORIS (SEQ ID NO: 34); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In yet another embodiment, provided herein is a method of treating breast cancer in a patient comprising administering to said patient a therapeutically effective amount of a unit dose of a breast cancer vaccine, wherein said unit dose comprises 6 compositions, wherein each composition comprises a cancer cell line selected from the group consisting of CAMA-1, AU565, HS-578T, MCF-7, T47D and DMS 53; wherein: (a) CAMA-1 is modified to (i) express GM-CSF (SEQ ID NO: 52), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55), and modPSMA (SEQ ID NO: 30); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) AU565 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ2 shRNA (SEQ ID NO: 55), modTERT (SEQ ID NO: 28), and peptides comprising one or more driver mutation sequences selected from the group consisting of Y220C, R248W and R273H of oncogene TP53, and N345K, E542K, E726K and H1047L of oncogene PIK3CA (SEQ ID NO: 122); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) HS-578T is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (d) MCF-7 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) T47D is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), modTBXT (SEQ ID NO: 34), and modBORIS (SEQ ID NO: 34); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52).

In yet another embodiment, provided herein is a method of preparing a composition comprising at least 1 modified cancer cell line capable of stimulating an immune response in a patient afflicted with cancer, wherein the cell line: (a) is known to express at least 5, 10, 15, or 20 or more TAAs associated with the cancer; and (b) is modified to (i) express or increase expression of at least 1 immunostimulatory factor, (ii) inhibit or decrease expression of at least 1 immunosuppressive factor. (iii) express or increase expression of at least 1 TAA that is either not expressed or minimally expressed by the cell line, optionally where the TAA or TAAs comprise one or more non-synonymous mutations (NSMs) or one or more neoepitopes. In still another embodiment, provided herein is a method of preparing a composition comprising at least 1 modified cancer cell line capable of stimulating an immune response in a patient afflicted with cancer, wherein the cell line: (a) is known to express at least 5, 10, 15, or 20 or more TAAs associated with the cancer; (b) is modified to (i) express or increase expression of at least 1 immunostimulatory factor, (ii) inhibit or decrease expression of at least 1 immunosuppressive factor, (iii) express or increase expression of at least 1 TAA that is either not expressed or minimally expressed by the cell line, optionally where the TAA or TAAs comprise one or more non-synonymous mutations (NSMs) or one or more neoepitopes; and optionally (c) is a cancer stem cell line. In still another embodiment, provided herein is a method of preparing a composition comprising at least 1 modified cancer cell line capable of stimulating an immune response in a patient afflicted with cancer, wherein the cell line: (a) is known to express at least 5, 10, 15, or 20 or more TAAs associated with the cancer; (b) is modified to (i) express or increase expression of at least 1 immunostimulatory factor, (ii) inhibit or decrease expression of at least 1 immunosuppressive factor, (iii) express or increase expression of at least 1 TAA that is either not expressed or minimally expressed by the cell line, optionally where the TAA or TAAs comprise one or more non-synonymous mutations (NSMs) or one or more neoepitopes; and optionally (c) is a cancer stem cell line; and optionally (d) is modified to express at least 1 peptide comprising at least 1 driver mutation; and optionally (e) is modified to express or increase expression of at least 1 peptide comprising at least 1 tumor fitness advantage mutation selected from the group consisting of an acquired tyrosine kinase inhibitor (TKI) resistance mutation, an EGFR activating mutation, and/or a modified ALK intracellular domain. In one embodiment, the cell line that is modified to express at least 1 peptide comprising at least 1 driver mutation is prepared according to the method of claim 28. In another embodiment, the at least one cell line is modified according to each of (a)-(d).

In other embodiments, an aforementioned method is provided further comprising administering to the subject a therapeutically effective dose of cyclophosphamide and/or a checkpoint inhibitor. In one embodiment, cyclophosphamide is administered orally at a dosage of 50 mg and the checkpoint inhibitor is pembrolizumab and is administered intravenously at a dosage of 200 mg.

The present disclosure provides, in one embodiment, a method of stimulating an immune response specific to tumor associated antigens (TAAs) associated with NSCLC in a human subject comprising: a. orally administering cyclophosphamide daily for one week at a dose of 50 mg/day; b. after said one week in (a), further administering a first dose of a vaccine comprising a first and second composition, wherein the first composition comprises therapeutically effective amounts of lung cancer cell lines NCI-H460, NCI-H520, and A549; wherein: (a) NCI-H460 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modBORIS (SEQ ID NO: 20), peptides comprising one or more TP53 driver mutations selected from the group consisting of R110L, C141Y, G154V, V157F, R158L, R175H, C176F, H214R, Y220C, Y234C, M237I, G245V, R249M, I251F, R273L, R337L, one or more PIK3CA driver mutations selected from the group consisting of E542K and H1047R, one or more KRAS driver mutations selected from the group consisting of G12A and G13C (SEQ ID NO: 79); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (b) A549 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modTBXT (SEQ ID NO: 18), modWT1 (SEQ ID NO: 18), peptides comprising one or more KRAS driver mutations selected from the group consisting of G12D and G12 (SEQ ID NO: 18), peptides comprising one or more EGFR activating mutations selected from the group consisting of D761 E762insEAFQ, A763 Y764insFQEA, A767 S768insSVA, S768 V769insVAS, V769 D770insASV, D770 N771insSVD, N771repGF, P772 H773insPR, H773 V774insH, V774 C775insHV, G719A, L858R and L861Q (SEQ ID NO: 82); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (c) NCI-H520 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and the second composition comprises therapeutically effective amounts of lung cancer cell lines DMS 53, LK-2, and NCI-H23; wherein (d) NCI-H23 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55), modMSLN (SEQ ID NO: 22), peptides comprising one or more EGFR tyrosine kinase inhibitor acquired resistance mutations selected from the group consisting of L692V, E709K, L718Q, G724S, T790M, C797S, L798I and L844V, one or more ALK tyrosine kinase inhibitor acquired resistance mutations selected from the group consisting of 1151Tins, C1156Y, I1171N, F1174L, V1180L, L1196M, G1202R, D1203N, S1206Y, F1245C, G1269A and R1275Q and modALK-IC (SEQ ID NO:94); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); (e) LK-2 is modified to (i) express GM-CSF (SEQ ID NO: 8), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), and TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); and (f) DMS 53 is modified to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 52); c. after said one week in (a), further administering via injection a first dose of a composition comprising pembrolizumab at a dosage of 200 mg; d. further administering subsequent doses of the first and second compositions at 3, 6, 9, 15, 21, and 27 weeks following administration of said first dose in (b), and wherein 50 mg of cyclophosphamide is orally administered for 7 days leading up to each subsequent dose; e. further administering intravenously subsequent doses of the composition comprising pembrolizumab at 3, 6, 9, 12, 15, 18, 21, 24, and 27 weeks following said first dose in (c) at a dosage of 200 mg; wherein the first composition is administered intradermally in the subject's arm, and the second composition is administered intradermally in the subject's thigh.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A-E show immune responses for seven HLA diverse donors to eight TP53 driver mutations encoded by five peptides (FIG. 1A), three PTEN driver mutations encoded by two peptides (FIG. 1B), one PIK3R1 driver mutation encoded by one peptide (FIG. 1C), two PIK3CA driver mutations encoded by one peptide (FIG. 1D), and one EGFR driver mutation encoded by one peptide expressed modified GB-1 compared to unmodified GB-1.

FIG. 2 shows immune responses for six HLA diverse donors to seven EGFR driver mutations encoded by seven peptides expressed by modified LN-229 compared to unmodified LN-229.

FIG. 3 A-C shows immune responses for six HLA diverse donors to three TP53 driver mutations encoded by three peptides, three SPOP driver mutations encoded by three peptides and three AR driver mutations encoded by three peptides expressed by modified PC3 compared to unmodified PC3.

FIGS. 4 A-D show endogenous expression of twenty-four prioritized NSCLC antigens (FIG. 4A) and nine NSCLC CSC-like markers (FIG. 4B) by NSCLC vaccine cell lines and expression of the twenty-four prioritized NSCLC antigens in patient tumor samples (FIG. 4C) and the number of NSCLC antigens expressed by the NSCLC vaccine cell lines also expressed by NSCLC patient tumors (FIG. 4D).

FIGS. 5 A-C show expression of modWT1 (FIG. 5A) and modTBXT (FIG. 5B) inserted in the NSCLC vaccine-A A549 cell line and modMSLN inserted into the NSCLC vaccine-B NCI-H23 cell line (FIG. 5C).

FIGS. 6A-B show immune responses for six HLA diverse donors to eight NSCLC TAAs induced by DMS 53 modified to reduce expression of CD276, reduce secretion of TGFβ2, and express GMCSF and membrane bound CD40L and DMS 53 modified to reduce expression of CD276, reduce secretion of TGFβ1 and TGFβ2, and express GM-CSF, membrane bound CD40L and IL-12 (6A) and the total antigen specific magnitude of IFNγ for individual donors summarized in FIG. 6A.

FIGS. 7 A-D show IFNγ responses to BORIS (FIG. 7A), TBXT (FIG. 7B), and WT1 (FIG. 7C) induced by NSCLC-vaccine A and MSLN (FIG. 7D) induced by NSCLC vaccine-B are higher in magnitude compared to unmodified controls.

FIGS. 8 A-G show IFNγ responses induced by NSCLC vaccine-A to neoepitopes included in the modBORIS (FIGS. 8A-C), modWT1 (FIG. 8D) and modTXT (FIGS. 8E-G) antigens compared to unmodified controls.

FIGS. 9 A-C show antigen specific IFNγ responses for six healthy donors induced by the unit dose of the NSCLC vaccine (FIG. 9A), NSCLC vaccine-A (FIG. 9B), and NSCLC vaccine-B (FIG. 9C) compared to unmodified controls.

FIG. 10 shows antigen specific IFNγ responses induced by the unit dose of the NSCLC vaccine in individual donors compared to unmodified controls summarized in FIG. 9A.

FIGS. 11 A-D show immune responses in eight HLA diverse donors to sixteen TP53 driver mutations encoded by nine peptides (FIG. 11A), two PIK3CA driver mutations encoded by two peptides (FIG. 11B), and two KRAS driver mutations encoded by one peptide (FIG. 11C) introduced into the NSCLC vaccine-A NCI-H460 cell line and two KRAS driver mutations encoded by two peptides introduced into the NSCLC vaccine-A A549 cell line (FIG. 11D) compared to unmodified controls.

FIG. 12 shows immune responses in eight HLA diverse donors to twelve EGFR activating mutations encoded by twelve peptides introduced into the NSCLC vaccine-A A549 cell line compared to unmodified controls.

FIG. 13 shows immune responses in eight HLA diverse donors to eight NSCLC EGFR TKI acquired resistance mutations encoded by five peptide sequences introduced into the NSCLC vaccine-B NCI-H23 cell line compared to unmodified controls.

FIG. 14 shows immune responses in eight HLA diverse donors to twelve NSCLC ALK TKI acquired resistance mutations encoded by five peptide sequences and modALK-IC introduced into the NSCLC vaccine-B NCI-H23 cell line compared to unmodified controls.

FIGS. 15 A-B show endogenous expression of twenty prioritized CRC antigens by vaccine cell lines (FIG. 15A) and the number of the twenty prioritized antigens expressed by the CRC vaccine also expressed by CRC patient tumors (FIG. 15B)

FIGS. 16 A-J show expression of and IFNγ responses to antigens introduced into CRC vaccine cell lines compared to unmodified controls. Expression of modPSMA by HuTu80 (FIG. 16A) and IFNγ responses to PSMA (FIG. 16F) in CRC-vaccine A. Expression of modTBXT, modWT1, KRAS G12D and KRAS G12V by HCT-116 (FIG. 16B-D) and IFNγ responses to TBXT (FIG. 2G), WT1 (FIG. 16H), KRAS G12D (FIG. 16I) and KRAS G12D (FIG. 16J) in CRC-vaccine B.

FIG. 17 A-C show antigen specific IFNγ responses for six HLA-diverse donors induced by the unit dose of the CRC vaccine (FIG. 17A), CRC vaccine-A (FIG. 17B) and CRC vaccine-B (FIG. 17C) compared to unmodified controls.

FIG. 18 shows antigen specific IFNγ responses induced by the unit dose of the CRC vaccine and unmodified controls for the six individual donors summarized in FIG. 17A.

FIG. 19 shows IFNγ responses for six HLA-diverse donors to three TP53 driver mutations encoded by two peptides, one KRAS driver mutation encoded by one peptide, three PIK3CA driver mutations encoded by two peptides, two FBXW7 driver mutations encoded by two peptides, one CTNNB1 driver mutation encoded by one peptide and one ERBB3 driver mutation encoded by one peptide expressed by modified RKO and unmodified RKO.

FIG. 20 shows IFNγ responses for six HLA-diverse donors to peptides encoding one TP53 driver mutation by one peptide, one PIK3CA driver mutation by one peptide, one FBXW7 driver mutation by one peptide, one SMAD4 driver mutation y one peptide, one GNAS driver mutation encoded by one peptide and one ATM driver mutation encoded by one peptide expressed by modified Hutu80 compared to unmodified Hutu80.

FIGS. 21 A-B show endogenous expression of prioritized twenty-two prioritized (FIG. 21A) by BRC vaccine cell lines and expression of these antigens by breast cancer patient tumors (FIG. 21B).

FIGS. 22 A-H show expression of modPSMA by CAMA-1 (FIG. 22A) and IFNγ responses to PSMA (FIG. 22E), show expression of modTERT by AU565 (FIG. 22B) and IFNγ responses to TERT (FIG. 22F), and show expression of modTBXT (FIG. 22C) and modBORIS (FIG. 22D) by T47D and IFNγ responses to TBXT (FIG. 22G) and BORIS (FIG. 22H).

FIGS. 23 A-C show antigen specific IFNγ responses for eight HLA-diverse donors induced by the unit dose of the BRC vaccine (FIG. 23A), BRC vaccine-A (FIG. 23B) and BRC vaccine-B (FIG. 23C) compared to unmodified controls.

FIG. 24 shows antigen specific IFNγ responses induced by the unit dose of the CRC vaccine and unmodified controls for the eight individual donors summarized in FIG. 23A.

FIGS. 25 A-B show IFNγ responses for six HLA-diverse donors to three TP53 driver mutations encoded by three peptides (FIG. 25A) and four PIK3CA driver mutations (FIG. 25B) encoded by four peptides expressed by modified AU565 compared to unmodified AU565.

DETAILED DESCRIPTION

Embodiments of the present disclosure provide a platform approach to cancer vaccination that provides both breadth, in terms of the types of cancer amenable to treatment by the compositions, methods, and regimens disclosed, and magnitude, in terms of the immune responses elicited by the compositions, methods, and regimens disclosed.

In various embodiments of the present disclosure, intradermal injection of an allogenic whole cancer cell vaccine induces a localized inflammatory response recruiting immune cells to the injection site. Without being bound to any theory or mechanism, following administration of the vaccine, antigen presenting cells (APCs) that are present locally in the skin (vaccine microenvironment, VME), such as Langerhans cells (LCs) and dermal dendritic cells (DCs), uptake vaccine cell components by phagocytosis and then migrate through the dermis to a draining lymph node. At the draining lymph node, DCs or LCs that have phagocytized the vaccine cell line components can prime naïve T cells and B cells. Priming of naïve T and B cells initiates an adaptive immune response to tumor associated antigens (TAAs) expressed by the vaccine cell lines. In some embodiments of the present disclosure, the priming occurs in vivo and not in vitro or ex vivo. In embodiments of the vaccine compositions provided herein, the multitude of TAAs expressed by the vaccine cell lines are also expressed a subject's tumor. Expansion of antigen specific T cells at the draining lymph node and the trafficking of these T cells to the tumor microenvironment (TME) can initiate a vaccine-induced anti-tumor response.

Immunogenicity of an allogenic vaccine can be enhanced through genetic modifications of the cell lines comprising the vaccine composition to introduce TAAs (native/wild-type or designed/mutated) as described herein. Immunogenicity of an allogenic vaccine can be enhanced through genetic modifications of the cell lines comprising the vaccine composition to express one or more tumor fitness advantage mutations, including but not limited to acquired tyrosine kinase inhibitor (TKI) resistance mutations, EGFR activating mutations, and/or modified ALK intracellular domain(s). Immunogenicity of an allogenic vaccine can be enhanced through genetic modifications of the cell lines comprising the vaccine composition to introduce driver mutations as described herein. Immunogenicity of an allogenic vaccine can be further enhanced through genetic modifications of the cell lines comprising the vaccine composition to reduce expression of immunosuppressive factors and/or increase the expression or secretion of immunostimulatory signals. Modulation of these factors can enhance the uptake of vaccine cell components by LCs and DCs in the dermis, facilitate the trafficking of DCs and LCs to the draining lymph node, and enhance effector T cell and B cell priming in the draining lymph node, thereby providing more potent anti-tumor responses.

In various embodiments, the present disclosure provides an allogeneic whole cell cancer vaccine platform that includes compositions and methods for treating cancer, and/or preventing cancer, and/or stimulating an immune response. Criteria and methods according to embodiments of the present disclosure include without limitation: (i) criteria and methods for cell line selection for inclusion in a vaccine composition, (ii) criteria and methods for combining multiple cell lines into a therapeutic vaccine composition, (iii) criteria and methods for making cell line modifications, and (iv) criteria and methods for administering therapeutic compositions with and without additional therapeutic agents. In some embodiments, the present disclosure provides an allogeneic whole cell cancer vaccine platform that includes, without limitation, administration of multiple cocktails comprising combinations of cell lines that together comprise one unit dose, wherein unit doses are strategically administered over time, and additionally optionally includes administration of other therapeutic agents such as cyclophosphamide and additionally optionally a checkpoint inhibitor and additionally optionally a retinoid (e.g., ATRA).

The present disclosure provides, in some embodiments, compositions and methods for tailoring a treatment regimen for a subject based on the subject's tumor type. In some embodiments, the present disclosure provides a cancer vaccine platform whereby allogeneic cell line(s) are identified and optionally modified and administered to a subject. In various embodiments, the tumor origin (primary site) of the cell line(s), the amount and number of TAAs expressed by the cell line(s), the number of cell line modifications, and the number of cell lines included in a unit dose are each customized based on the subject's tumor type, stage of cancer, and other considerations. As described herein, the tumor origin of the cell lines may be the same or different than the tumor intended to be treated. In some embodiments, the cancer cell lines may be cancer stem cell lines.

Definitions

In this disclosure, “comprises”, “comprising”, “containing”, “having”, and the like have the meaning ascribed to them in U.S. patent law and mean “includes”, “including”, and the like; the terms “consisting essentially of” or “consists essentially” likewise have the meaning ascribed in U.S. patent law and these terms are open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited are not changed by the presence of more than that which is recited, but excluding prior art embodiments.

Unless specifically otherwise stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

The terms “cell”, “cell line”, “cancer cell line”, “tumor cell line”, and the like as used interchangeably herein refers to a cell line that originated from a cancerous tumor as described herein, and/or originates from a parental cell line of a tumor originating from a specific source/organ/tissue. In some embodiments the cancer cell line is a cancer stem cell line as described herein. In certain embodiments, the cancer cell line is known to express or does express multiple tumor-associated antigens (TAAs) and/or tumor specific antigens (TSAs). In some embodiments of the disclosure, a cancer cell line is modified to express, or increase expression of, one or more TAAs. In certain embodiments, the cancer cell line includes a cell line following any number of cell passages, any variation in growth media or conditions, introduction of a modification that can change the characteristics of the cell line such as, for example, human telomerase reverse transcriptase (hTERT) immortalization, use of xenografting techniques including serial passage through xenogenic models such as, for example, patient-derived xenograft (PDX) or next generation sequencing (NGS) mice, and/or co-culture with one or more other cell lines to provide a mixed population of cell lines. As used herein, the term “cell line” includes all cell lines identified as having any overlap in profile or segment, as determined, in some embodiments, by Short Tandem Repeat (STR) sequencing, or as otherwise determined by one of skill in the art. As used herein, the term “cell line” also encompasses any genetically homogeneous cell lines, in that the cells that make up the cell line(s) are clonally derived from a single cell such that they are genetically identical. This can be accomplished, for example, by limiting dilution subcloning of a heterogeneous cell line. The term “cell line” also encompasses any genetically heterogeneous cell line, in that the cells that make up the cell line(s) are not expected to be genetically identical and contain multiple subpopulations of cancer cells. Various examples of cell lines are described herein. Unless otherwise specifically stated, the term “cell line” or “cancer cell line” encompasses the plural “cell lines.”

As used herein, the term “tumor” refers to an accumulation or mass of abnormal cells. Tumors may be benign (non-cancerous), premalignant (pre-cancerous, including hyperplasia, atypia, metaplasia, dysplasia and carcinoma in situ), or malignant (cancerous). It is well known that tumors may be “hot” or “cold”. By way of example, melanoma and lung cancer, among others, demonstrate relatively high response rates to checkpoint inhibitors and are commonly referred to as “hot” tumors. These are in sharp contrast to tumors with low immune infiltrates called “cold” tumors or non-T-cell-inflamed cancers, such as those from the prostate, pancreas, glioblastoma, and bladder, among others. In some embodiments, the compositions and methods provided herein are useful to treat or prevent cancers with associated hot tumors. In some embodiments, the compositions and methods provided herein are useful to treat or prevent cancers with cold tumors. Embodiments of the vaccine compositions of the present disclosure can be used to convert cold (i.e., treatment-resistant or refractory) cancers or tumors to hot (i.e., amenable to treatment, including a checkpoint inhibition-based treatment) cancers or tumors. Immune responses against cold tumors are dampened because of the lack of neoepitopes associated with low mutational burden. In various embodiments, the compositions described herein comprise a multitude of potential neoepitopes arising from point-mutations that can generate a multitude of exogenous antigenic epitopes. In this way, the patients' immune system can recognize these epitopes as non-self, subsequently break self-tolerance, and mount an anti-tumor response to a cold tumor, including induction of an adaptive immune response to wide breadth of antigens (See Leko, V. et al. J Immunol (2019)).

Cancer stem cells are responsible for initiating tumor development, cell proliferation, and metastasis and are key components of relapse following chemotherapy and radiation therapy. In certain embodiments, a cancer stem cell line or a cell line that displays cancer stem cell characteristics is included in one or more of the vaccine compositions. As used herein, the phrase “cancer stem cell” (CSC) or “cancer stem cell line” refers to a cell or cell line within a tumor that possesses the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor. CSCs are highly resistant to traditional cancer therapies and are hypothesized to be the leading driver of metastasis and tumor recurrence. To clarify, a cell line that displays cancer stem cell characteristics is included within the definition of a “cancer stem cell”. Exemplary cancer stem cell markers identified by primary tumor site are provided in Table 2 and described herein. Cell lines expressing one or more of these markers are encompassed by the definition of “cancer stem cell line”. Exemplary cancer stem cell lines are described herein, each of which are encompassed by the definition of “cancer stem cell line”.

As used herein, the phrase “each cell line or a combination of cell lines” refers to, where multiple cell lines are provided in a combination, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more or the combination of the cell lines. As used herein, the phrase “each cell line or a combination of cell lines have been modified” refers to, where multiple cell lines are provided in combination, modification of one, some, or all cell lines, and also refers to the possibility that not all of the cell lines included in the combination have been modified. By way of example, the phrase “a composition comprising a therapeutically effective amount of at least 2 cancer cell lines, wherein each cell line or a combination of the cell lines comprises cells that have been modified . . . ” means that each of the two cell lines has been modified or one of the two cell lines has been modified. By way of another example, the phrase “a composition comprising a therapeutically effective amount of at least 3 cancer cell lines, wherein each cell line or a combination of the cell lines comprises cells that have been modified . . . ” means that each (i.e., all three) of the cell lines have been modified or that one or two of the three cell lines have been modified.

The term “oncogene” as used herein refers to a gene involved in tumorigenesis. An oncogene is a mutated (i.e., changed) form of a gene that contributes to the development of a cancer. In their normal, unmutated state, oncogenes are called proto-oncogenes, and they play roles in the regulation of normal cell growth and cell division.

The term “driver mutation” as used herein, for example in the context of an oncogene, refers to a somatic mutation that initiates, alone or in combination with other mutations, tumorogenesis and/or confers a fitness advantage to tumor cells. Driver mutations typically occur early in cancer evolution and are therefore found in all or a subset of tumor cells across cancer pateints (i.e., at a high frequency). The phrase “wherein the oncogene driver mutation is in one or more oncogenes” as used herein means the driver mutation (e.g., the missense mutation) occurs within the polynucleotide sequence (and thus the corresponding amino acid sequence) of the oncogene or oncogenes.

The term “tumor fitness advantage mutation” as used herein refers to one or more mutations that result in or cause a rapid expansion of a tumor (e.g., a collection of tumor cells) or tumor cell (e.g., tumor cell clone) harboring such mutations. In some embodiments, tumor fitness advantage mutations include, but are not limited to, (oncogene) driver mutations as described herein, acquired tyrosine kinase inhibitor (TKI) resistance mutations as described herein, and activating mutations as described herein. The term “acquired tyrosine kinase inhibitor (TKI) resistance mutation” as used herein refers to mutations that account for TKI resistance and cause tumor cells to effectively escape TKI treatment. In some embodiments provided herein, the mutation or mutations occur in the ALK gene (i.e., “ALK acquired tyrosine kinase inhibitor (TKI) resistance mutation”) and/or in the EGFR gene (i.e., “EGFR acquired tyrosine kinase inhibitor (TKI) resistance mutation”). The term “EGFR activating mutation” as used herein refers to a mutation resulting in constitutive activation of EGFR. Exemplary driver/acquired resistance/activating mutations (e.g., point mutations, substitutions, etc.) are provided herein.

The term “modified ALK intracellular domain (modALK-IC)” as used herein refers to neoepitope-containing ALK C-terminus intracelluar tyrosine kinase domain, which mediates the ligand-dependent dimerization and/or oligomerization of ALK, resulting in constitutive kinase activity and promoting downstream signaling pathways involved in the proliferation and survival of tumor cells.

As used herein, the phrase “identifying one or more . . . mutations” for example in the process for preparing compositions useful for stimulating an immune response or treating cancer as described herein, refers to newly identifying, identifying within a database or dataset or otherwise using a series of criteria or one or more components thereof as described herein and, optionally, selecting the oncogene or mutation for use or inclusion in a vaccine composition as described herein.

The phrase “ . . . cells that express at least [n] tumor associated antigens (TAAs) associated with a cancer of a subject intended to receive said composition.” as used herein refers to cells that express, either natively or by way of genetic modification, the designated number of TAAs and wherein said same TAAs are expressed or known to be expressed by cells of a patient's tumor. The expression of specific TAAs by cells of a patient's tumor may be determined by assay, surgical procedures (e.g., biopsy), or other methods known in the art. In other embodiments, a clinician may consult the Cancer Cell Line Encyclopedia (CCLE) and other known resources to identify a list of TAAs known to be expressed by cells of a particular tumor type.

As used herein, the phrase “ . . . wherein the cell lines comprise cells that collectively express at least [15] tumor associated antigens (TAAs) associated with the cancer . . . ” refers to a composition or method employing multiple cell lines and wherein the combined total of TAAs expressed by the multiple cell lines is at least the recited number.

As used herein, the phrase “ . . . that is either not expressed or minimally expressed . . . ” means that the referenced gene or protein (e.g., a TAA or an immunosuppressive protein or an immunostimulatory protein) is not expressed by a cell line or is expressed at a low level, where such level is inconsequential to or has a limited impact on immunogenicity. For example, it is readily appreciated in the art that a TAA may be present or expressed in a cell line in an amount insufficient to have a desired impact on the therapeutic effect of a vaccine composition including said cell line. In such a scenario, the present disclosure provides compositions and methods to increase expression of such a TAA. Assays for determining the presence and amount of expression are well known in the art and described herein.

As used herein, the term “equal” generally means the same value+/−10%. In some embodiments, a measurement, such as number of cells, etc., can be +/−1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. Similarly, as used herein and as related to amino acid position or nucleotide position, the term “approximately” refers to within 1, 2, 3, 4, or 5 such residues. With respect to the number of cells, the term “approximately” refers to +/−1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%.

As used herein, the phrase “ . . . wherein said composition is capable of stimulating a 1.3-fold increase in IFNγ production compared to unmodified cancer cell lines . . . ” means, when compared to a composition of the same cell line or cell lines that has/have not been modified, the composition comprising a modified cell line or modified cell lines is capable of stimulating at least 1.3-fold more IFNγ production. In this example, “at least 1.3” means 1.3, 1.4, 1.5, etc., or higher. This definition is used herein with respect to other values of IFNγ production, including, but not limited to, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 4.0, or 5.0-fold or higher increase in IFNγ production compared to unmodified cancer cell lines (e.g., a modified cell line compared to an modified cell line, a composition of 2 or 3 modified cell lines (e.g., a vaccine composition) compared cell lines to the same composition comprising unmodified cell lines, or a unit dose comprising 6 modified cell lines compared to the same unit dose comprising unmodified cell lines). In other embodiments, the IFNγ production is increased by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25-fold or higher compared to unmodified cancer cell lines. Similarly, in various embodiments, the present disclosure provides compositions of modified cells or cell lines that are compared to unmodified cells or cell lines on the basis of TAA expression, immunostimulatory factor expression, immunosuppressive factor expression, and/or immune response stimulation using the methods provided herein and the methods known in the art including, but not limited to, ELISA, IFNγ ELISpot, and flow cytometry.

As used herein, the phrase “fold increase” refers to the change in units of expression or units of response relative to a control. By way of example, ELISA fold change refers to the level of secreted protein detected for the modified cell line divided by the level of secreted protein detected, or the lower limit of detection, by the unmodified cell line. In another example, fold change in expression of an antigen by flow cytometry refers to the mean fluorescence intensity (MFI) of expression of the protein by a modified cell line divided by the MFI of the protein expression by the unmodified cell line. IFNγ ELISpot fold change refers to the average IFNγ spot-forming units (SFU) induced across HLA diverse donors by the test variable divided by the average IFNγ SFU induced by the control variable. For example, the average total antigen specific IFNγ SFU across donors by a composition of three modified cell lines divided by the IFNγ SFU across the same donors by a composition of the same three unmodified cell lines.

In some embodiments, the fold increase in IFNγ production will increase as the number of modifications (e.g., the number of immunostimulatory factors and the number of immunosuppressive factors) is increased in each cell line. In some embodiments, the fold increase in IFNγ production will increase as the number of cell lines (and thus, the number of TAAs), whether modified or unmodified, is increased. The fold increase in IFNγ production, in some embodiments, is therefore attributed to the number of TAAs and the number of modifications.

As used herein, the term “modified” means genetically modified or changed to express, overexpress, increase, decrease, or inhibit the expression of one or more protein or nucleic acid. As described herein, exemplary proteins include, but are not limited to immunostimulatory factors. Exemplary nucleic acids include sequences that can be used to knockdown (KD) (i.e., decrease expression of) or knockout (KO) (i.e., completely inhibit expression of) immunosuppressive factors. As used herein, the term “decrease” is synonymous with “reduce” or “partial reduction” and may be used in association with gene knockdown. Likewise, the term “inhibit” is synonymous with “complete reduction” and may be used in the context of a gene knockout to describe the complete excision of a gene from a cell.

Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive.

As used herein, the terms “patient”, “subject”, “recipient”, and the like are used interchangeably herein to refer to any mammal, including humans, non-human primates, domestic and farm animals, and other animals, including, but not limited to dogs, horses, cats, cattle, sheep, pigs, mice, rats, and goats. Exemplary subjects are humans, including adults, children, and the elderly. In some embodiments, the subject can be a donor.

The terms “treat”, “treating”, “treatment”, and the like, as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting the process of disease, disorder or condition to which such term applies, or one or more symptoms of such disease, disorder or condition and includes the administration of any of the compositions, pharmaceutical compositions, or dosage forms described herein, to prevent the onset of the symptoms or the complications, alleviate the symptoms or the complications, or eliminate the disease, condition, or disorder. As used herein, treatment can be curative or ameliorating.

As used herein, “preventing” means preventing in whole or in part, controlling, reducing, or halting the production or occurrence of the thing or event to which such term applies, for example, a disease, disorder, or condition to be prevented.

Embodiments of the methods and compositions provided herein are useful for preventing a tumor or cancer, meaning the occurrence of the tumor is prevented or the onset of the tumor is significantly delayed. In some embodiments, the methods and compositions are useful for treating a tumor or cancer, meaning that tumor growth is significantly inhibited as demonstrated by various techniques well-known in the art such as, for example, by a reduction in tumor volume. Tumor volume may be determined by various known procedures, (e.g., obtaining two dimensional measurements with a dial caliper). Preventing and/or treating a tumor can result in the prolonged survival of the subject being treated.

As used herein, the term “stimulating”, with respect to an immune response, is synonymous with “promoting”, “generating”, and “eliciting” and refers to the production of one or more indicators of an immune response. Indicators of an immune response are described herein. Immune responses may be determined and measured according to the assays described herein and by methods well-known in the art.

The phrases “therapeutically effective amount”, “effective amount”, “immunologically effective amount”, “anti-tumor effective amount”, and the like, as used herein, indicate an amount necessary to administer to a subject, or to a cell, tissue, or organ of a subject, to achieve a therapeutic effect, such as an ameliorating or a curative effect. The therapeutically effective amount is sufficient to elicit the biological or medical response of a cell, tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, clinician, or healthcare provider. For example, a therapeutically effective amount of a composition is an amount of cell lines, whether modified or unmodified, sufficient to stimulate an immune response as described herein. In certain embodiments, a therapeutically effective amount of a composition is an amount of cell lines, whether modified or unmodified, sufficient to inhibit the growth of a tumor as described herein. Determination of the effective amount or therapeutically effective amount is, in certain embodiments, based on publications, data or other information such as, for example, dosing regimens and/or the experience of the clinician.

The terms “administering”, “administer”, “administration”, and the like, as used herein, refer to any mode of transferring, delivering, introducing, or transporting a therapeutic agent to a subject in need of treatment with such an agent. Such modes include, but are not limited to, oral, topical, intravenous, intraarterial, intraperitoneal, intramuscular, intratumoral, intradermal, intranasal, and subcutaneous administration.

As used herein, the term “vaccine composition” refers to any of the vaccine compositions described herein containing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) cell lines. As described herein, one or more of the cell lines in the vaccine composition may be modified. In certain embodiments, one or more of the cell lines in the vaccine composition may not be modified. The terms “vaccine”, “tumor cell vaccine”, “cancer vaccine”, “cancer cell vaccine”, “whole cancer cell vaccine”, “vaccine composition”, “composition”, “cocktail”, “vaccine cocktail”, and the like are used interchangeably herein. In some embodiments, the vaccine compositions described herein are useful to treat or prevent cancer. In some embodiments, the vaccine compositions described herein are useful to stimulate or elicit an immune response. In such embodiments, the term “immunogenic composition” is used. In some embodiments, the vaccine compositions described herein are useful as a component of a therapeutic regimen to increase immunogenicity of said regimen.

The terms “dose” or “unit dose” as used interchangeably herein refer to one or more vaccine compositions that comprise therapeutically effective amounts of one more cell lines. As described herein, a “dose” or “unit dose” of a composition may refer to 1, 2, 3, 4, 5, or more distinct compositions or cocktails. In some embodiments, a unit dose of a composition refers to 2 distinct compositions administered substantially concurrently (i.e., immediate series). In exemplary embodiments, one dose of a vaccine composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 separate vials, where each vial comprises a cell line, and where cell lines, each from a separate vial, are mixed prior to administration. In some embodiments, a dose or unit dose includes 6 vials, each comprising a cell line, where 3 cell lines are mixed and administered at one site, and the other 3 cell lines are mixed and administered at a second site. Subsequent “doses” may be administered similarly. In still other embodiments, administering 2 vaccine cocktails at 2 sites on the body of a subject for a total of 4 concurrent injections is contemplated.

As used herein, the term “cancer” refers to diseases in which abnormal cells divide without control and are able to invade other tissues. Thus, as used herein, the phrase “ . . . associated with a cancer of a subject” refers to the expression of tumor associated antigens, neoantigens, or other genotypic or phenotypic properties of a subject's cancer or cancers. TAAs associated with a cancer are TAAs that expressed at detectable levels in a majority of the cells of the cancer. Expression level can be detected and determined by methods described herein. There are more than 100 different types of cancer. Most cancers are named for the organ or type of cell in which they start; for example, cancer that begins in the colon is called colon cancer; cancer that begins in melanocytes of the skin is called melanoma. Cancer types can be grouped into broader categories. In some embodiments, cancers may be grouped as solid (i.e., tumor-forming) cancers and liquid (e.g., cancers of the blood such as leukemia, lymphoma and myeloma) cancers. Other categories of cancer include: carcinoma (meaning a cancer that begins in the skin or in tissues that line or cover internal organs, and its subtypes, including adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma); sarcoma (meaning a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue); leukemia (meaning a cancer that starts in blood-forming tissue (e.g., bone marrow) and causes large numbers of abnormal blood cells to be produced and enter the blood; lymphoma and myeloma (meaning cancers that begin in the cells of the immune system); and central nervous system cancers (meaning cancers that begin in the tissues of the brain and spinal cord). The term myelodysplastic syndrome refers to a type of cancer in which the bone marrow does not make enough healthy blood cells (white blood cells, red blood cells, and platelets) and there are abnormal cells in the blood and/or bone marrow. Myelodysplastic syndrome may become acute myeloid leukemia (AML). By way of non-limiting examples, the compositions and methods described herein are used to treat and/or prevent the cancer described herein, including in various embodiments, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), prostate cancer, breast cancer, triple negative breast cancer, metastatic breast cancer, ductal carcinoma in situ, invasive breast cancer, inflammatory breast cancer, Paget disease, breast angiosarcoma, phyllodes tumor, testicular cancer, colorectal cancer, bladder cancer, gastric cancer, head and neck cancer, liver cancer, renal cancer, glioma, gliosarcoma, astrocytoma, ovarian cancer, neuroendocrine cancer, pancreatic cancer, esophageal cancer, endometrial cancer, melanoma, mesothelioma, and/or hepatocellular cancers.

Examples of carcinomas include, without limitation, giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in an adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor; branchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; non-encapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease; mammary acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; sertoli cell carcinoma; embryonal carcinoma; and choriocarcinoma.

Examples of sarcomas include, without limitation, glomangiosarcoma; sarcoma; fibrosarcoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyo sarcoma; alveolar rhabdomyo sarcoma; stromal sarcoma; carcinosarcoma; synovial sarcoma; hemangiosarcoma; kaposi's sarcoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; myeloid sarcoma; and mast cell sarcoma.

Examples of leukemias include, without limitation, leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; and hairy cell leukemia.

Examples of lymphomas and myelomas include, without limitation, malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; and multiple myeloma.

Examples of brain/spinal cord cancers include, without limitation, pinealoma, malignant; chordoma; glioma, gliosarcoma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; and neurilemmoma, malignant.

Examples of other cancers include, without limitation, a thymoma; an ovarian stromal tumor; a thecoma; a granulosa cell tumor; an androblastoma; a leydig cell tumor; a lipid cell tumor; a paraganglioma; an extra-mammary paraganglioma; a pheochromocytoma; blue nevus, malignant; fibrous histiocytoma, malignant; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; mesothelioma, malignant; dysgerminoma; teratoma, malignant; struma ovarii, malignant; mesonephroma, malignant; hemangioendothelioma, malignant; hemangiopericytoma, malignant; chondroblastoma, malignant; granular cell tumor, malignant; malignant histiocytosis; and immunoproliferative small intestinal disease.

All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

Vaccine Compositions

The present disclosure is directed to a platform approach to cancer vaccination that provides breadth, with regard to the scope of cancers and tumor types amenable to treatment with the compositions, methods, and regimens disclosed, as well as magnitude, with regard to the level of immune responses elicited by the compositions and regimens disclosed. Embodiments of the present disclosure provide compositions comprising cancer cell lines. In some embodiments, the cell lines have been modified as described herein.

The compositions of the disclosure are designed to increase immunogenicity and/or stimulate an immune response. For example, in some embodiments, the vaccines provided herein increase IFNγ production and the breadth of immune responses against multiple TAAs (e.g., the vaccines are capable of targeting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more TAAs, indicating the diversity of T cell receptor (TCR) repertoire of these anti-TAA T cell precursors. In some embodiments, the immune response produced by the vaccines provided herein is a response to more than one epitope associated with a given TAA (e.g., the vaccines are capable of targeting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 epitopes or more on a given TAA), indicating the diversity of TCR repertoire of these anti-TAA T cell precursors.

This can be accomplished in certain embodiments by selecting cell lines that express numerous TAAs associated with the cancer to be treated; knocking down or knocking out expression of one or more immunosuppressive factors that facilitates tumor cell evasion of immune system surveillance; expressing or increasing expression of one or more immunostimulatory factors to increase immune activation within the vaccine microenvironment (VME); increasing expression of one or more tumor-associated antigens (TAAs) to increase the scope of relevant antigenic targets that are presented to the host immune system, optionally where the TAA or TAAs are designed or enhanced (e.g., modified by mutation) and comprise, for example, non-synonymous mutations (NSMs) and/or neoepitopes; administering a vaccine composition comprising at least 1 cancer stem cell; and/or any combination thereof.

As described herein, in some embodiments the cell lines are optionally additionally modified to express tumor fitness advantage mutations, including but not limited to acquired tyrosine kinase inhibitor (TKI) resistance mutations, EGFR activating mutations, and/or modified ALK intracellular domain(s), and/or driver mutations.

The one or more cell lines of the vaccine composition can be modified to reduce production of more than one immunosuppressive factor (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more immunosuppressive factors). The one or more cell lines of a vaccine can be modified to increase production of more than one immunostimulatory factor (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more immunostimulatory factors). The one or more cell lines of the vaccine composition can naturally express, or be modified to express more than one TAA, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more TAAs.

The vaccine compositions can comprise cells from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cell lines. Further, as described herein, cell lines can be combined or mixed, e.g., prior to administration. In some embodiments, production of one or more immunosuppressive factors from one or more or the combination of the cell lines can be reduced or eliminated. In some embodiments, production of one or more immunostimulatory factors from one or more or the combination of the cell lines can be added or increased. In certain embodiments, the one or more or the combination of the cell lines can be selected to express a heterogeneity of TAAs. In some embodiments, the cell lines can be modified to increase the production of one or more immunostimulatory factors, TAAs, and/or neoantigens. In some embodiments, the cell line selection provides that a heterogeneity of HLA supertypes are represented in the vaccine composition. In some embodiments, the cells lines are chosen for inclusion in a vaccine composition such that a desired complement of TAAs are represented.

In various embodiments, the vaccine composition comprises a therapeutically effective amount of cells from at least one cancer cell line, wherein the cell line or the combination of cell lines expresses more than one of the TAAs of Tables 9-25. In some embodiments, a vaccine composition is provided comprising a therapeutically effective amount of cells from at least two cancer cell lines, wherein each cell line or the combination of cell lines expresses at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten of the TAAs of Tables 9-25. In some embodiments, a vaccine composition is provided comprising a therapeutically effective amount of cells from at least one cancer cell line, wherein the at least one cell line is modified to express at least one of the immunostimulatory factors of Table 4, at least two of the immunostimulatory factors of Table 4, or at least three of the immunostimulatory factors of Table 4. In further embodiments, a vaccine composition is provided comprising a therapeutically effective amount of cells from at least one cancer cell line, wherein each cell line or combination of cell lines is modified to reduce at least one of the immunosuppressive factors of Table 8, or at least two of the immunosuppressive factors of Table 8.

In embodiments where the one or more cell lines are modified to increase the production of one or more TAAs, the expressed TAAs may or may not have the native coding sequence of DNA/protein. That is, expression may be codon optimized or modified. Such optimization or modification may enhance certain effects (e.g., may lead to reduced shedding of a TAA protein from the vaccine cell membrane). As described herein, in some embodiments the expressed TAA protein is a designed antigen comprising one or more nonsynonymous mutations (NSMs) identified in cancer patients. In some embodiments, the NSMs introduces CD4, CD8, or CD4 and CD8 neoepitopes.

Any of the vaccine compositions described herein can be administered to a subject in order to treat cancer, prevent cancer, prolong survival in a subject with cancer, and/or stimulate an immune response in a subject.

Cell Lines

In various embodiments of the disclosure, the cell lines comprising the vaccine compositions and used in the methods described herein originate from parental cancer cell lines.

Cell lines are available from numerous sources as described herein and are readily known in the art. For example, cancer cell lines can be obtained from the American Type Culture Collection (ATCC, Manassas, Va.), Japanese Collection of Research Bioresources cell bank (JCRB, Kansas City, Mo.), Cell Line Service (CLS, Eppelheim, Germany), German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), RI KEN BioResource Research Center (RCB, Tsukuba, Japan), Korean Cell Line Bank (KCLB, Seoul, South Korea), NIH AIDS Reagent Program (NIH-ARP/Fisher BioServices, Rockland, Md.), Bioresearch Collection and Research Center (BCRC, Hsinchu, Taiwan), Interlab Cell Line Collection (ICLC, Genova, Italy), European Collection of Authenticated Cell Cultures (ECACC, Salisbury, United Kingdom), Kunming Cell Bank (KCB, Yunnan, China), National Cancer Institute Development Therapeutics Program (NCI-DTP, Bethesda, Md.), Rio de Janeiro Cell Bank (BCRJ, Rio de Janeiro, Brazil), Experimental Zooprophylactic Institute of Lombardy and Emilia Romagna (IZSLER, Milan, Italy), Tohoku University cell line catalog (TKG, Miyagi, Japan), and National Cell Bank of Iran (NCBI, Tehran, Iran). In some embodiments, cell lines are identified through an examination of RNA-seq data with respect to TAAs, immunosuppressive factor expression, and/or other information readily available to those skilled in the art.

In various embodiments, the cell lines in the compositions and methods described herein are from parental cell lines of solid tumors originating from the lung, prostate, testis, breast, urinary tract, colon, rectum, stomach, head and neck, liver, kidney, nervous system, endocrine system, mesothelium, ovaries, pancreas, esophagus, uterus or skin. In certain embodiments, the parental cell lines comprise cells of the same or different histology selected from the group consisting of squamous cells, adenocarcinoma cells, adenosquamous cells, large cell cells, small cell cells, sarcoma cells, carcinosarcoma cells, mixed mesodermal cells, and teratocarcinoma cells. In related embodiments, the sarcoma cells comprise osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma, mesothelioma, fibrosarcoma, angiosarcoma, liposarcoma, glioma, gliosarcoma, astrocytoma, myxosarcoma, mesenchymous or mixed mesodermal cells.

In certain embodiments, the cell lines comprise cancer cells originating from lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), prostate cancer, glioblastoma, colorectal cancer, breast cancer including triple negative breast cancer (TNBC), bladder or urinary tract cancer, squamous cell head and neck cancer (SCCHN), liver hepatocellular (HCC) cancer, kidney or renal cell carcinoma (RCC) cancer, gastric or stomach cancer, ovarian cancer, esophageal cancer, testicular cancer, pancreatic cancer, central nervous system cancers, endometrial cancer, melanoma, and mesothelium cancer.

According to various embodiments, the cell lines are allogeneic cell lines (i.e., cells that are genetically dissimilar and hence immunologically incompatible, although from individuals of the same species.) In certain embodiments, the cell lines are genetically heterogeneous allogeneic. In other embodiments, the cell lines are genetically homogeneous allogeneic.

Allogeneic cell-based vaccines differ from autologous vaccines in that they do not contain patient-specific tumor antigens. Embodiments of the allogeneic vaccine compositions disclosed herein comprise laboratory-grown cancer cell lines known to express TAAs of a specific tumor type. Embodiments of the allogeneic cell lines of the present disclosure are strategically selected, sourced, and modified prior to use in a vaccine composition. Vaccine compositions of embodiments of the present disclosure can be readily mass-produced. This efficiency in development, manufacturing, storage, and other areas can result in cost reductions and economic benefits relative to autologous-based therapies.

Tumors are typically made up of a highly heterogeneous population of cancer cells that evolve and change over time. Therefore, it can be hypothesized that a vaccine composition comprising only autologous cell lines that do not target this cancer evolution and progression may be insufficient in the elicitation of a broad immune response required for effective vaccination. As described in embodiments of the vaccine composition disclosed herein, use of one or more strategically selected allogeneic cell lines with certain genetic modification(s) addresses this disparity.

In some embodiments, the allogeneic cell-based vaccines are from cancer cell lines of the same type (e.g., breast, prostate, lung) of the cancer sought to be treated. In other embodiments, various types of cell lines (i.e., cell lines from different primary tumor origins) are combined (e.g., stem cell, prostate, testes). In some embodiments, the cell lines in the vaccine compositions are a mixture of cell lines of the same type of the cancer sought to be treated and cell lines from different primary tumor origins.

Exemplary cancer cell lines, including, but not limited to those provided in Table 1, below, are contemplated for use in the compositions and methods described herein. The Cell Line Sources identified herein are for exemplary purposes only. The cell lines described in various embodiments herein may be available from multiple sources.

TABLE 1
Exemplary vaccine composition cell lines per indication
Anatomical Site of	Cell Line	Cell Line	Cell Line Source
Primary Tumor	Common Name	Source	Identification
Lung	ABC-1	JCRB	JCRB0815
(Small Cell	Calu-1	ATCC	HTB-54
and Non-	LOU-NH91	DSMZ	ACC-393
Small Cell)	NCI-H1581	ATCC	CRL-5878
	NCI-H1703	ATCC	CRL-5889
	NCI-H460	ATCC	HTB-177
	NCI-H520	ATCC	HTB-182
	A549	ATCC	CCL-185
	LK-2	JCRB	JCRB0829
	NCI-H23	ATCC	CRL-5800
	NCI-H2066	ATCC	CRL-5917
	NCI-H2009	ATCC	CRL-5911
	NCI-H2023	ATCC	CRL-5912
	RERF-LC-Ad1	JCRB	JCRB1020
	SK-LU-1	ATCC	HTB-57
	NCI-H2172	ATCC	CRL-5930
	NCI-H292	ATCC	CRL-1848
	NCI-H661	ATCC	HTB-183
	SQ-1	RCB	RCB1905
	RERF-LC-KJ	JCRB	JCRB0137
	SW900	ATCC	HTB-59
	NCI-H838	ATCC	CRL-5844
	NCI-H1693	ATCC	CRL-5887
	HCC2935	ATCC	CRL-2869
	NCI-H226	ATCC	CRL-5826
	HCC4006	ATCC	CRL-2871
	DMS 53	ATCC	CRL-2062
	DMS 114	ATCC	CRL-2066
	NCI-H196	ATCC	CRL-5823
	NCI-H1092	ATCC	CRL-5855
	SBC-5	JCRB	JCRB0819
	NCI-H510A	ATCC	HTB-184
	NCI-H889	ATCC	CRL-5817
	NCI-H1341	ATCC	CRL-5864
	NCIH-1876	ATCC	CRL-5902
	NCI-H2029	ATCC	CRL-5913
	NCI-H841	ATCC	CRL-5845
	NCI-H1694	ATCC	CRL-5888
	DMS 79	ATCC	CRL-20496
	HCC33	DSMZ	ACC-487
	NCI-H1048	ATCC	CRL-5853
	NCI-H1105	ATCC	CRL-5856
	NCI-H1184	ATCC	CRL-5858
	NCI-H128	ATCC	HTB-120
	NCI-H1436	ATCC	CRL-5871
	DMS 153	ATCC	CRL-2064
	NCI-H1836	ATCC	CRL-5898
	NCI-H1963	ATCC	CRL-5982
	NCI-H2081	ATCC	CRL-5920
	NCI-H209	ATCC	HTB-172
	NCI-H211	ATCC	CRL-524
	NCI-H2171	ATCC	CRL-5929
	NCI-H2196	ATCC	CRL-5932
	NCI-H2227	ATCC	CRL-5934
	NCI-H446	ATCC	HTB-171
	NCI-H524	ATCC	CRL-5831
	NCI-H526	ATCC	CRL-5811
	NCI-H69	ATCC	HTB-119
	NCI-H82	ATCC	HTB-175
	SHP-77	ATCC	CRL-2195
	SW1271	ATCC	CRL-2177
Prostate or	PC3	ATCC	CRL-1435
Testis	DU145	ATCC	HTB-81
	LNCaP clone	ATCC	CRL-1740
	FGC
	NCCIT	ATCC	CRL-2073
	NEC-8	JCRB	JCRB0250
	NTERA-2cl-D1	ATCC	CRL-1973
	NCI-H660	ATCC	CRL-5813
	VCaP	ATCC	CRL-2876
	MDA-PCa-2b	ATCC	CRL-2422
	22Rv1	ATCC	CRL-2505
	E006AA	Millipore	SCC102
	NEC14	JCRB	JCRB0162
	SuSa	DSMZ	ACC-747
	833K-E	ECACC	06072611
Colorectal	LS123	ATCC	CCL-255
	HCT15	ATCC	CCL-225
	SW1463	ATCC	CCL-234
	RKO	ATCC	CRL-2577
	HUTU80	ATCC	HTB-40
	HCT116	ATCC	CCL-247
	LOVO	ATCC	CCL-229
	T84	ATCC	CCL-248
	LS411N	ATCC	CRL-2159
	SW48	ATCC	CCL-231
	C2BBe1	ATCC	CRL-2102
	Caco-2	ATCC	HTB-37
	SNU-1033	KCLB	01033
	COLO 201	ATCC	CCL-224
	GP2d	ECACC	95090714
	CL-14	DSMZ	ACC-504
	SW403	ATCC	CCL-230
	SW1116	ATCC	CCL-233
	SW837	ATCC	CCL-235
	SK-CO-1	ATCC	HTB-39
	CL-34	DSMZ	ACC-520
	NCI-H508	ATCC	CCL-253
	CCK-81	JCRB	JCRB0208
	SNU-C2A	ATCC	CCL-250.1
	GP2d	ECACC	95090714
	HT-55	ECACC	85061105
	MDST8	ECACC	99011801
	RCM-1	JCRB	JCRB0256
	CL-40	DSMZ	ACC-535
	COLO 678	DSMZ	ACC-194
	LS180	ATCC	CL-187
Breast	BT20	ATCC	HTB-19
	BT549	ATCC	HTB-122
	MDA-MB-231	ATCC	HTB-26
	HS578T	ATCC	HTB-126
	AU565	ATCC	CRL-2351
	CAMA1	ATCC	HTB-21
	MCF7	ATCC	HTB-22
	T-47D	ATCC	HTB-133
	ZR-75-1	ATCC	CRL-1500
	MDA-MB-415	ATCC	HTB-128
	CAL-51	DSMZ	ACC-302
	CAL-120	DSMZ	ACC-459
	HCC1187	ATCC	CRL-2322
	HCC1395	ATCC	CRL-2324
	SK-BR-3	ATCC	HTB-30
	HDQ-P1	DSMZ	ACC-494
	HCC70	ATCC	CRL-2315
	HCC1937	ATCC	CRL-2336
	MDA-MB-436	ATCC	HTB-130
	MDA-MB-468	ATCC	HTB-132
	MDA-MB-157	ATCC	HTB-24
	HMC-1-8	JCRB	JCRB0166
	Hs 274.T	ATCC	CRL-7222
	Hs 281.T	ATCC	CRL-7227
	JIMT-1	ATCC	ACC-589
	Hs 343.T	ATCC	CRL-7245
	Hs 606.T	ATCC	CRL-7368
	UACC-812	ATCC	CRL-1897
	UACC-893	ATCC	CRL-1902
Urinary Tract	UM-UC-3	ATCC	CRL-1749
	5637	ATCC	HTB-9
	J82	ATCC	HTB-1
	T24	ATCC	HTB-4
	HT-1197	ATCC	CRL-1473
	TCCSUP	ATCC	HTB-5
	HT-1376	ATCC	CRL-1472
	SCaBER	ATCC	HTB-3
	RT4	ATCC	HTB-2
	CAL-29	DSMZ	ACC-515
	AGS	ATCC	CRL-1739
	KMBC-2	JCRB	JCRB1148
	253J	KCLB	080001
	253J-BV	KCLB	080002
	SW780	ATCC	CRL-2169
	SW1710	DSMZ	ACC-426
	VM-CUB-1	DSMZ	ACC-400
	BC-3C	DSMZ	ACC-450
	U-BLC1	ECACC	U-BLC1
	KMBC-2	JCRB	JCRB1148
	RT112/84	ECACC	85061106
	UM-UC-1	ECACC	06080301
	RT-112	DSMZ	ACC-418
	KU-19-19	DSMZ	ACC-395
	639V	DSMZ	ACC-413
	647V	DSMZ	ACC-414
Kidney	A-498	ATCC	HTB-44
	A-704	ATCC	HTB-45
	769-P	ATCC	CRL-1933
	786-O	ATCC	CRL-1932
	ACHN	ATCC	CRL-1611
	KMRC-1	JCRB	JCRB1010
	KMRC-2	JCRB	JCRB1011
	VMRC-RCZ	JCRB	JCRB0827
	VMRC-RCW	JCRB	JCRB0813
	UO-31	NCI-DTP	UO-31
	Caki-1	ATCC	HTB-46
	Caki-2	ATCC	HTB-47
	OS-RC-2	RCB	RCB0735
	TUHR-4TKB	RCB	RCB1198
	RCC-10RGB	RCB	RCB1151
	SNU-1272	KCLB	01272
	SNU-349	KCLB	00349
	TUHR-14TKB	RCB	RCB1383
	TUHR-10TKB	RCB	RCB1275
	BFTC-909	DSMZ	ACC-367
	CAL-54	DSMZ	ACC-365
	KMRC-3	JCRB	JCRB1012
	KMRC-20	JCRB	JCRB1071
Upper	HSC-4	JCRB	JCRB0624
Aerodigestive	DETROIT 562	ATCC	CCL-138
Tract (Head	SCC-9	ATCC	CRL-1629
and Neck)	SCC-4	ATCC	CRL-1624
	OSC-19	JCRB	JCRB0198
	KON	JCRB	JCRB0194
	HO-1-N-1	JCRB	JCRB0831
	OSC-20	JCRB	JCRB0197
	HSC-3	JCRB	JCRB0623
	SNU-1066	KCLB	01066
	SNU-1041	KCLB	01041
	SNU-1076	KCLB	01076
	BICR 18	ECACC	06051601
	CAL-33	DSMZ	ACC-447
	YD-8	KCLB	60501
	FaDu	ATCC	HTB-43
	2A3	ATCC	CRL-3212
	CAL-27	ATCC	CRL-2095
	SCC-25	ATCC	CRL-1628
	SCC-15	ATCC	CRL-1623
	HO-1-u-1	JCRB	JCRB0828
	KOSC-2	JCRB	JCRB0126.1
	RPMI-2650	ATCC	CCL-30
	SCC-90	ATCC	CRL-3239
	SKN-3	JCRB	JCRB1039
	HSC-2	JCRB	JCRB0622
	Hs 840.T	ATCC	CRL-7573
	SAS	JCRB	JCRB0260
	SAT	JCRB	JCRB1027
	SNU-46	KCLB	00046
	YD-38	KCLB	60508
	SNU-899	KCLB	00899
	HN	DSMZ	ACC-417
	BICR 10	ECACC	04072103
	BICR 78	ECACC	04072111
Ovaries	OVCAR-3	ATCC	HTB-161
	TOV-112D	ATCC	CRL-11731
	ES-2	ATCC	CRL-1978
	TOV-21G	ATCC	CRL-11730
	OVTOKO	JCRB	JCRB1048
	KURAMOCHI	JCRB	JCRB0098
	MCAS	JCRB	JCRB0240
	TYK-nu	JCRB	JCRB0234.0
	OVSAHO	JCRB	JCRB1046
	OVMANA	JCRB	JCRB1045
	JHOM-2B	RCB	RCB1682
	OV56	ECACC	96020759
	JHOS-4	RCB	RCB1678
	JHOC-5	RCB	RCB1520
	OVCAR-4	NCI-DTP	OVCAR-4
	JHOS-2	RCB	RCB1521
	EFO-21	DSMZ	ACC-235
	OV-90	ATCC	CRL-11732
	OVKATE	JCRB	JCRB1044
	SK-OV-3	ATCC	HTB-77
	Caov-4	ATCC	HTB-76
	Coav-3	ATCC	HTB-75
	JHOM-1	RCB	RCB1676
	COV318	ECACC	07071903
	OVK-18	RCB	RCB1903
	SNU-119	KCLB	00119
	SNU-840	KCLB	00840
	SNU-8	KCLB	0008
	COV362	ECACC	07071910
	COV434	ECACC	07071909
	COV644	ECACC	07071908
	OV7	ECACC	96020764
	OAW-28	ECACC	85101601
	OVCAR-8	NCI-DTP	OVCAR-8
	59M	ECACC	89081802
	EFO-27	DSMZ	ACC-191
Pancreas	PANC-1	ATCC	CRL-1469
	HPAC	ATCC	CRL-2119
	KP-2	JCRB	JCRB0181
	KP-3	JCRB	JCRB0178.0
	KP-4	JCRB	JCRB0182
	HPAF-II	ATCC	CRL-1997
	SUIT-2	JCRB	JCRB1094
	AsPC-1	ATCC	CRL-1682
	PSN1	ATCC	CRL-3211
	CFPAC-1	ATCC	CRL-1918
	Capan-1	ATCC	HTB-79
	Panc 02.13	ATCC	CRL-2554
	Panc 03.27	ATCC	CRL-2549
	BxPC-3	ATCC	CRL-1687
	SU.86.86	ATCC	CRL-1837
	Hs 766T	ATCC	HTB-134
	Panc 10.05	ATCC	CRL-2547
	Panc 04.03	ATCC	CRL-2555
	PaTu 8988s	DSMZ	ACC-204
	PaTu 8988t	DSMZ	ACC-162
	SW1990	ATCC	CRL-2172
	SNU-324	KCLB	00324
	SNU-213	KCLB	00213
	DAN-G	DSMZ	ACC-249
	Panc 02.03	ATCC	CRL-2553
	PaTu 8902	DSMZ	ACC-179
	Capan-2	ATCC	HTB-80
	MIA PaCa-2	ATCC	CRL-1420
	YAPC	DSMZ	ACC-382
	HuP-T3	DSMZ	ACC-259
	T3M-4	RCB	RCB1021
	PK-45H	RCB	RCB1973
	Panc 08.13	ATCC	CRL-2551
	PK-1	RCB	RCB1972
	PK-59	RCB	RCB1901
	HuP-T4	DSMZ	ACC-223
	Panc 05.04	ATCC	CRL-2557
Stomach	RERF-GC-1B	JCRB	JCRB1009
	Fu97	JCRB	JCRB1074
	MKN74	JCRB	JCRB0255
	NCI-N87	ATCC	CRL-5822
	NUGC-2	JCRB	JCRB0821
	MKN45	JCRB	JCRB0254
	OCUM-1	JCRB	JCRB0192
	MKN7	JCRB	JCRB1025
	MKN1	JCRB	JCRB0252
	ECC10	RCB	RCB0983
	TGBC-11-TKB	RCB	RCB1148
	SNU-620	KCLB	00620
	GSU	RCB	RCB2278
	KE-39	RCB	RCB1434
	HuG1-N	RCB	RCB1179
	NUGC-4	JCRB	JCRB0834
	SNU-16	ATCC	CRL-5974
	Hs 746.T	ATCC	HTP-135
	LMSU	RCB	RCB1062
	SNU-520	KCLB	00520
	GSS	RCB	RCB2277
	ECC12	RCB	RCB1009
	GCIY	RCB	RCB0555
	SH-10-TC	RCB	RCB1940
	HGC-27	BCRJ	0310
	HuG1-N	RCB	RCB1179
	SNU-601	KCLB	KCLB00601
	SNU-668	KCLB	00668
	NCC-StC-K140	JCRB	JCRB1228
	SNU-719	KCLB	00719
	SNU-216	KCLB	00216
	NUGC-3	JCRB	JCRB0822
Liver	Hep-G2	ATCC	HB-8065
	JHH-2	JCRB	JCRB1028
	JHH-4	JCRB	JCRB0435
	JHH-6	JCRB	JCRB1030
	Li7	RCB	RCB1941
	HLF	JCRB	JCRB0405
	HuH-6	RCB	BRC1367
	JHH-5	JCRB	JCRB1029
	HuH-7	JCRB	JCRB0403
	SNU-182	ATCC	CRL-2235
	JHH-7	JCRB	JCRB1031
	SK-HEP-1	ATCC	HTB-52
	Hep3B2.1-7	ATCC	HB-8064
	SNU-449	ATCC	CRL-2234
	SNU-761	KCLB	KCLB
	JHH-1	JCRB	JCRB1062
	SNU-398	ATCC	CRL-2233
	SNU-423	ATCC	CRL-2238
	SNU-387	ATCC	CRL-2237
	SNU-475	ATCC	CRL-2236
	SNU-886	KCLB	KCLB 00886
	SNU-878	KCLB	KCLB 00878
	NCI-H684	KCLB	KCLB 90684
	PLC/PRF/5	ATCC	CRL-8024
	HuH-1	JCRB	JCRB0199
	HLE	JCRB	JCRB0404
	C3A	ATCC	HB-8065
Central	DBTRG-05MG	ATCC	CRL-2020
Nervous	LN-229	ATCC	CRL-2611
System	SF-126	JCRB	IFO50286
	M059K	ATCC	CRL-2365
	M059KJ	ATCC	CRL-2366
	U-251 MG	JCRB	IFO50288
	A-172	ATCC	CRL-1620
	YKG-1	ATCC	JCRB0746
	GB-1	ATCC	IFO50489
	KNS-60	ATCC	IFO50357
	KNS-81	JCRB	IFO50359
	TM-31	RCB	RCB1731
	NMC-G1	JCRB	IFO50467
	SNU-201	KCLB	00201
	SW1783	ATCC	HTB-13
	GOS-3	DSMZ	ACC-408
	KNS-81	JCRB	IFO50359
	KG-1-C	JCRB	JCRB0236
	AM-38	JCRB	IFO50492
	CAS-1	ILCL	HTL97009
	H4	ATCC	HTB-148
	D283 Med	ATCC	HTB-185
	DK-MG	DSMZ	ACC-277
	U-118MG	ATCC	HTB-15
	SNU-489	KCLB	00489
	SNU-466	KCLB	00426
	SNU-1105	KCLB	01105
	SNU-738	KCLB	00738
	SNU-626	KCLB	00626
	Daoy	ATCC	HTB-186
	D341 Med	ATCC	HTB-187
	SW1088	ATCC	HTB-12
	Hs 683	ATCC	HTB-138
	ONS-76	JCRB	IFO50355
	LN-18	ATCC	CRL-2610
	T98G	ATCC	CRL-1690
	GMS-10	DSMZ	ACC-405
	42-MG-BA	DSMZ	ACC-431
	GaMG	DSMZ	ACC-242
	8-MG-BA	DSMZ	ACC-432
	IOMM-Lee	ATCC	CRL-3370
	SF268	NCI-DTP	SF-268
	SF539	NCI-DTP	SF-539
	SNB75	NCI-DTP	SNB-75
Esophagus	TE-10	RCB	RCB2099
	TE-6	RCB	RCB1950
	TE-4	RCB	RCB2097
	EC-GI-10	RCB	RCB0774
	OE33	ECACC	96070808
	TE-9	RCB	RCB1988
	TT	JCRB	JCRB0262
	TE-11	RCB	RCB2100
	OE19	ECACC	96071721
	OE21	ECACC	96062201
	KYSE-450	JCRB	JCRB1430
	TE-14	RCB	RCB2101
	TE-8	RCB	RCB2098
	KYSE-410	JCRB	JCRB1419
	KYSE-140	DSMZ	ACC-348
	KYSE-180	JCRB	JCRB1083
	KYSE-520	JCRB	JCRB1439
	KYSE-270	JCRB	JCRB1087
	KYSE-70	JCRB	JCRB0190
	TE-1	RCB	RCB1894
	TE-5	RCB	RCB1949
	TE-15	RCB	RCB1951
	KYSE-510	JCRB	JCRB1436
	KYSE-30	ECACC	94072011
	KYSE-150	DSMZ	ACC-375
	COLO 680N	DSMZ	ACC-182
	KYSE-450	JCRB	JCRB1430
	TE-10	RCB	RCB2099
	ESO-26	ECACC	11012009
	ESO-51	ECACC	11012010
	FLO-1	ECACC	11012001
	KYAE-1	ECACC	11012002
	KYSE-220	JCRB	JCRB1086
	KYSE-50	JCRB	JCRB0189
	OACM5.1 C	ECACC	11012006
	OACP4 C	ECACC	11012005
Endometrium	SNG-M	JCRB	IFO50313
	HEC-1-B	ATCC	HTB-113
	JHUEM-3	Riken RCB	RCB1552
	RL95-2	ATCC	CRL-1671
	MFE-280	ECACC	98050131
	MFE-296	ECACC	98031101
	TEN	Riken RCB	RCB1433
	JHUEM-2	Riken RCB	RCB1551
	AN3-CA	ATCC	HTB-111
	KLE	ATCC	CRL-1622
	Ishikawa	ECACC	99040201
	HEC-151	JCRB	JCRB1122
	SNU-1077	KCLB	01077
	MFE-319	DSMZ	ACC-423
	EFE-184	DSMZ	ACC-230
	HEC-108	JCRB	JCRB1123
	HEC-265	JCRB	JCRB1142
	HEC-6	JCRB	JCRB1118
	HEC-50B	JCRB	JCRB1145
	JHUEM-1	RCB	RCB1548
	HEC-251	JCRB	JCRB1141
	COLO 684	ECACC	87061203
	SNU-685	KCLB	00685
	HEC-59	JCRB	JCRB1120
	EN	DSMZ	ACC-564
	ESS-1	DSMZ	ACC-461
	HEC-1A	ATCC	HTB-112
	JHUEM-7	RCB	RCB1677
	HEC-1	JCRB	JCRB0042
Skin	RPMI-7951	ATCC	HTB-66
	MeWo	ATCC	HTB-65
	Hs 688(A).T	ATCC	CRL-7425
	COLO 829	ATCC	CRL-1974
	C32	ATCC	CRL-1585
	A-375	ATCC	CRL-1619
	Hs 294T	ATCC	HTB-140
	Hs 695T	ATCC	HTB-137
	Hs 852T	ATCC	CRL-7585
	A2058	ATCC	CRL-11147
	RVH-421	DSMZ	ACC-127
	Hs 895.T	ATCC	CRL-7637
	Hs 940.T	ATCC	CRL-7691
	SK-MEL-1	ATCC	HTB-67
	SK-MEL-28	ATCC	HTB-72
	SH-4	ATCC	CRL-7724
	COLO 800	ECACC	93051123
	COLO 783	DSMZ	ACC-257
	MDA-MB-435S	ATCC	HTB-129
	IGR-1	CLS	300219/
			p483_IGR-1
	IGR-39	DSMZ	ACC-239
	HT-144	ATCC	HTB-63
	SK-MEL-31	ATCC	HTB-73
	Hs 839.T	ATCC	CRL-7572
	Hs 600.T	ATCC	CRL-7360
	A101D	ATCC	CRL-7898
	IPC-298	DSMZ	ACC-251
	SK-MEL-24	ATCC	HTB-71
	SK-MEL-3	ATCC	HTB-69
	HMCB	ATCC	CRL-9607
	Malme-3M	ATCC	HTB-64
	Mel JuSo	DSMZ	ACC-74
	COLO 679	RCB	RCB0989
	COLO 741	ECACC	93052621
	SK-MEL-5	ATCC	HTB-70
	WM266-4	ATCC	CRL-1676
	IGR-37	DSMZ	ACC-237
	Hs 934.T	ATCC	CRL-7684
	UACC-257	NCI-DTP	UACC-257
Mesothelium	NCI-H28	ATCC	CRL-5820
	MSTO-211H	ATCC	CRL-2081
	IST-Mes1	ICLC	HTL01005
	ACC-MESO-1	RCB	RCB2292
	NCI-H2052	ATCC	CRL-5951
	NCI-H2452	ATCC	CRL-2081
	MPP 89	ICLC	HTL00012
	IST-Mes2	ICLC	HTL01007
	RS-5	DSMZ	ACC-604
	DM-3	DSMZ	ACC-595
	JL-1	DSMZ	ACC-596
	COR-L321	ECACC	96020756

In addition to the cell lines identified in Table 1, the following cell lines are also contemplated in various embodiments.

In various embodiments, one or more non-small cell lung (NSCLC) cell lines are prepared and used according to the disclosure. By way of example, the following NSCLC cell lines are contemplated: NCI-H460, NCI-H520, A549, DMS 53, LK-2, and NCI-H23. Additional NSCLC cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising NSCLC cell lines is also contemplated.

In some embodiments, one or more prostate cancer cell lines are prepared and used according to the disclosure. By way of example, the following prostate cancer cell lines are contemplated: PC3, DU-145, LNCAP, NEC8, and NTERA-2cl-D1. Additional prostate cancer cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising prostate cancer cell lines is also contemplated.

In some embodiments, one or more colorectal cancer (CRC) cell lines are prepared and used according to the disclosure. By way of example, the following colorectal cancer cell lines are contemplated: HCT-15, RKO, HuTu-80, HCT-116, and LS411N. Additional colorectal cancer cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising CRC cell lines is also contemplated.

In some embodiments, one or more breast cancer or triple negative breast cancer (TNBC) cell lines are prepared and used according to the disclosure. By way of example, the following TNBC cell lines are contemplated: Hs-578T, AU565, CAMA-1, MCF-7, and T-47D. Additional breast cancer cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising breast and/or TNBC cancer cell lines is also contemplated.

In some embodiments, one or more bladder or urinary tract cancer cell lines are prepared and used according to the disclosure. By way of example, the following urinary tract or bladder cancer cell lines are contemplated: UM-UC-3, J82, TCCSUP, HT-1376, and SCaBER. Additional bladder cancer cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising bladder or urinary tract cancer cell lines is also contemplated.

In some embodiments, one or more stomach or gastric cancer cell lines are prepared and used according to the disclosure. By way of example, the following stomach or gastric cancer cell lines are contemplated: Fu97, MKN74, MKN45, OCUM-1, and MKN1. Additional stomach cancer cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising stomach or gastric cancer cell lines is also contemplated.

In some embodiments, one or more squamous cell head and neck cancer (SCCHN) cell lines are prepared and used according to the disclosure. By way of example, the following SCCHN cell lines are contemplated: HSC-4, Detroit 562, KON, HO-1-N-1, and OSC-20. Additional SCCHN cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising SCCHN cancer cell lines is also contemplated.

In some embodiments, one or more small cell lung cancer (SCLC) cell lines are prepared and used according to the disclosure. By way of example, the following SCLC cell lines are contemplated: DMS 114, NCI-H196, NCI-H1092, SBC-5, NCI-H510A, NCI-H889, NCI-H1341, NCIH-1876, NCI-H2029, NCI-H841, and NCI-H1694. Additional SCLC cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising SCLC cell lines is also contemplated.

In some embodiments, one or more liver or hepatocellular cancer (HCC) cell lines are prepared and used according to the disclosure. By way of example, the following HCC cell lines are contemplated: Hep-G2, JHH-2, JHH-4, JHH-6, Li7, HLF, HuH-6, JHH-5, and HuH-7. Additional HCC cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising liver or HCC cancer cell lines is also contemplated.

In some embodiments, one or more kidney cancer such as renal cell carcinoma (RCC) cell lines are prepared and used according to the disclosure. By way of example, the following RCC cell lines are contemplated: A-498, A-704, 769-P, 786-O, ACHN, KMRC-1, KMRC-2, VMRC-RCZ, and VMRC-RCW. Additional RCC cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising kidney or RCC cancer cell lines is also contemplated.

In some embodiments, one or more glioblastoma (GBM) cancer cell lines are prepared and used according to the disclosure. By way of example, the following GBM cell lines are contemplated: DBTRG-05MG, LN-229, SF-126, GB-1, and KNS-60. Additional GBM cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising GBM cancer cell lines is also contemplated.

In some embodiments, one or more ovarian cancer cell lines are prepared and used according to the disclosure. By way of example, the following ovarian cell lines are contemplated: TOV-112D, ES-2, TOV-21G, OVTOKO, and MCAS. Additional ovarian cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising ovarian cancer cell lines is also contemplated.

In some embodiments, one or more esophageal cancer cell lines are prepared and used according to the disclosure. By way of example, the following esophageal cell lines are contemplated: TE-10, TE-6, TE-4, EC-GI-10, OE33, TE-9, TT, TE-11, OE19, OE21. Additional esophageal cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising esophageal cancer cell lines is also contemplated.

In some embodiments, one or more pancreatic cancer cell lines are prepared and used according to the disclosure. By way of example, the following pancreatic cell lines are contemplated: PANC-1, KP-3, KP-4, SUIT-2, and PSN1. Additional pancreatic cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising pancreatic cancer cell lines is also contemplated.

In some embodiments, one or more endometrial cancer cell lines are prepared and used according to the disclosure. By way of example, the following endometrial cell lines are contemplated: SNG-M, HEC-1-B, JHUEM-3, RL95-2, MFE-280, MFE-296, TEN, JHUEM-2, AN3-CA, and Ishikawa. Additional endometrial cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising endometrial cancer cell lines is also contemplated.

In some embodiments, one or more melanoma cancer cell lines are prepared and used according to the disclosure. By way of example, the following melanoma cell lines are contemplated: RPMI-7951, MeWo, Hs 688(A).T, COLO 829, C32, A-375, Hs 294T, Hs 695T, Hs 852T, and A2058. Additional melanoma cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising melanoma cancer cell lines is also contemplated.

In some embodiments, one or more mesothelioma cancer cell lines are prepared and used according to the disclosure. By way of example, the following mesothelioma cell lines are contemplated: NCI-H28, MSTO-211H, IST-Mes1, ACC-MESO-1, NCI-H2052, NCI-H2452, MPP 89, and IST-Mes2. Additional mesothelioma cell lines are also contemplated by the present disclosure. As described herein, inclusion of a cancer stem cell line such as DMS 53 in a vaccine comprising mesothelioma cancer cell lines is also contemplated.

Embodiments of vaccine compositions according to the disclosure are used to treat and/or prevent various types of cancer. In some embodiments, a vaccine composition may comprise cancer cell lines that originated from the same type of cancer. For example, a vaccine composition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NSCLC cell lines, and such a composition may be useful to treat or prevent NSCLC. According to certain embodiments, the vaccine composition comprising NCSLC cell lines may be used to treat or prevent cancers other than NSCLC, examples of which are described herein.

In some embodiments, a vaccine composition may comprise cancer cell lines that originated from different types of cancer. For example, a vaccine composition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NSCLC cell lines, plus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more SCLC cancer cell lines, optionally plus one or other cancer cell lines, such as cancer stem cell lines, and so on, and such a composition may be useful to treat or prevent NSCLC, and/or prostate cancer, and/or breast cancer including triple negative breast cancer (TNBC), and so on. According to some embodiments, the vaccine composition comprising different cancer cell lines as described herein may be used to treat or prevent various cancers. In some embodiments, the targeting of a TAA or multiple TAAs in a particular tumor is optimized by using cell lines derived from different tissues or organs within a biological system to target a cancer of primary origin within the same system. By way of non-limiting examples, cell lines derived from tumors of the reproductive system (e.g., ovaries, fallopian tubes, uterus, vagina, mammary glands, testes, vas deferens, seminal vesicles, and prostate) may be combined; cell lines derived from tumors of the digestive system (e.g., salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestines, rectum, and anus) may be combined; cell lines from tumors of the respiratory system (e.g., pharynx, larynx, bronchi, lungs, and diaphragm) may be combined; and cell lines derived from tumors of the urinary system (e.g., kidneys, ureters, bladder, and urethra) may be combined.

According to various embodiments of the vaccine compositions, the disclosure provides compositions comprising a combination of cell lines. By way of non-limiting examples, cell line combinations are provided below. In each of the following examples, cell line DMS 53, whether modified or unmodified, is combined with 5 other cancer cell lines in the associated list. One or more of the cell lines within each recited combination may be modified as described herein. In some embodiments, none of the cell lines in the combination of cell lines are modified. In some embodiments, DMS 53 is modified to reduce expression of CD276, reduce secretion of TGFβ1 and TGFβ2, and express GM-CSF, membrane bound CD40L and IL-12. In other embodiments, DMS 53 is modified to reduce expression of CD276, reduce secretion of TGFβ2, and express GM-CSF and membrane bound CD40L.

(1) NCI-H460, NCI-H520, A549, DMS 53, LK-2, and NCI-H23 for the treatment and/or prevention of NSCLC;

(2) DMS 114, NCI-H196, NCI-H1092, SBC-5, NCI-H510A, NCI-H889, NCI-H1341, NCIH-1876, NCI-H2029, NCI-H841, DMS 53, and NCI-H1694 for the treatment and/or prevention of SCLC;

(3) DMS 53, PC3, DU-145, LNCAP, NEC8, and NTERA-2cl-D1 for the treatment and/or prevention of prostate cancer;

(4) DMS 53, HCT-15, RKO, HuTu-80, HCT-116, and LS411N for the treatment and/or prevention of colorectal cancer;

(5) DMS 53, Hs-578T, AU565, CAMA-1, MCF-7, and T-47D for the treatment and/or prevention of breast cancer including triple negative breast cancer (TNBC);

(6) DMS 53, UM-UC-3, J82, TCCSUP, HT-1376, and SCaBER for the treatment and/or prevention of bladder cancer;

(7) DMS 53, HSC-4, Detroit 562, KON, HO-1-N-1, and OSC-20 for the treatment and/or prevention of head and/or neck cancer;

(8) DMS 53, Fu97, MKN74, MKN45, OCUM-1, and MKN1 for the treatment and/or prevention of stomach cancer;

(9) DMS 53, Hep-G2, JHH-2, JHH-4, JHH-6, Li7, HLF, HuH-6, JHH-5, and HuH-7 for the treatment and/or prevention of liver cancer;

(10) DMS 53, DBTRG-05MG, LN-229, SF-126, GB-1, and KNS-60 for the treatment and/or prevention of glioblastoma;

(11) DMS 53, TOV-112D, ES-2, TOV-21G, OVTOKO, and MCAS for the treatment and/or prevention of ovarian cancer;

(12) DMS 53, TE-10, TE-6, TE-4, EC-GI-10, OE33, TE-9, TT, TE-11, OE19, and OE21 for the treatment and/or prevention of esophageal cancer;

(13) DMS 53, A-498, A-704, 769-P, 786-O, ACHN, KMRC-1, KMRC-2, VMRC-RCZ, and VMRC-RCW for the treatment and/or prevention of kidney cancer;

(14) DMS 53, PANC-1, KP-3, KP-4, SUIT-2, and PSN1 for the treatment and/or prevention of pancreatic cancer;

(15) DMS 53, SNG-M, HEC-1-B, JHUEM-3, RL95-2, MFE-280, MFE-296, TEN, JHUEM-2, AN3-CA, and Ishikawa for the treatment and/or prevention of endometrial cancer;

(16) DMS 53, RPMI-7951, MeWo, Hs 688(A).T, COLO 829, C32, A-375, Hs 294T, Hs 695T, Hs 852T, and A2058 for the treatment and/or prevention of skin cancer; and

(17) DMS 53, NCI-H28, MSTO-211H, IST-Mes1, ACC-MESO-1, NCI-H2052, NCI-H2452, MPP 89, and IST-Mes2 for the treatment and/or prevention of mesothelioma.

In some embodiments, the cell lines in the vaccine compositions and methods described herein include one or more cancer stem cell (CSC) cell lines, whether modified or unmodified. One example of a CSC cell line is small cell lung cancer cell line DMS 53, whether modified or unmodified. CSCs display unique markers that differ depending on the anatomical origin of the tumor. Exemplary CSC markers include: prominin-1 (CD133), A2B5, aldehyde dehydrogenase (ALDH1), polycomb protein (Bmi-1), integrin-β1 (CD29), hyaluronan receptor (CD44), Thy-1 (CD90), SCF receptor (CD117), TRA-1-60, nestin, Oct-4, stage-specific embryonic antigen-1 (CD15), GD3 (CD60a), stage-specific embryonic antigen-1 (SSEA-1) or (CD15), stage-specific embryonic antigen-4 (SSEA-4), stage-specific embryonic antigen-5 (SSEA-5), and Thomsen-Friedenreich antigen (CD176).

Expression markers that identify cancer cell lines with greater potential to have stem cell-like properties differ depending on various factors including anatomical origin, organ, or tissue of the primary tumor. Exemplary cancer stem cell markers identified by primary tumor site are provided in Table 2 and are disclosed across various references (e.g., Gilbert, C A & Ross, AH. J Cell Biochem. (2009); Karsten, U & Goletz, S. SpringerPlus (2013); Zhao, Wet al. Cancer Transl Med. (2017)).

Exemplary cell lines expressing one or more markers of cancer stem cell-like properties specific for the anatomical site of the primary tumor from which the cell line was derived are listed in Table 2. Exemplary cancer stem cell lines are provided in Table 3. Expression of CSC markers was determined using RNA-seq data from the Cancer Cell Line Encyclopedia (CCLE) (retrieved from www.broadinstitute.org/ccle on Nov. 23, 2019; Barretina, J et al. Nature. (2012)). The HUGO Gene Nomenclature Committee gene symbol was entered into the CCLE search and mRNA expression downloaded for each CSC marker. The expression of a CSC marker was considered positive if the RNA-seq value (FPKM) was greater than 0.

TABLE 2
Exemplary CSC markers by primary tumor anatomical origin
	Anatomical Site of	CSC Marker	CSC Marker
	Primary Tumor	Common Name	Gene Symbol
	Ovaries	Endoglin, CD105	ENG
		CD117, cKIT	KIT
		CD44	CD44
		CD133	PROM1
		SALL4	SAL4
		Nanog	NANOG
		Oct-4	POU5F1
	Pancreas	ALDH1A1	ALDH1A1
		c-Myc	MYC
		EpCAM, TROP1	EPCAM
		CD44	CD44
		Cd133	PROM1
		CXCR4	CXCR4
		Oct-4	POU5F1
		Nestin	NES
		BMI-1	BMI1
	Skin	CD27	CD27
		ABCB5	ABCB5
		ABCG2	ABCG2
		CD166	ALCAM
		Nestin	NES
		CD133	PROM1
		CD20	MS4A1
		NGFR	NGFR
	Lung	ALDH1A1	ALDH1A1
		EpCAM, TROP1	EPCAM
		CD90	THY1
		CD117, cKIT	KIT
		CD133	PROM1
		ABCG2	ABCG2
		SOX2	SOX2
	Liver	Nanog	NANOG
		CD90/thy1	THY1
		CD133	PROM1
		CD13	ANPEP
		EpCAM, TROP1	EPCAM
		CD117, cKIT	KIT
		SALL4	SAL4
		SOX2	SOX2
	Upper	ABCG2	ABCG2
	Aerodigestive	ALDH1A1	ALDH1A1
	Tract (Head	Lgr5, GPR49	LGR5
	and Neck)	BMI-1	BMI1
		CD44	CD44
		cMET	MET
	Central	ALDH1A1	ALDH1A1
	Nervous	ABCG2	ABCG2
	System	BMI-1	BMI1
		CD15	FUT4
		CD44	CD44
		CD49f, Integrin α6	ITGA6
		CD90	THY1
		CD133	PROM1
		CXCR4	CXCR4
		CX3CR1	CX3CR1
		SOX2	SOX2
		c-Myc	MYC
		Musashi-1	MSI1
		Nestin	NES
	Stomach	ALDH1A1	ALDH1A1
		ABCB1	ABCB1
		ABCG2	ABCG2
		CD133	PROM1
		CD164	CD164
		CD15	FUT4
		Lgr5, GPR49	LGR5
		CD44	CD44
		MUC1	MUC1
		DLL4	DLL4
	Colon	ALDH1A1	ALDH1A1
	(Large and	c-myc	MYC
	Small Intestines)	CD44	CD44
		CD133	PROM1
		Nanog	NANOG
		Musashi-1	MSI1
		EpCAM, TROP1	EPCAM
		Lgr5, GPR49	LGR5
		SALL4	SAL4
	Breast	ABCG2	ABCG2
		ALDH1A1	ALDH1A1
		BMI-1	BMI1
		CD133	PROM1
		CD44	CD44
		CD49f, Integrin a6	ITGA6
		CD90	THY1
		c-myc	MYC
		CXCR1	CXCR1
		CXCR4	CXCR4
		EpCAM, TROP1	EPCAM
		KLF4	KLF4
		MUC1	MUC1
		Nanog	NANOG
		SALL4	SAL4
		SOX2	SOX2
	Urinary Tract	ALDH1A1	ALDH1A1
		CEACAM6, CD66c	CEACAM6
		Oct4	OCT4
		CD44	CD44
		YAP1	YAP1
	Hematopoietic and	BMI-1	BMI1
	Lymphoid Tissue	CD117, c-kit	KIT
		CD20	MS4A1
		CD27, TNFRSF7	CD27
		CD34	CD34
		CD38	CD38
		CD44	CD44
		CD96	CD96
		GLI-1	GLI1
		GLI-2	GLI2
		IL-3Rα	IL3RA
		MICL	CLEC12A
		Syndecan-1, CD138	SDC1
		TIM-3	HAVCR2
	Bone	ABCG2	ABCG2
		CD44	CD44
		Endoglin, CD105	ENG
		Nestin	NES

TABLE 3
Cell lines expressing CSC markers
Anatomical Site of	Cell Line	Cell Line	Cell Line Source
Primary Tumor	Common Name	Source	Identification
Ovaries	JHOM-2B	RCB	RCB1682
	OVCAR-3	ATCC	HTB-161
	OV56	ECACC	96020759
	JHOS-4	RCB	RCB1678
	JHOC-5	RCB	RCB1520
	OVCAR-4	NCI-DTP	OVCAR-4
	JHOS-2	RCB	RCB1521
	EFO-21	DSMZ	ACC-235
Pancreas	CFPAC-1	ATCC	CRL-1918
	Capan-1	ATCC	HTB-79
	Panc 02.13	ATCC	CRL-2554
	SUIT-2	JCRB	JCRB1094
	Panc 03.27	ATCC	CRL-2549
Skin	SK-MEL-28	ATCC	HTB-72
	RVH-421	DSMZ	ACC-127
	Hs 895.T	ATCC	CRL-7637
	Hs 940.T	ATCC	CRL-7691
	SK-MEL-1	ATCC	HTB-67
	Hs 936.T	ATCC	CRL-7686
	SH-4	ATCC	CRL-7724
	COLO 800	DSMZ	ACC-193
	UACC-62	NCI-DTP	UACC-62
Lung	NCI-H2066	ATCC	CRL-5917
	NCI-H1963	ATCC	CRL-5982
	NCI-H209	ATCC	HTB-172
	NCI-H889	ATCC	CRL-5817
	COR-L47	ECACC	92031915
	NCI-H1092	ATCC	CRL-5855
	NCI-H1436	ATCC	CRL-5871
	COR-L95	ECACC	96020733
	COR-L279	ECACC	96020724
	NCI-H1048	ATCC	CRL-5853
	NCI-H69	ATCC	HTB-119
	DMS 53	ATCC	CRL-2062
Liver	HuH-6	RCB	RCB1367
	Li7	RCB	RCB1941
	SNU-182	ATCC	CRL-2235
	JHH-7	JCRB	JCRB1031
	SK-HEP-1	ATCC	HTB-52
	Hep 3B2.1-7	ATCC	HB-8064
Upper	SNU-1066	KCLB	01066
Aerodigestive	SNU-1041	KCLB	01041
Tract (Head	SNU-1076	KCLB	01076
and Neck)	BICR 18	ECACC	06051601
	CAL-33	DSMZ	ACC-447
	DETROIT 562	ATCC	CCL-138
	HSC-3	JCRB	JCRB0623
	HSC-4	JCRB	JCRB0624
	SCC-9	ATCC	CRL-1629
	YD-8	KCLB	60501
Urinary Tract	CAL-29	DSMZ	ACC-515
	KMBC-2	JCRB	JCRB1148
	253J	KCLB	80001
	253J-BV	KCLB	80002
	SW780	ATCC	CRL-2169
	SW1710	DSMZ	ACC-426
	VM-CUB-1	DSMZ	ACC-400
	BC-3C	DSMZ	ACC-450
Central	KNS-81	JCRB	IFO50359
Nervous	TM-31	RCB	RCB1731
System	NMC-G1	JCRB	IFO50467
	GB-1	JCRB	IFO50489
	SNU-201	KCLB	00201
	DBTRG-05MG	ATCC	CRL-2020
	YKG-1	JCRB	JCRB0746
Stomach	ECC10	RCB	RCB0983
	RERF-GC-1B	JCRB	JCRB1009
	TGBC-11-TKB	RCB	RCB1148
	SNU-620	KCLB	00620
	GSU	RCB	RCB2278
	KE-39	RCB	RCB1434
	HuG1-N	RCB	RCB1179
	NUGC-4	JCRB	JCRB0834
	MKN-45	JCRB	JCRB0254
	SNU-16	ATCC	CRL-5974
	OCUM-1	JCRB	JCRB0192
Colon (Large	C2BBe1	ATCC	CRL-2102
and Small	Caco-2	ATCC	HTB-37
Intestines)	SNU-1033	KCLB	01033
	SW1463	ATCC	CCL-234
	COLO 201	ATCC	CCL-224
	GP2d	ECACC	95090714
	LoVo	ATCC	CCL-229
	SW403	ATCC	CCL-230
	CL-14	DSMZ	ACC-504
Breast	HCC2157	ATCC	CRL-2340
	HCC38	ATCC	CRL-2314
	HCC1954	ATCC	CRL-2338
	HCC1143	ATCC	CRL-2321
	HCC1806	ATCC	CRL-2335
	HCC1599	ATCC	CRL-2331
	MDA-MB-415	ATCC	HTB-128
	CAL-51	DSMZ	ACC-302
Hematopoietic and	KO52	JCRB	JCRB0123
Lymphoid Tissue	SKNO-1	JCRB	JCRB1170
	Kasumi-1	ATCC	CRL-2724
	Kasumi-6	ATCC	CRL-2775
	MHH-CALL-3	DSMZ	ACC-339
	MHH-CALL-2	DSMZ	ACC-341
	JVM-2	ATCC	CRL-3002
	HNT-34	DSMZ	ACC-600
Bone	HOS	ATCC	CRL-1543
	OUMS-27	JCRB	IFO50488
	T1-73	ATCC	CRL-7943
	Hs 870.T	ATCC	CRL-7606
	Hs 706.T	ATCC	CRL-7447
	SJSA-1	ATCC	CRL-2098
	RD-ES	ATCC	HTB-166
	U2OS	ATCC	HTB-96
	SaOS-2	ATCC	HTB-85
	SK-ES-1	ATCC	HTB-86

In certain embodiments, the vaccine compositions comprising a combination of cell lines are capable of stimulating an immune response and/or preventing cancer and/or treating cancer. The present disclosure provides compositions and methods of using one or more vaccine compositions comprising therapeutically effective amounts of cell lines.

The amount (e.g., number) of cells from the various individual cell lines in a cocktail or vaccine compositions can be equal (as defined herein) or different. In various embodiments, the number of cells from a cell line or from each cell line (in the case where multiple cell lines are administered) in a vaccine composition, is approximately 1.0×10⁶, 2.0×10⁶, 3.0×10⁶, 4.0×10⁶, 5.0×10⁶, 6.0×10⁶, 7.0×10⁶, 8×10⁶, 9.0×10⁶, 1.0×10⁷, 2.0×10⁷, 3.0×10⁷, 4.0×10⁷, 5.0×10⁷, 6.0×10⁷, 8.0×10⁷, or 9.0×10⁷ cells.

The total number of cells administered to a subject, e.g., per administration site, can range from 1.0×10⁶ to 9.0×10⁷. For example, 2.0×10⁶, 3.0×10⁶, 4.0×10⁶, 5.0×10⁶, 6.0×10⁶, 7.0×10⁶, 8×10⁶, 9.0×10⁶, 1.0×10⁷, 2.0×10⁷, 3.0×10⁷, 4.0×10⁷, 5.0×10⁷, 6.0×10⁷, 8.0×10⁷, 8.6×10⁷, 8.8×10⁷, or 9.0×10⁷ cells are administered.

In certain embodiments, the number of cell lines included in each administration of the vaccine composition can range from 1 to 10 cell lines. In some embodiments, the number of cells from each cell line are not equal and different ratios of cell lines are used. For example, if one cocktail contains 5.0×10⁷ total cells from 3 different cell lines, there could be 3.33×10⁷ cells of one cell line and 8.33×10⁶ of the remaining 2 cell lines.

HLA Diversity

HLA mismatch occurs when the subject's HLA molecules are different from those expressed by the cells of the administered vaccine compositions. The process of HLA matching involves characterizing 5 major HLA loci, which include the HLA alleles at three Class I loci HLA-A, —B and —C and two class II loci HLA-DRB1 and -DQB1. Every individual expresses two alleles at each loci so the degree of HLA match or mismatch is calculated on a scale of 10, with 10/10 being a perfect match at all 10 alleles.

The response to mismatched HLA loci is mediated by both innate and adaptive cells of the immune system. Within the cells of the innate immune system, recognition of mismatches in HLA alleles is mediated to some extent by monocytes. Without being bound to any theory or mechanism, the sensing of “non-self” by monocytes triggers infiltration of monocyte-derived DCs, followed by their maturation, resulting in efficient antigen presentation to naïve T cells. Alloantigen-activated DCs produce increased amounts of IL-12 as compared to DCs activated by matched syngeneic antigens, and this increased IL-12 production results in the skewing of responses to Th1 T cells and increased IFN gamma production. HLA mismatch recognition by the adaptive immune system is driven to some extent by T cells. Without being bound to any theory or mechanism, 1-10% of all circulating T cells are alloreactive and respond to HLA molecules that are not present in self. This is several orders of magnitude greater than the frequency of endogenous T cells that are reactive to a conventional foreign antigen. The ability of the immune system to recognize these differences in HLA alleles and generate an immune response is a barrier to successful transplantation between donors and patients and has been viewed an obstacle in the development of cancer vaccines.

As many as 945 different HLA-A and -B alleles can be assigned to one of the nine supertypes based on the binding affinity of the HLA molecule to epitope anchor residues. In some embodiments, the vaccine compositions provided herein exhibit a heterogeneity of HLA supertypes, e.g., mixtures of HLA-A supertypes, and HLA-B supertypes. As described herein, various features and criteria may be considered to ensure the desired heterogeneity of the vaccine composition including, but not limited to, an individual's ethnicity (with regard to both cell donor and subject receiving the vaccine). Additional criteria are described in Example 25 of WO/2021/113328 and herein. In certain embodiments, a vaccine composition expresses a heterogeneity of HLA supertypes, wherein at least two different HLA-A and at least two HLA-B supertypes are represented.

In some embodiments, a composition comprising therapeutically effective amounts of multiple cell lines are provided to ensure a broad degree of HLA mismatch on multiple class I and class II HLA molecules between the tumor cell vaccine and the recipient.

In some embodiments, the vaccine composition expresses a heterogeneity of HLA supertypes, wherein the composition expresses a heterogeneity of major histocompatibility complex (MHC) molecules such that two of HLA-A24, HLA-A03, HLA-A01, and two of HLA-B07, HLA-B08, HLA-B27, and HLA-B44 supertypes are represented. In some embodiments, the vaccine composition expresses a heterogeneity HLA supertypes, wherein the composition expresses a heterogeneity of MHC molecules and at least the HLA-A24 is represented. In some exemplary embodiments, the composition expresses a heterogeneity of MHC molecules such that HLA-A24, HLA-A03, HLA-A01, HLA-B07, HLA-B27, and HLA-B44 supertypes are represented. In other exemplary embodiments, the composition expresses a genetic heterogeneity of MHC molecules such that HLA-A01, HLA-A03, HLA-B07, HLA-B08, and HLA-B44 supertypes are represented.

Patients display a wide breadth of HLA types that act as markers of self. A localized inflammatory response that promotes the release of cytokines, such as IFNγ and IL-2, is initiated upon encountering a non-self cell. In some embodiments, increasing the heterogeneity of HLA-supertypes within the vaccine cocktail has the potential to augment the localized inflammatory response when the vaccine is delivered conferring an adjuvant effect. As described herein, in some embodiments, increasing the breadth, magnitude, and immunogenicity of tumor reactive T cells primed by the cancer vaccine composition is accomplished by including multiple cell lines chosen to have mismatches in HLA types, chosen, for example, based on expression of certain TAAs. Embodiments of the vaccine compositions provided herein enable effective priming of a broad and effective anti-cancer response in the subject with the additional adjuvant effect generated by the HLA mismatch. Various embodiments of the cell line combinations in a vaccine composition express the HLA-A supertypes and HLA-B supertypes. Non-limiting examples are provided in Example 25 of WO/2021/113328 and herein.

Cell Line Modifications

In certain embodiments, the vaccine compositions comprise cells that have been modified. Modified cell lines can be clonally derived from a single modified cell, i.e., genetically homogenous, or derived from a genetically heterogenous population.

Cell lines can be modified to express or increase expression (e.g., relative to an unmodified cell) of one or more immunostimulatory factors, to inhibit or decrease expression of one or more immunosuppressive factors (e.g., relative to an unmodified cell), and/or to express or increase expression of one or more TAAs (e.g., relative to an unmodified cell), including optionally TAAs that have been mutated in order to present neoepitopes (e.g., designed or enhanced antigens with NSMs) as described herein. Additionally, cell lines can be modified to express or increase expression of factors that can modulate pathways indirectly, such expression or inhibition of microRNAs. Further, cell lines can be modified to secrete non-endogenous or altered exosomes. As described herein, in some embodiments the cell lines are optionally additionally modified to express tumor fitness advantage mutations, including but not limited to acquired tyrosine kinase inhibitor (TK I) resistance mutations, EGFR activating mutations, and/or modified ALK intracellular domain(s), and/or driver mutations.

In addition to modifying cell lines to express a TAA or immunostimulatory factor, the present disclosure also contemplates co-administering one or more TAAs (e.g., an isolated TAA or purified and/or recombinant TAA) or immunostimulatory factors (e.g., recombinantly produced therapeutic protein) with the vaccines described herein.

Thus, in various embodiments, the present disclosure provides a unit dose of a vaccine comprising (i) a first composition comprising a therapeutically effective amount of at least 1, 2, 3, 4, 5 or 6 cancer cell lines, wherein the cell line or a combination of the cell lines comprises cells that express at least 5, 10, 15, 20, 25, 30, 35, or 40 tumor associated antigens (TAAs) associated with a cancer of a subject intended to receive said composition, and wherein the composition is capable of eliciting an immune response specific to the at least 5, 10, 15, 20, 25, 30, 35, or 40 TAAs, and (ii) a second composition comprising one or more isolated TAAs. In other embodiments, the first composition comprises a cell line or cell lines that is further modified to (a) express or increase expression of at least 1 immunostimulatory factor, and/or (ii) inhibit or decrease expression of at least 1 immunosuppressive factor.

Mutations Providing a Fitness Advantage to Tumor Cells

Cancers arise as a result of changes that have occurred in genome sequences of cells. Oncogenes as described in detail herein are genes that are involved in tumorigenesis. In tumor cells, oncogenes are often mutated and/or expressed at high levels. The term “driver mutations” as used herein, refers to somatic mutations that confer a growth advantage to the tumor cells carrying them and that have been positively selected during the evolution of the cancer. Driver mutations frequently represent a large fraction of the total mutations in oncogenes, and often dictate cancer phenotype.

As described herein, cancer vaccine platforms can, in some embodiments, be designed to target tumor associated antigens (TAAs) that are overexpressed in tumor cells. Neoepitopes are non-self epitopes generated from somatic mutations arising during tumor growth. The targeting of neoepitopes is a beneficial component of the cancer vaccine platform as described in various embodiments herein at least because neoepitopes are tumor specific and not subject to central tolerance in the thymus.

Based on the information on the number of alleles harboring the mutation and the fraction of tumor cells with the mutation, mutations can be classified as clonal (truncal mutations, present in all tumor cells sequenced) and subclonal (shared and private mutations, present in a subset of regions or cells within a single biopsy) (McGranahan N. et al., Sci. Trans. Med. 7(283): 283ra54, 2015). Unlike the majority of neoepitopes that are private mutations and not found in more than one patient, driver mutations in known driver genes typically occur early in the evolution of the cancer and are found in all or a subset of tumor cells across patients (Jamal-Hanjani, M. et al. Clin Cancer Res. 21(6), 1258-66, 2015). Driver mutations show a tendency to be clonal and give a fitness advantage to the tumor cells that carry them and are crucial for the tumor's transformation, growth and survival (Schumacher T., et al. Science 348:69-74, 2015). As described herein, targeting driver mutations is an effective strategy to overcome intra- and inter-tumor neoantigen heterogeneity and tumor escape. Inclusion of a pool of driver mutations that occur at high frequency in a vaccine can potentially promote potent anti-tumor immune responses.

Mutations that confer a tumor fitness advantage can also occur as the result of targeted therapies. For example, a subset of NSCLC tumors contain tumorigenic amplifications of EGFR or ALK that may be initially treatable with tyrosine kinase inhibitors. NSCLC tumors treated with tyrosine kinase inhibitors often develop mutations resulting in resistance to these therapies enabling tumor growth. (Ricordel, C. et al. Annals of Oncology. 29 (Supplement 1): i28-i37, 2018; Lin, J et al., Cancer Discovery, 7(2):137-155, 2017).

Table 4 describes exemplary tumor fitness advantage mutations that can provide a fitness advantage to solid tumors. Some exemplary mutations are specific the anatomical origin of the tumor, such as prostate cancer mutations in SPOP, while some exemplary mutations, such as some mutations in TP53, can provide a fitness advantage to tumors originating from more than one ananatomical site.

TABLE 4
Exemplary mutations providing a fitness advantage
to solid tumors by mutated gene and indication
Gene (Gene ID)	Mutation	Anantomical origin of the tumor
AR (367)	H875Y	Prostate
	L702H	Prostate
	W742C	Prostate
ATM (472)	R337C	Colorectal
CDH1 (999)	D254Y	Stomach
CDKN2A (1029)	H83Y	Pancreas
CTNNB1 (1499)	S45F	Colorectal
EGFR (1956)	A289D	Central Nervous System
	G598V	Central Nervous System
	G63R	Central Nervous System
	H304Y	Central Nervous System
	R108K	Central Nervous System
	R252C	Central Nervous System
	S645C	Central Nervous System
	V774M	Central Nervous System
EP300 (2033)	D1399N	Upper Aerodigestive Tract
ERBB2 (2064)	R678Q	Stomach
	S310F	Stomach, Bladder
	V842I	Stomach, Bladder
ERBB3 (2065)	D297Y	Stomach
	V104L	Bladder
	V104M	Stomach, Colorectal
ERBB4 (2066)	S1289A	Bladder
ERCC2 (2068)	E86Q	Bladder
	N238S	Bladder
	S44L	Bladder
FBXW7 (55294)	R465H	Stomach, Colorectal
	R479Q	Stomach
	R505C	Colorectal
	R505G	Bladder
	S582L	Colorectal
FGFR3 (2261)	G370C	Bladder
	S249C	Bladder
	Y373C	Bladder
GNAS (2778)	R201H	Colorectal
HRAS (3265)	G13R	Bladder
	Q61R	Bladder
KRAS (3845)	A59T	Stomach
	G12A	Lung
	G12C	Pancreas, Colorectal
	G12D	Lung, Pancreas
	G12V	Lung, Pancreas
	G13C	Lung
	Q61R	Pancreas
PIK3CA (5290)	E542K	Stomach, Bladder, Colorectal,
		Breast, Upper Aerodigestive
		Tract, Lung
	E726K	Bladder, Breast
	H1047L	Breast, Upper Aerodigestive Tract
	H1047R	Stomach, Bladder, Central
		Nervous System, Lung
	H1047Y	Colorectal
	M1043I	Colorectal
	M1043V	Central Nervous System
	N345K	Stomach, Breast
	R88Q	Stomach, Bladder, Colorectal
PIK3R1 (5295)	G376R	Central Nervous System
PTEN (5728)	R130Q	Central Nervous System
	G132D	Central Nervous System
	R173H	Central Nervous System
RHOA1 (387)	L57V	Stomach
SMAD4 (4089)	R361H	Colorectal, Pancreas
SPOP (8405)	F102V	Prostate
	F133L	Prostate
	Y87C	Prostate
TP53 (7157)	C141Y	Lung
	C176F	Stomach, Lung
	C176Y	Ovaries
	C238Y	Ovaries, Pancreas
	C275Y	Central Nervous System, Ovaries
	E285K	Bladder
	G154V	Lung
	G245S	Stomach, Central Nervous System,
		Colorectal, Upper Aerodigestive
		Tract, Pancreas
	G245V	Central Nervous System
	G266R	Ovaries
	H179R	Central Nervous System
	H193L	Upper Aerodigestive Tract
	H193Y	Ovaries
	H214R	Pancreas, Lung
	I195T	Ovaries
	I251F	Lung
	K132N	Bladder
	L194R	Ovaries
	M237I	Stomach, Lung
	P278S	Upper Aerodigestive Tract
	R110L	Upper Aerodigestive Tract, Lung
	R158H	Central Nervous System
	R158L	Lung
	R175H	Stomach, Bladder, Central Nervous
		System, Colorectal, Prostate,
		Pancreas, Lung
	R248W	Stomach, Bladder, Central Nervous
		System, Colorectal, Breast, Ovaries,
		Upper Aerodigestive Tract, Pancreas
	R249M	Lung
	R273C	Pancreas, Prostate, Colorectal,
		Bladder, Stomach
	R273H	Central Nervous System, Breast,
		Upper Aerodigestive Tract
	R273L	Ovaries, Lung
	R280K	Bladder
	R337L	Lung
	S241F	Bladder
	V157F	Ovaries, Upper Aerodigestive Tract,
		Lung
	V216M	Central Nervous System, Ovaries
	V272M	Ovaries
	Y163C	Ovaries, Upper Aerodigestive Tract
	Y220C	Stomach, Prostate, Breast, Ovaries,
		Pancreas, Lung
	Y234C	Lung, Ovaries

Exemplary EGFR activating mutations, EGFR TKI acquired resistance mutations, ALK TKI acquired resistance mutations, and mutations that can be introduced into the intracellular tyrosine kinase domain of ALK are provided in Table 4-33, Table 4-38 and Table 4-41.

As described herein, one or more cell lines of the cancer vaccines are modified to express one or more peptides comprising one or more driver mutation sequences. The driver mutation modification design process is described in detail herein. In general, the design process includes identifying frequently mutated oncogenes for a given indication, identifying driver mutations in selected oncogenes, and selecting driver mutations to be engineered into a component of the vaccine platform based on, for example, the presence of CD4, CD8 or CD4 and CD8 epitopes. Additional steps may also be performed as provided herein.

“Frequently mutated oncogenes” as used herein can refer to, for example, oncogenes that contain more mutations relative to other known oncogenes in a set of patient tumor samples for a specific tumor type. Mutations in the oncogene may occur at the same amino acid position in multiple tumor samples. Some or all of the oncogene mutations may be private mutations and occur at different amino acid locations. The frequency of oncogene mutations varies based on the tumor mutational burden of the specific tumor type. Immunologically “cold” tumors in general tend to have fewer oncogenes with lower frequency of mutations, while immunologically “hot” tumors generally tend to have more oncogenes with greater frequency of mutations. Frequently mutated oncogenes may be similar for different tumor indications, such as TP53, or be indication specific, such as SPOP in prostate cancer. Among the 10 indications specifically described herein, the highest frequency of mutated oncogene is 69.7% (TP53, Ovarian). Oncogenes with lower than 5% mutation frequency are unlikely to possess an individual mutation occurring in greater than 0.5% of profiled patient tumor samples, and thus in one embodiment of the present disclosure, a mutation frequency of greater than or equal to 5% mutation is observed and selected. In various embodiments, a frequency of greater than or equal to 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% mutation is provided.

A list of frequently mutated oncogenes (>5%) is provided in Table 5.

TABLE 5
Frequently mutated oncogenes in solid tumors
Anatomical Site of Primary
Tumor                            NCBI Gene Symbol (Gene ID)
Central Nervous System (Glioma)  ATRX (546)
                                 EGFR (1956)
                                 NF1 (4763)
                                 PCLO (27445)
                                 PIK3CA (5290)
                                 PIK3R1 (5295)
                                 PTEN (5728)
                                 RB1 (5925)
                                 TP53 (7157)
Prostate                         AR (367)
                                 FOXA1 (3169)
                                 KMT2C (58508)
                                 KMT2D (8085)
                                 SPOP (8405)
                                 TP53 (7157)
Lung (non-small cell lung cancer) ALK (238)
                                 ARID1A (8289)
                                 ATM (472)
                                 CDKN2A (1029)
                                 CPS1 (1373)
                                 CREBBP (1387)
                                 EGFR (1956)
                                 EP400 (57634)
                                 EPHA3 (2042)
                                 EPHA5 (2044)
                                 EPHA7 (2045)
                                 ERBB4 (2066)
                                 FAT1 (2195)
                                 FAT4 (79633)
                                 GRIN2A (2903)
                                 HGF (3082)
                                 KDR (3791)
                                 KEAP1 (9817)
                                 KMT2C (58508)
                                 KMT2D (8085)
                                 KRAS (3845)
                                 LRP1B (53353)
                                 LRRK2 (120892)
                                 MGA (23269)
                                 MGAM (8972)
                                 NF1 (4763)
                                 NFE2L2 (4780)
                                 NOTCH1 (4851)
                                 NTRK3 (4916)
                                 PCLO (27445)
                                 PDE4DIP (9659)
                                 PDGFRA (5156)
                                 PIK3CA (5290)
                                 PIK3CG (5294)
                                 POLE (5426)
                                 POLQ (10721)
                                 PREX2 (80243)
                                 PRKDC (5591)
                                 PTPRB (5787)
                                 PTPRC (5788)
                                 PTPRD (5789)
                                 PTPRT (11122)
                                 RB1 (5925)
                                 RELN (5649)
                                 RNF213 (57674)
                                 ROS1 (6098)
                                 RUNX1T1 (862)
                                 SETBP1 (26040)
                                 SMARCA4 (6597)
                                 STK11 (6794)
                                 TP53 (7157)
                                 TPR (7175)
                                 TRRAP (8295)
                                 ZFHX3 (463)
                                 ZNF521 (25925)
Colorectal                       ACVR2A (92)
                                 AFDN (4301)
                                 ALK (238)
                                 AMER1 (139285)
                                 ANKRD11 (29123)
                                 APC (324)
                                 ARID1A (8289)
                                 ARID1B (57492)
                                 ARID2 (196528)
                                 ASXL1 (171023)
                                 ATM (472)
                                 ATRX (546)
                                 AXIN2 (8313)
                                 B2M (567)
                                 BCL9 (607)
                                 BCL9L (283149)
                                 BCORL1 (63035)
                                 BRAF (673)
                                 BRCA2 (675)
                                 CACNA1D (776)
                                 CAD (790)
                                 CAMTA1 (23261)
                                 CARD11 (84433)
                                 CHD4 (1108)
                                 CIC (23152)
                                 COL1A1 (1277)
                                 CREBBP (1387)
                                 CTNNB1 (1499)
                                 CUX1 (1523)
                                 DICER1 (23405)
                                 EP300 (2033)
                                 EP400 (57634)
                                 EPHA5 (2044)
                                 ERBB3 (2065)
                                 ERBB4 (2066)
                                 FAT1 (2195)
                                 FAT4 (79633)
                                 FBXW7 (55294)
                                 FLT4 (2324)
                                 GNAS (2778)
                                 GRIN2A (2903)
                                 IRS1 (3667)
                                 IRS4 (8471)
                                 KDM2B (84678)
                                 KMT2A (4297)
                                 KMT2B (9757)
                                 KMT2C (58508)
                                 KMT2D (8085)
                                 KRAS (3845)
                                 LARP4B (23185)
                                 LRP1B (53353)
                                 LRP5 (4041)
                                 LRRK2 (120892)
                                 MGA (23269)
                                 MKI67 (4288)
                                 MTOR (2475)
                                 MYH11 (4629)
                                 MYH9 (4627)
                                 MYO5A (4644)
                                 NCOR2 (9612)
                                 NF1 (4763)
                                 NOTCH1 (4851)
                                 NOTCH3 (4854)
                                 NUMA1 (4926)
                                 PCLO (27445)
                                 PDE4DIP (9659)
                                 PIK3CA (5290)
                                 PIK3CG (5294)
                                 PIK3R1 (5295)
                                 PLCG2 (5336)
                                 POLE (5426)
                                 POLQ (10721)
                                 PREX2 (80243)
                                 PRKDC (5591)
                                 PTEN (5728)
                                 PTPRC (5788)
                                 PTPRD (5789)
                                 PTPRK (5796)
                                 PTPRS (5802)
                                 PTPRT (11122)
                                 RANBP2 (5903)
                                 RELN (5649)
                                 RNF213 (57674)
                                 RNF43 (54894)
                                 ROBO1 (6091)
                                 ROS1 (6098)
                                 SETBP1 (26040)
                                 SETD1A (9739)
                                 SLX4 (84464)
                                 SMAD4 (4089)
                                 SMARCA4 (6597)
                                 SOX9 (6662)
                                 SPEN (23013)
                                 TCF7L2 (6934)
                                 TP53 (7157)
                                 TP53BP1 (7158)
                                 TRRAP (8295)
                                 UBR5 (51366)
                                 ZBTB20 (26137)
                                 ZFHX3 (463)
                                 ZNF521 (25925)
Head and Neck                    CASP8 (841)
                                 CDKN2A (1029)
                                 EP300 (2033)
                                 FAT1 (2195)
                                 FAT4 (79633)
                                 KMT2C (58508)
                                 KMT2D (8085)
                                 LRP1B (53353)
                                 NOTCH1 (4851)
                                 NSD1 (64324)
                                 PCLO (27445)
                                 PIK3CA (5290)
                                 RELN (5649)
                                 TP53 (7157)
Bladder                          ARID1A (8289)
                                 APC (324)
                                 ARID2 (196528)
                                 ATM (472)
                                 ATR (545)
                                 BRCA1 (672)
                                 BRCA2 (675)
                                 CDK12 (51755)
                                 CDKN1A (1026)
                                 CREBBP (1387)
                                 ELF3 (1999)
                                 EP300 (2033)
                                 ERBB2 (2064)
                                 ERBB3 (2065)
                                 ERBB4 (2066)
                                 ERCC2 (2068)
                                 FAT1 (2195)
                                 FAT4 (79633)
                                 FBXW7 (55294)
                                 FGFR3 (2261)
                                 HRAS (3265)
                                 KDM6A (7403)
                                 KMT2A (4297)
                                 KMT2C (58508)
                                 KMT2D (8085)
                                 LRP1B (53353)
                                 LRRK2 (120892)
                                 MKI67 (4288)
                                 MYH9 (4627)
                                 NCOR1 (9611)
                                 NF1 (4763)
                                 PCLO (27445)
                                 PDE4DIP (9659)
                                 PIK3CA (5290)
                                 PTPRD (5789)
                                 RB1 (5925)
                                 RICTOR (253260)
                                 RNF213 (57674)
                                 SETD2 (29072)
                                 SMARCA4 (6597)
                                 STAG2 (10735)
                                 TP53 (7157)
                                 TRRAP (8295)
                                 TSC1 (7248)
                                 UBR5 (51366)
                                 ZFP36L1 (677)
Breast                           CDH1 (999)
                                 GATA3 (2625)
                                 KMT2C (58508)
                                 KMT2D (8085)
                                 MAP3K1 (4214)
                                 PIK3CA (5290)
                                 TP53 (7157)
Ovarian                          NF1 (4763)
                                 TP53 (7157)
Pancreas                         ARID1A (8289)
                                 CDKN2A (1029)
                                 KRAS (3845)
                                 MEN1 (4221)
                                 RNF43 (54894)
                                 SMAD4 (4089)
                                 TP53 (7157)
Stomach                          ACVR2A (92)
                                 ANKRD11 (29123)
                                 APC (324)
                                 AR (367)
                                 ARID1A (8289)
                                 ARID2 (196528)
                                 ATM (472)
                                 ATR (545)
                                 BCL9L (283149)
                                 BCOR (54880)
                                 BRCA2 (675)
                                 CACNA1D (776)
                                 CARD11 (84433)
                                 CDH1 (999)
                                 CDH11 (1009)
                                 CHD4 (1108)
                                 CIC (23152)
                                 CREBBP (1387)
                                 CTNNB1 (1499)
                                 EP400 (57634)
                                 EPHA3 (2042)
                                 EPHA5 (2044)
                                 EPHB1 (2047)
                                 ERBB2 (2064)
                                 ERBB3 (2065)
                                 ERBB4 (2066)
                                 FAT1 (2195)
                                 FAT4 (79633)
                                 FBXW7 (55294)
                                 GNAS (2778)
                                 GRIN2A (2903)
                                 KAT6A (7994)
                                 KMT2A (4297)
                                 KMT2B (9757)
                                 KMT2C (58508)
                                 KMT2D (8085)
                                 KRAS (3845)
                                 LARP4B (23185)
                                 LRP1B (53353)
                                 LRP5 (4041)
                                 LRRK2 (120892)
                                 MDC1 (9656)
                                 MGA (23269)
                                 MKI67 (4288)
                                 MYH11 (4629)
                                 MYH9 (4627)
                                 NCOA2 (10499)
                                 NCOR2 (9612)
                                 NF1 (4763)
                                 NFATC2 (4773)
                                 NIN (51199)
                                 NOTCH1 (4851)
                                 NOTCH2 (4853)
                                 NSD1 (64324)
                                 NUMA1 (4926)
                                 PBRM1 (55193)
                                 PCLO (27445)
                                 PDE4DIP (9659)
                                 PDS5B (23047)
                                 PIK3CA (5290)
                                 POLE (5426)
                                 POLQ (10721)
                                 PREX2 (80243)
                                 PRKDC (5591)
                                 PTEN (5728)
                                 PTPRD (5789)
                                 PTPRS (5802)
                                 PTPRT (11122)
                                 RELN (5649)
                                 RHOA (387)
                                 RNF213 (57674)
                                 RNF43 (54894)
                                 ROBO1 (6091)
                                 ROS1 (6098)
                                 RPL22 (6146)
                                 SETBP1 (26040)
                                 SMAD4 (4089)
                                 SMARCA4 (6597)
                                 SPEN (23013)
                                 TGFBR2 (7048)
                                 TP53 (7157)
                                 TRRAP (8295)
                                 UBR5 (51366)
                                 ZBTB20 (26137)
                                 ZFHX3 (463)
                                 ZNF521 (25925)

Following identification of one or more frequently mutated oncogenes, driver mutations within the oncogenes are identified and selected. In various embodiments, driver mutations occurring in the same amino acid position in 0.5% of profiled patient tumor samples in each mutated oncogene are selected. In various embodiments, driver mutations occurring in the same amino acid position in 0.75, 1.0 or 1.5% of profiled patient tumor samples in each mutated oncogene are selected.

In various embodiments, the driver mutation is a missense (substitution), insertion, in-frame insertion, deletion, in-frame deletion, or gene amplification mutation. In various embodiments, one or more driver mutation sequences, once identified and prioritized as described herein, are inserted into a vector. In some embodiments, the vector is a lentiviral vector (lentivector).

In various embodiments of the present disclosure, a peptide sequence containing MHC class I and II epitopes and a given driver mutation that is 28-35 amino acid in length is generated to induce a potent driver mutation-specific immune response (e.g., cytotoxic and T helper cell responses). In some embodiments, a respective driver mutation is placed in the middle of a 28-35-mer peptide, flanked by roughly 15 aa on either side taken from the respective non-mutated, adjacent, natural human protein backbone. In some embodiments, when two (or more) driver mutations occur within 9 amino acids of a protein sequence, a long peptide sequence containing two (or more) driver mutations is also generated so long as there are at least 8 amino acids before and after each driver mutation. In various embodiments, up to 20 driver mutation-containing long peptides are assembled into one insert, separated by the furin and/or P2A cleavage site.

In some embodiments, the cell lines of the vaccine composition can be modified (e.g., genetically modified) to express, overexpress, or increase the expression of one or more peptides comprising one or more of the driver mutations in one or more of the oncogenes selected from Table 5. For example, at least one (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the cancer cell lines in any of the vaccine compositions described herein may be genetically modified to express, overexpress, or increase the expression of one or more peptides comprising one or more of the driver mutations in one or more of the oncogenes selected from Table 5. The driver mutations expressed by the cells within the composition may all be the same, may all be different, or any combination thereof.

In some embodiments, a vaccine composition comprises a therapeutically effective amount of cells from at least one cancer cell line, wherein the at least one cell line is modified to express, overexpress, or increase the expression of one or more peptides comprising one or more of the driver mutations in one or more of the oncogenes selected from Table 5. In some embodiments, the composition comprises a therapeutically effective amount of cells from 2, 3, 4, 5, 6, 7, 8, 9, or 10 cancer cell lines.

In various embodiments, the cell line or cell lines modified to express, overexpress, or increase the expression of one or more peptides comprising one or more of the driver mutations in one or more of the oncogenes selected from Table 5 are (a) non-small cell lung cancer cell lines (NSCLC) and/or small cell lung cancer (SCLC) cell lines selected from the group consisting of NCI-H460, NCI H520, A549, DMS 53, LK-2, and NCI-H23; (b) small cell lung cancer cell lines selected from the group consisting of DMS 114, NCI-H196, NCI-H1092, SBC-5, NCI-H510A, NCI-H889, NCI-H1341, NCIH-1876, NCI-H2029, NCI-H841, DMS 53, and NCI-H1694; (c) prostate cancer cell lines and/or testicular cancer cell lines selected from the group consisting of PC3, DU-145, LNCAP, NEC8, and NTERA-2cl-D1; (d) colorectal cancer cell lines selected from the group consisting of HCT-15, RKO, HuTu-80, HCT-116, and LS411N; (e) breast and/or triple negative breast cancer cell lines selected from the group consisting of Hs 578T, AU565, CAMA-1, MCF-7, and T-47D; (f) bladder and/or urinary tract cancer cell lines selected from the group consisting of UM-UC-3, J82, TCCSUP, HT-1376, and SCaBER; (g) head and/or neck cancer cell lines selected from the group consisting of HSC-4, Detroit 562, KON, HO-1-N-1, and OSC-20; (h) gastric and/or stomach cancer cell lines selected from the group consisting of Fu97, MKN74, MKN45, OCUM-1, and MKN1; (i) liver cancer and/or hepatocellular cancer (HCC) cell lines selected from the group consisting of Hep-G2, JHH-2, JHH-4, JHH-5, JHH-6, Li7, HLF, HuH-1, HuH-6, and HuH-7; (j) glioblastoma cancer cell lines selected from the group consisting of DBTRG-05MG, LN-229, SF-126, GB-1, and KNS-60; (k) ovarian cancer cell lines selected from the group consisting of TOV-112D, ES-2, TOV-21G, OVTOKO, and MCAS: (l) esophageal cancer cell lines selected from the group consisting of TE-10, TE-6, TE-4, EC-GI-10, OE33, TE-9, TT, TE-11, OE19, and OE21; (m) kidney and/or renal cell carcinoma cancer cell lines selected from the group consisting of A-498, A-704, 769-P, 786-O, ACHN, KMRC-1, KMRC-2, VMRC-RCZ, and VMRC-RCW; (n) pancreatic cancer cell lines selected from the group consisting of PANC-1, KP-3, KP-4, SUIT-2, and PSN11; (o) endometrial cancer cell lines selected from the group consisting of SNG-M, HEC-1-B, JHUEM-3, RL95-2, MFE-280, MFE-296, TEN, JHUEM-2, AN3-CA, and Ishikawa; (p) skin and/or melanoma cancer cell lines selected from the group consisting of RPMI-7951, MeWo, Hs 688(A).T, COLO 829, C32, A-375, Hs 294T, Hs 695T, Hs 852T, and A2058; or (q) mesothelioma cancer cell lines selected from the group consisting of NCI-H28, MSTO-211H, IST-Mes1, ACC-MESO-1, NCI-H2052, NCI-H2452, MPP 89, and IST-Mes2.

In some embodiments, a vaccine composition comprises a therapeutically effective amount of cells from at least one cancer cell line, wherein the at least one cell line is modified to express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more peptides comprising one or more driver mutation sequences. In some embodiments, the composition comprises a therapeutically effective amount of cells from 2, 3, 4, 5, 6, 7, 8, 9, or 10 cancer cell lines. In some embodiments, the at least one cell line is modified to increase the production of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 peptides comprising one or more driver mutation sequences.

In some embodiments, a driver mutation may satisfy the selection criteria described in the methods herein but is already present in a given cell or has been added to a cell line (e.g., via an added TAA) and are optionally included or optionally not included among the cell line modifications for a given vaccine.

Immunostimulatory Factors

An immunostimulatory protein is one that is membrane bound, secreted, or both that enhances and/or increases the effectiveness of effector T cell responses and/or humoral immune responses. Without being bound to any theory, immunostimulatory factors can potentiate antitumor immunity and increase cancer vaccine immunogenicity. There are many factors that potentiate the immune response. For example, these factors may impact the antigen-presentation mechanism or the T cell mechanism. Insertion of the genes for these factors may enhance the responses to the vaccine composition by making the vaccine more immunostimulatory of anti-tumor response.

Without being bound to any theory or mechanism, expression of immunostimulatory factors by the combination of cell lines included in the vaccine in the vaccine microenvironment (VME) can modulate multiple facets of the adaptive immune response. Expression of secreted cytokines such as GM-CSF and IL-15 by the cell lines can induce the differentiation of monocytes, recruited to the inflammatory environment of the vaccine delivery site, into dendritic cells (DCs), thereby enriching the pool of antigen presenting cells in the VME. Expression of certain cytokines can also mature and activate DCs and Langerhans cells (LCs) already present. Expression of certain cytokines can promote DCs and LCs to prime T cells towards an effector phenotype. DCs that encounter vaccine cells expressing IL-12 in the VME should prime effector T cells in the draining lymph node and mount a more efficient anti-tumor response. In addition to enhancing DC maturation, engagement of certain immunostimulatory factors with their receptors on DCs can promote the priming of T cells with an effector phenotype while suppressing the priming of T regulatory cells (Tregs). Engagement of certain immunostimulatory factors with their receptors on DCs can promote migration of DCs and T cell mediated acquired immunity.

In some embodiments of the vaccine compositions provided herein, modifications to express the immunostimulatory factors are not made to certain cell lines or, in other embodiments, all of the cell lines present in the vaccine composition.

Provided herein are embodiments of vaccine compositions comprising a therapeutically effective amount of cells from at least one cancer cell line, wherein the cell line is modified to increase production of at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) immunostimulatory factors. In some embodiments, the immunostimulatory factors are selected from those presented in Table 6. Also provided are exemplary NCBI Gene IDs that can be utilized by a skilled artisan to determine the sequences to be introduced in the vaccine compositions of the disclosure. These NCBI Gene IDs are exemplary only.

TABLE 6
Exemplary immunostimulatory factors
Factor                NCBI Gene Symbol (Gene ID)
CCL5                  CCL5 (6352)
XCL1                  XCL1 (6375)
Soluble CD40L (CD154) CD40LG (959)
Membrane-bound CD40L  CD40LG (959)
CD36                  CD36 (948)
GITR                  TNFRSF18 (8784)
GM-CSF                CSF2 (1437)
OX-40                 TNFRSF4 (7293)
OX-40L                TNFSF4 (7292)
CD137 (41BB)          TNFRSF9 (13604)
CD80 (B7-1)           CD80 (941)
IFNγ                  IFNG (3458)
IL-Iβ                 IL1B (3553)
IL-2                  IL2 (3558)
IL-6                  IL6 (3569)
IL-7                  IL7 (3574)
IL-9                  IL9 (3578)
IL-12                 IL12A (3592) IL12B (3593)
IL-15                 IL15 (3600)
IL-18                 IL-18 (3606)
IL-21                 IL21 (59067)
IL-23                 IL23A (51561) IL12B (3593)
TNFα                  TNF (7124)

In some embodiments, the cell lines of the vaccine composition can be modified (e.g., genetically modified) to express, overexpress, or increase the expression of one or more immunostimulatory factors selected from Table 6. In certain embodiments, the immunostimulatory sequence can be a native human sequence. In some embodiments, the immunostimulatory sequence can be a genetically engineered sequence. The genetically engineered sequence may be modified to increase expression of the protein through codon optimization, or to modify the cellular location of the protein (e.g., through mutation of protease cleavage sites).

For example, at least one (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the cancer cell lines in any of the vaccine compositions described herein may be genetically modified to express or increase expression of one or more immunostimulatory factors. The immunostimulatory factors expressed by the cells within the composition may all be the same, may all be different, or any combination thereof.

In some embodiments, a vaccine composition comprises a therapeutically effective amount of cells from at least one cancer cell line, wherein the at least one cell line is modified to express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the immunostimulatory factors of Table 6. In some embodiments, the composition comprises a therapeutically effective amount of cells from 2, 3, 4, 5, 6, 7, 8, 9, or 10 cancer cell lines. In some embodiments, the at least one cell line is modified to increase the production of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunostimulatory factors of Table 7. In some embodiments, the composition comprises a therapeutically effective amount of cells from 2, 3, 4, 5, 6, 7, 8, 9, or 10 cancer cell lines, and each cell line is modified to increase the production of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunostimulatory factors of Table 6.

In some embodiments, the composition comprises a therapeutically effective amount of cells from 3 cancer cells lines wherein 1, 2, or all 3 of the cell lines have been modified to express or increase expression of GM-CSF, membrane bound CD40L, and IL-12.

Exemplary combinations of modifications, e.g., where a cell line or cell lines have been modified to express or increase expression of more than one immunostimulatory factor include but are not limited to: GM-CSF+IL-12; CD40L+IL-12; GM-CSF+CD40L; GM-CSF+IL-12+CD40L; GM-CSF+IL-15; CD40L+IL-15; GM-CSF+CD40L; and GM-CSF+IL-15+CD40L, among other possible combinations.

In certain instances, tumor cells express immunostimulatory factors including the IL-12A (p35 component of IL-12), GM-CSF (kidney cell lines), and CD40L (leukemia cell lines). Thus, in some embodiments, cell lines may also be modified to increase expression of one or more immunostimulatory factors.

In some embodiments, the cell line combination of or cell lines that have been modified as described herein to express or increase expression of one or more immunostimulatory factors will express the immunostimulatory factor or factors at least 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more relative to the same cell line or combination of cell lines that have not been modified to express or increase expression of the one or more immunostimulatory factors.

Methods to increase immunostimulatory factors in the vaccine compositions described herein include, but are not limited to, introduction of the nucleotide sequence to be expressed by way of a viral vector or DNA plasmid. The expression or increase in expression of the immunostimulatory factors can be stable expression or transient expression.

In some embodiments, the cancer cells in any of the vaccine compositions described herein are genetically modified to express CD40 ligand (CD40L). In some embodiments, the CD40L is membrane bound. In some embodiments, the CD40L is not membrane bound. Unless stated otherwise, as used herein CD40L refers to membrane bound CD40L. In some embodiments, the cancer cells in any of the vaccine compositions described herein are genetically modified to express GM-CSF, membrane bound CD40L, GITR, IL-12, and/or IL-15. Exemplary amino acid and nucleotide sequences useful for expression of the one or more of the immunostimulatory factors provided herein are presented in Table 7.

TABLE 7
Sequences of exemplary immunostimulatory factors
Factor                 Sequence
CD154 (CD40L)          atgatcgaaacatacaaccaaacttctccccgatctgcggccactggactgcccatcagcatgaaaatttttatgtatttacttactgtttttcttat
(membrane bound) (SEQ  cacccagatgattgggtcagcactttttgctgtgtatcttcatagaaggttggacaagatagaagatgaaaggaatcttcatgaagattttgtatt
ID NO: 1)              catgaaaacgatacagagatgcaacacaggagaaagatccttatccttactgaactgtgaggagattaaaagccagtttgaaggctttgtg
                       aaggatataatgttaaacaaagaggagacgaagaaagaaaacagctttgaaatgcctcgtggtgaagaggatagtcaaattgcggcac
                       atgtcataagtgaggccagcagtaaaacaacatctgtgttacagtgggctgaaaaaggatactacaccatgagcaacaacttggtaaccc
                       tggaaaatgggaaacagctgaccgttaaaagacaaggactctattatatctatgcccaagtcaccttctgttccaatcgggaagcttcgagt
                       caagctccatttatagccagcctctgcctaaagtcccccggtagattcgagagaatcttactcagagctgcaaatacccacagttccgccaa
                       accttgcgggcaacaatccattcacttgggaggagtatttgaattgcaaccaggtgcttcggtgtttgtcaatgtgactgatccaagccaagtg
                       agccatggcactggcttcacgtcctttggcttactcaaactctga
CD154 (CD40L)          Atgatcgaaacctacaaccagacctcaccacgaagtgccgccaccggactgcctattagtatgaaaatctttatgtacctgctgacagtgtt
(membrane bound)       cctgatcacccagatgatcggctccgccctgtttgccgtgtacctgcaccggagactggacaagatcgaggatgagcggaacctgcacga
(codon-optimized) (SEQ ggacttcgtgtttatgaagaccatccagcggtgcaacacaggcgagagaagcctgtccctgctgaattgtgaggagatcaagagccagtt
ID NO: 2)              cgagggctttgtgaaggacatcatgctgaacaaggaggagacaaagaaggagaacagcttcgagatgcccagaggcgaggaggatt
                       cccagatcgccgcccacgtgatctctgaggccagctccaagaccacaagcgtgctgcagtgggccgagaagggctactataccatgtct
                       aacaatctggtgacactggagaacggcaagcagctgaccgtgaagaggcagggcctgtactatatctatgcccaggtgacattctgcagc
                       aatcgcgaggcctctagccaggccccctttatcgccagcctgtgcctgaagagccctggcaggttcgagcgcatcctgctgagagccgcc
                       aacacccactcctctgccaagccatgcggacagcagtcaatccacctgggaggcgtgttcgagctgcagccaggagcaagcgtgttcgt
                       gaatgtgactgacccatcacaggtgtctcacggcactggattcacatcatttggactgctgaaactgtga
CD154 (CD40L)          MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTI
(membrane bound)       QRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMPRGEEDSQIAAHVISEASSKTTS
(SEQ ID NO: 3)         VLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFE
                       RILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL
GITR (SEQ ID NO: 4)    Atggctcagcatggggctatgggggccttcagggctctgtgcggactggctctgctgtgcgctctgtcactggggcagagaccaacagga
                       ggaccaggatgcggacctggcaggctgctgctgggcaccggcacagacgcaaggtgctgtagagtgcacaccacaaggtgctgtcgc
                       gactaccctggcgaggagtgctgttctgagtgggattgcatgtgcgtgcagccagagtttcactgtggcgatccctgctgtaccacatgccgc
                       caccacccatgtccacctggacagggagtgcagtctcagggcaagttcagctttggcttccagtgcatcgactgtgcaagcggcaccttttc
                       cggaggacacgagggacactgcaagccctggaccgattgtacacagtttggcttcctgaccgtgttccctggcaacaagacacacaatgc
                       cgtgtgcgtgcctggctccccaccagcagagcccctgggctggctgaccgtggtgctgctggccgtggcagcatgcgtgctgctgctgaca
                       agcgcccagctgggactgcacatctggcagctgcggtcccagtgtatgtggccaagagagacccagctgctgctggaggtgcctccatcc
                       acagaggacgcccggtcttgccagttccccgaagaggagaggggggaaagaagtgccgaagaaaagggaaggctgggagacctgt
                       gggtg
GITR (SEQ ID NO: 5)    MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPG
                       EECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHE
                       GHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTWLLAVAACVLLLTSAQLGLHIW
                       QLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDL1NV
GM-CSF (SEQ ID NO: 6)  atgtggctgcagagcctgctgctcttgggcactgtggcctgcagcatctctgcacccgcccgctcgcccagccccagcacgcagccctggg
                       agcatgtgaatgccatccaggaggcccggcgtctcctgaacctgagtagagacactgctgctgagatgaatgaaacagtagaagtcatct
                       cagaaatgtttgacctccaggagccgacctgcctacagacccgcctggagctgtacaagcagggcctgcggggcagcctcaccaagct
                       caagggccccttgaccatgatggccagccactacaagcagcactgccctccaaccccggaaacttcctgtgcaacccagattatcaccttt
                       gaaagtttcaaagagaacctgaaggactttctgcttgtcatcccctttgactgctgggagccagtccaggagtga
GM-CSF                 atgtggctgcagtctctgctgctgctgggcaccgtcgcctgttctatttccgcacccgctcgctccccttctccctcaactcagccttgggagcac
(codon-optimized) (SEQ gtgaacgccatccaggaggcccggagactgctgaatctgtcccgggacaccgccgccgagatgaacgagacagtggaagtgatctctg
ID NO: 7)              agatgttcgatctgcaggagcccacctgcctgcagacaaggctggagctgtacaagcagggcctgcgcggctctctgaccaagctgaag
                       ggcccactgacaatgatggccagccactataagcagcactgcccccctacccccgagacaagctgtgccacccagatcatcacattcga
                       gtcctttaaggagaacctgaaggactttctgctggtcattccatttgattgttgggagcccgtgcaggagtga
GM-CSF (SEQ ID NO: 8)  MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDL
                       QEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLV
                       IPFDCWEPVQE
IL-12 (SEQ ID NO: 9)   atgtgccatcagcaactggttatatcttggttcagtctcgtctttctcgcgtcacccttggtcgctatctgggagcttaaaaaagatgtctacgtcgt
                       tgaacttgattggtaccctgatgctccgggggaaatggtggttttgacttgcgatacgccagaagaggatggcataacgtggacactggacc
                       agtcttcagaggttctcgggtctggtaagacactcactatacaggtgaaggagtttggtgacgcaggacaatatacttgccataaaggcggc
                       gaggtgctctcccatagccttctgctccttcataaaaaagaggacgggatatggtcaactgacattctgaaggatcagaaagaaccgaag
                       aacaaaactttcctcagatgcgaggcaaagaactattcaggccgctttacttgctggtggctcactaccatcagcactgacctcactttcagc
                       gtcaagagcagtagaggctcaagtgacccacaaggggttacatgcggggccgctacgttgtctgccgagcgagtcaggggagataata
                       aggaatatgagtatagcgttgaatgccaagaagattcagcctgcccagccgcagaagagagtcttcccatagaagttatggtggacgcag
                       ttcataaactgaagtatgagaactatacatcttccttctttattcgcgatatcataaagcctgatcctccgaaaaacttgcaactcaagccgttga
                       agaatagccgacaggtcgaggtctcttgggagtatccagatacgtggtctaccccgcactcctatttcagtctcaccttctgtgtgcaggtgca
                       ggggaaaagtaagcgggaaaaaaaggaccgggtatttactgataagacctccgctacagtgatttgtagaaagaacgcctctatcagcg
                       tgagagcccaggatagatattattctagtagttggtctgagtgggcctccgtcccttgttccggaagcggagccacgaacttctctctgttaaag
                       caagcaggagatgttgaagaaaaccccgggcctatgtgtccagcgcgcagcctcctccttgtggctaccctggtcctcctggaccacctca
                       gtttggcccgaaacctgccggtcgctacacccgatcctggaatgtttccctgccttcatcacagccagaatctgctgagggcagtcagtaac
                       atgctgcagaaggcgcggcaaactctggagttctatccatgtacctccgaggaaattgatcacgaggacattactaaggataaaacaagt
                       acagtagaagcctgtttgcctcttgagctcactaaaaatgagtcatgcttgaacagtcgagagacgagttttatcactaacggttcatgcttgg
                       cgtccaggaagacaagctttatgatggcgctctgcctgtcttctatatatgaagaccttaaaatgtaccaagttgagtttaagaccatgaacgc
                       caaacttttgatggaccccaagaggcagatcttccttgatcagaatatgttggcggtgatcgatgaacttatgcaagctttgaacttcaacagt
                       gagacagtgcctcagaaaagttccttggaggaaccggacttctataagaccaagatcaaactgtgcattttgctgcatgcatttagaattcga
                       gccgttacaatcgaccgggtgatgtcatatttgaatgcatcataa
IL-12 SEQ ID NO: 10)   MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMWLTCDTPEEDGITWTLDQSS
                       EVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEA
                       KNYSGRFTCIMNLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDS
                       ACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWST
                       PHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSG
                       SGATNFSLLKQAGDVEENPGPMCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLL
                       RAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLA
                       SRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVP
                       QKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS
IL-15 (SEQ ID NO: 11)  atgtataggatgcagctgctgtcatgtatcgcactgtccctggcactggtgactaactctaactgggtgaatgtgatctccgacctgaagaag
                       atcgaggacctgatccagtctatgcacatcgatgccaccctgtacacagagtccgacgtgcacccctcttgcaaggtgaccgccatgaagt
                       gtttcctgctggagctgcaggtcatcagcctggagagcggcgacgcatccatccacgataccgtggagaacctgatcatcctggccaaca
                       atagcctgagctccaacggcaatgtgacagagtccggctgcaaggagtgtgaggagctggaggagaagaatatcaaagagttcctgca
                       gtcattcgtccatatcgtccagatgtttatcaataccagt
IL-15 (SEQ ID NO: 12)  MYRMQLLSCIALSLALVTNSNINVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQ
                       VISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFlNTS
IL-23 (SEQ ID NO: 13)  atgtgccatcagcagctggtcattagttggtttagcctggtctttctggcctcacccctggtcgcaatctgggaactgaagaaggacgtgtacgt
                       ggtggagctggactggtatccagatgcaccaggagagatggtggtgctgacctgcgacacacctgaggaggatggcatcacctggaca
                       ctggatcagagctccgaggtgctgggcagcggcaagaccctgacaatccaggtgaaggagttcggcgacgccggccagtacacatgtc
                       acaagggcggcgaggtgctgtcccactctctgctgctgctgcacaagaaggaggacggcatctggtccacagacatcctgaaggatcag
                       aaggagccaaagaacaagaccttcctgcggtgcgaggccaagaattatagcggccggttcacctgttggtggctgaccacaatctccac
                       cgatctgacattttctgtgaagtctagcaggggctcctctgacccccagggagtgacatgcggagcagccaccctgagcgccgagcgggt
                       gagaggcgataacaaggagtacgagtattctgtggagtgccaggaggacagcgcctgtccagcagcagaggagtccctgcctatcgaa
                       gtgatggtggatgccgtgcacaagctgaagtacgagaattatacaagctccttctttatcagggacatcatcaagccagatccccctaagaa
                       cctgcagctgaagcccctgaagaatagccgccaggtggaggtgtcctgggagtaccctgacacctggtccacaccacactcttatttcagc
                       ctgaccttttgcgtgcaggtgcagggcaagagcaagagggagaagaaggaccgcgtgttcaccgataagacatccgccaccgtgatctg
                       tcggaagaacgccagcatctccgtgagggcccaggatcgctactattctagctcctggagcgagtgggcctccgtgccatgctctggagga
                       ggaggcagcggcggaggaggctccggaggcggcggctctggcggcggcggctccctgggctctcgggccgtgatgctgctgctgctgct
                       gccctggaccgcacagggaagagccgtgccaggaggctctagcccagcatggacacagtgccagcagctgtcccagaagctgtgcac
                       cctggcatggtctgcccaccctctggtgggccacatggacctgagagaggagggcgatgaggagaccacaaacgacgtgcctcacatc
                       cagtgcggcgacggctgtgatccacagggcctgagggacaattctcagttctgtctgcagcgcatccaccagggcctgatcttctacgaga
                       agctgctgggcagcgatatctttacaggagagcccagcctgctgcctgactccccagtgggacagctgcacgcctctctgctgggcctgag
                       ccagctgctgcagccagagggacaccactgggagacccagcagatcccttctctgagcccatcccagccttggcagcggctgctgctgc
                       ggttcaagatcctgagaagcctgcaggcattcgtcgcagtcgcagccagggtgttcgcccacggagccgctactctgagccca
IL-23 (SEQ ID NO: 14)  MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMWLTCDTPEEDGITWTLDQSS
                       EVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEA
                       KNYSGRFTCIMNLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDS
                       ACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWST
                       PHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSG
                       GGGSGGGGSGGGGSGGGGSLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLA
                       WSAHPLVGHMDLREEGDEETTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIF
                       TGEPSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAV
                       AARVFAHGAATLSP
XCL1 (SEQ ID NO: 15)   atgaggctgctgattctggcactgctgggcatctgctctctgaccgcttacatcgtggaaggagtcggctctgaagtctctgacaagcgcaca
                       tgcgtgtctctgaccacacagcgcctgcccgtgagccggatcaagacctacacaatcaccgagggcagcctgagagccgtgatcttcatc
                       acaaagaggggcctgaaggtgtgcgccgaccctcaggcaacctgggtgcgggacgtggtgagaagcatggataggaagtccaacac
                       ccggaacaatatgatccagacaaaacccacaggaacccagcagagcactaatacagccgtgacactgaccggg
XCL1 (SEQ ID NO: 16)   MRLLILALLGICSLTAYIVEGVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCA
                       DPQATINVRDWRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTG

Provided herein is a GITR protein comprising the amino acid sequence of SEQ ID NO: 4, or a nucleic acid sequence encoding the same, e.g., SEQ ID NO: 5. Provided herein is a vaccine composition comprising one or more cell lines expressing the same. Provided herein is a GM-CSF protein comprising the amino acid sequence of SEQ ID NO: 8, or a nucleic acid sequence encoding the same, e.g., SEQ ID NO: 6 or SEQ ID NO: 7. Provided herein is a vaccine composition comprising one or more cell lines expressing the same. Provided herein is an IL-12 protein comprising the amino acid sequence of SEQ ID NO: 10, or a nucleic acid sequence encoding the same, e.g., SEQ ID NO: 9. Provided herein is a vaccine composition comprising one or more cell lines expressing the same. Provided herein is an IL-15 protein comprising the amino acid sequence of SEQ ID NO: 12, or a nucleic acid sequence encoding the same, e.g., SEQ ID NO: 11. Provided herein is a vaccine composition comprising one or more cell lines expressing the same. Provided herein is an IL-23 protein comprising the amino acid sequence of SEQ ID NO: 14, or a nucleic acid sequence encoding the same, e.g., SEQ ID NO: 13. Provided herein is a vaccine composition comprising one or more cell lines expressing the same. Provided herein is a XCL1 protein comprising the amino acid sequence of SEQ ID NO: 16, or a nucleic acid sequence encoding the same, e.g., SEQ ID NO: 15. Provided herein is a vaccine composition comprising one or more cell lines expressing the same.

In some embodiments, the cancer cells in any of the vaccine compositions described herein are genetically modified to express one or more of CD28, B7-H2 (ICOS LG), CD70, CX3CL1, CXCL10 (IP10), CXCL9, LFA-1 (ITGB2), SELP, ICAM-1, ICOS, CD40, CD27 (TNFRSF7), TNFRSF14 (HVEM), BTN3A1, BTN3A2, ENTPD1, GZMA, and PERF1.

In some embodiments, vectors contain polynucleotide sequences that encode immunostimulatory molecules. Exemplary immunostimulatory molecules may include any of a variety of cytokines. The term “cytokine” as used herein refers to a protein released by one cell population that acts on one or more other cells as an intercellular mediator. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and —II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1 through IL-36, including, IL-1, IL-1alpha, IL-2, IL-3, IL-7, IL-8, IL-9, IL-11, IL-12; IL-15, IL-18, IL-21, IL-23, IL-27, TNF; and other polypeptide factors including LIF and kit ligand (KL). Other immunomodulatory molecules contemplated for use herein include IRF3, B7.1, B7.2, 4-1BB, CD40 ligand (CD40L), drug-inducible CD40 (iCD40), and the like.

In certain embodiments, polynucleotides encoding the immunostimulatory factors are under the control of one or more regulatory elements that direct the expression of the coding sequences. In various embodiments, more than one (i.e., 2, 3, or 4) immunostimulatory factors are encoded on one expression vector. In some embodiments, more than one (i.e., 2, 3, 4, 5, or 6) immunostimulatory factors are encoded on separate expression vectors. Lentivirus containing a gene or genes of interest (e.g., GM-CSF, CD40L, or IL-12 and other immunostimulatory molecules as described herein) are produced in various embodiments by transient co-transfection of 293T cells with lentiviral transfer vectors and packaging plasmids (OriGene) using LipoD293™ In Vitro DNA Transfection Reagent (SignaGen Laboratories).

For lentivirus infection, in some embodiments, cell lines are seeded in a well plate (e.g., 6-well, 12-well) at a density of 1-10×10⁵ cells per well to achieve 50-80% cell confluency on the day of infection. Eighteen-24 hours after seeding, cells are infected with lentiviruses in the presence of 10 μg/mL of polybrene. Eighteen-24 hours after lentivirus infection, cells are detached and transferred to larger vessel. After 24-120 hours, medium is removed and replaced with fresh medium supplemented with antibiotics.

Immunosuppressive Factors

An immunosuppressive factor is a protein that is membrane bound, secreted, or both and capable of contributing to defective and reduced cellular responses. Various immunosuppressive factors have been characterized in the context of the tumor microenvironment (TME). In addition, certain immunosuppressive factors can negatively regulate migration of LCs and DCs from the dermis to the draining lymph node.

TGFβ1 is a suppressive cytokine that exerts its effects on multiple immune cell subsets in the periphery as well as in the TME. In the VME, TGFβ1 negatively regulates migration of LCs and DCs from the dermis to the draining lymph node. Similarly, TGFβ2 is secreted by most tumor cells and exerts immunosuppressive effects similar to TGFβ1. Modification of the vaccine cell lines to reduce TGFβ1 and/or TGFβ2 secretion in the VME ensures the vaccine does not further TGFβ-mediated suppression of LC or DC migration.

Within the TME, CD47 expression is increased on tumor cells as a mode of tumor escape by preventing macrophage phagocytosis and tumor clearance. DCs also express SIRPα, and ligation of SIRPα on DCs can suppress DC survival and activation. Additional immunosuppressive factors in the vaccine that could play a role in the TME and VME include CD276 (B7-H3) and CTLA4. DC contact with a tumor cell expressing CD276 or CTLA4 in the TME dampens DC stimulatory capabilities resulting in decreased T cell priming, proliferation, and/or promotes proliferation of T cells. Expression of CTLA4 and/or CD276 on the vaccine cell lines could confer the similar suppressive effects on DCs or LCs in the VME.

In certain embodiments of the vaccine compositions, production of one or more immunosuppressive factors can be inhibited or decreased in the cells of the cell lines contained therein. In some embodiments, production (i.e., expression) of one or more immunosuppressive factors is inhibited (i.e., knocked out or completely eliminated) in the cells of the cell lines contained in the vaccine compositions. In some embodiments, the cell lines can be genetically modified to decrease (i.e., reduce) or inhibit expression of the immunosuppressive factors. In some embodiments, the immunosuppressive factor is excised from the cells completely. In some embodiments, one or more of the cell lines are modified such that one or more immunosuppressive factor is produced (i.e., expressed) at levels decreased or reduced (e.g., relative to an unmodified cell) by at least 5, 10, 15, 20, 25, or 30% (i.e., at least 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). In some embodiments, the one or more immunosuppressive factors is selected from the group presented in Table 8.

Simultaneously, production of one or more immunostimulatory factors, TAAs, and/or neoantigens can be increased in the vaccine compositions as described herein. In some embodiments of the vaccine compositions, in addition to the partial reduction or complete (e.g., excision and/or expression at undetectable levels) inhibition of expression of one or more immunosuppressive factors by the cell, one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the cell types within the compositions also can be genetically modified to increase the immunogenicity of the vaccine, e.g., by ensuring the expression of certain immunostimulatory factors, and/or TAAs.

Any combinations of these actions, modifications, and/or factors can be used to generate the vaccine compositions described herein. By way of non-limiting example, the combination of decreasing or reducing expression of immunosuppressive factors by at least 5, 10, 15, 20, 25, or 30% and increasing expression of immunostimulatory factors at least 2-fold higher than an unmodified cell line may be effective to improve the anti-tumor response of tumor cell vaccines. By way of another non-limiting example, the combination of reducing immunosuppressive factors by at least 5, 10, 15, 20, 25, or 30% and modifying cells to express certain TAAs in the vaccine composition, may be effective to improve the anti-tumor response of tumor cell vaccines.

In some embodiments, a cancer vaccine comprises a therapeutically effective amount of cells from at least one cancer cell line, wherein the cell line is modified to reduce production of at least one immunosuppressive factor by the cell line, and wherein the at least one immunosuppressive factor is CD47 or CD276. In some embodiments, expression of CTLA4, HLA-E, HLA-G, TGFβ1, and/or TGFβ2 are also reduced. In some embodiments, one or more, or all, cell lines in a vaccine composition are modified to inhibit or reduce expression of CD276, TGFβ1, and TGFβ2. In another embodiment, a vaccine composition is provided comprising three cell lines that have each been modified to inhibit (e.g., knockout) expression of CD276, and reduce expression of (e.g., knockdown) TGFβ1 and TGFβ2.

In some embodiments, a cancer vaccine composition comprises a therapeutically effective amount of cells from a cancer cell line wherein the cell line is modified to reduce expression of at least CD47. In some embodiments, the CD47 is excised from the cells or is produced at levels reduced by at least 5, 10, 15, 20, 25, or 30% (i.e., at least 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). In some embodiments, CD47 is excised from the cells or is produced at levels reduced by at least 90%. Production of additional immunosuppressive factors can be reduced in one or more cell lines. In some embodiments, expression of CD276, CTLA4, HLA-E, HLA-G, TGFβ1, and/or TGFβ2 are also reduced or inhibited. Production of one or more immunostimulatory factors, TAAs, or neoantigens can be increased in one or more cell lines in these vaccine compositions.

In some embodiments, provided herein is a cancer vaccine composition comprising a therapeutically effective amount of cells from a cancer cell line wherein the cell line is modified to reduce production of at least CD276. In some embodiments, the CD276 is excised from the cells or is produced at levels reduced by at least 5, 10, 15, 20, 25, or 30% (i.e., at least 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). In some embodiments, CD276 is excised from the cells or is produced at levels reduced by at least 90%. Production of additional immunosuppressive factors can be reduced in one or more cell lines. In some embodiments, expression of CD47, CTLA4, HLA-E, HLA-G, TGFβ1, and/or TGFβ2 are also reduced or inhibited. Production of one or more immunostimulatory factors, TAAs, or neoantigens can be increased in one or more cell lines in these vaccine compositions.

In some embodiments, provided herein is a cancer vaccine composition comprising a therapeutically effective amount of cells from a cancer cell line wherein the cell line is modified to reduce production of at least HLA-G. In some embodiments, the HLA-G is excised from the cells or is produced at levels reduced by at least 5, 10, 15, 20, 25, or 30% (i.e., at least 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). In some embodiments, HLA-G is excised from the cells or is produced at levels reduced by at least 90%. Production of additional immunosuppressive factors can be reduced in one or more cell lines. In some embodiments, expression of CD47, CD276, CTLA4, HLA-E, TGFβ1, and/or TGFβ2 are also reduced or inhibited. Production of one or more immunostimulatory factors, TAAs, or neoantigens can be increased in one or more cell lines in these vaccine compositions.

In some embodiments, provided herein is a cancer vaccine composition comprising a therapeutically effective amount of cells from a cancer cell line wherein the cell line is modified to reduce production of at least CTLA4. In some embodiments, the CTLA4 is excised from the cells or is produced at levels reduced by at least 5, 10, 15, 20, 25, or 30% (i.e., at least 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). In some embodiments, CTLA4 is excised from the cells or is produced at levels reduced by at least 90%. Production of additional immunosuppressive factors can be reduced in one or more cell lines. In some embodiments, expression of CD47, CD276, HLA-E, TGFβ1, and/or TGFβ2 are also reduced or inhibited. Production of one or more immunostimulatory factors, TAAs, or neoantigens can be increased in one or more cell lines in these vaccine compositions.

In some embodiments, provided herein is a cancer vaccine composition comprising a therapeutically effective amount of cells from a cancer cell line wherein the cell line is modified to reduce production of at least HLA-E. In some embodiments, the HLA-E is excised from the cells or is produced at levels reduced by at least 5, 10, 15, 20, 25, or 30% (i.e., at least 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). In some embodiments, HLA-E is excised from the cells or is produced at levels reduced by at least 90%. Production of additional immunosuppressive factors can be reduced in one or more cell lines. In some embodiments, expression of CD47, CD276, CTLA4, TGFβ1, and/or TGFβ2 are also reduced or inhibited. Production of one or more immunostimulatory factors, TAAs, or neoantigens can be increased in one or more cell lines in these vaccine compositions.

In some embodiments, provided herein is a cancer vaccine composition comprising a therapeutically effective amount of cells from a cancer cell line wherein the cell line is modified to reduce production of TGFβ1, TGFβ2, or both TGFβ1 and TGFβ2. In some embodiments, TGFβ1, TGFβ2, or both TGFβ1 and TGFβ2 is excised from the cells or is produced at levels reduced by at least 5, 10, 15, 20, 25, or 30% (i.e., at least 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). In some embodiments of the vaccine composition, TGFβ1, TGFβ2, or both TGFβ1 and TGFβ2 is excised from the cells or is produced at levels reduced by at least 90%.

In some embodiments, TGFβ1, TGFβ2, or both TGFβ1 and TGFβ2 expression is reduced via a short hairpin RNA (shRNA) delivered to the cells using a lentiviral vector. Production of additional immunosuppressive factors can be reduced. In some embodiments, expression of CD47, CD276, CTLA4, HLA-E, and/or HLA-G are also reduced in one or more cell lines where TGFβ1, TGFβ2, or both TGFβ1 and TGFβ2 expression is reduced. Production of one or more immunostimulatory factors, TAAs, or neoantigens can also be increased in one or more cell lines in embodiments of these vaccine compositions.

In some embodiments, the immunosuppressive factor selected for knockdown or knockout may be encoded by multiple native sequence variants. Accordingly, the reduction or inhibition of immunosuppressive factors can be accomplished using multiple gene editing/knockdown approaches known to those skilled in the art. As described herein, in some embodiments, complete knockout of one or more immunosuppressive factors may be less desirable than knockdown. For example, TGFβ1 contributes to the regulation of the epithelial-mesenchymal transition, so complete lack of TGFβ1 (e.g., via knockout) may induce a less immunogenic phenotype in tumor cells.

Table 8 provides exemplary immunosuppressive factors that can be incorporated or modified as described herein, and combinations of the same. Also provided are exemplary NCBI Gene IDs that can be utilized for a skilled artisan to determine the sequence to be targeted for knockdown strategies. These NCBI Gene IDs are exemplary only.

TABLE 8
Exemplary immunosuppressive factors
Factor                           NCBI Gene Symbol (Gene ID)
B7-H3 (CD276)                    CD276 (80381)
BST2 (CD317)                     BST2 (684)
CD200                            CD200 (4345)
CD39 (ENTPD1)                    ENTPD1 (953)
CD47                             CD47 (961)
CD73 (NT5E)                      NT5E (4907)
COX-2                            PTGS2 (5743)
CTLA4                            CTLA4 (1493)
HLA-E                            HLA-E (3133)
HLA-G                            HLA-G (3135)
IDO (indoleamine 2,3-dioxygenase) IDO1 (3620)
IL-10                            IL10 (3586)
PD-L1 (CD274)                    CD274 (29126)
TGFβ1                            TGFB1 (7040)
TGFβ2                            TGFB2 (7042)
TGFβ3                            TGFB3 (7043)
VISTA (VSIR)                     VSIR (64115)
M-CSF                            CSF1 (1435)
B7S1 (B7H4)                      VTCN1 (79679)
PTPN2                            PTPN2 (5771)

In exemplary embodiments, the production of the following combination of immunosuppressive factors is reduced or inhibited in the vaccine composition: CD47+TGFβ1, CD47+TGFβ2, or CD47+TGFβ1+TGFβ2. In exemplary embodiments, the production of the following combination of immunosuppressive factors is reduced or inhibited in the vaccine composition: CD276+TGFβ1, CD276+TGFβ2, or CD276+TGFβ1+TGFβ2. In exemplary embodiments, the production of the following combination of immunosuppressive factors is reduced or inhibited in the vaccine composition: CD47+TGFβ1+CD276, CD47+TGFβ2+CD276, or CD47+TGFβ1+TGFβ2+CD276. In exemplary embodiments, the production of the following combination of immunosuppressive factors is reduced or inhibited in the vaccine composition: CD47+TGFβ1+B7-H3, CD47+TGFβ2+CD276, or CD47+TGFβ1+TGFβ2+CD276. In exemplary embodiments, the production of the following combination of immunosuppressive factors is reduced or inhibited in the vaccine composition: CD47+TGFβ1+CD276+BST2, CD47+TGFβ2+CD276+BST2, or CD47+TGFβ1+TGFβ2+CD276+BST2. In exemplary embodiments, the production of the following combination of immunosuppressive factors is reduced or inhibited in the vaccine composition: CD47+TGFβ1+CD276+CTLA4, CD47+TGFβ2+CD276+CTLA4, or CD47+TGFβ1+TGFβ2+CD276+CTLA4. In exemplary embodiments, the production of the following combination of immunosuppressive factors is reduced or inhibited in the vaccine composition: CD47+TGFβ1+CD276+CTLA4, CD47+TGFβ2+CD276+CTLA4, or CD47+TGFβ1+TGFβ2+CD276+CTLA4.

In exemplary embodiments, the production of the following combination of immunosuppressive factors is reduced or inhibited in the vaccine composition: CD47+TGFβ1+CD276+CTLA4, CD47+TGFβ2+CD276+CTLA4, or CD47+TGFβ1+TGFβ2+CD276+CTLA4, CD47+TGFβ2 or TGFβ1+CTLA4, or CD47+TGFβ1+TGFβ2+CD276+HLA-E or CD47+TGFβ1+TGFβ2+CD276+HLA-G, or CD47+TGFβ1+TGFβ2+CD276+HLA-G+CTLA-4, or CD47+TGFβ1+TGFβ2+CD276+HLA-E+CTLA-4.

In still other embodiments, the production of the following combination of immunosuppressive factors is reduced or inhibited in the vaccine composition: TGFβ1+TGFβ2+CD276, TGFβ1+CD276, or TGFβ2+CD276.

Those skilled in the art will recognize that in embodiments of the vaccine compositions described herein, at least one (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the cell lines within the composition has a knockdown or knockout of at least one immunosuppressive factor (e.g., one or more of the factors listed in Table 8). The cell lines within the composition may have a knockdown or knockout of the same immunosuppressive factor, or a different immunosuppressive factor for each cell line, or of some combination thereof.

Optionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the cell lines within the composition may be further genetically modified to have a knockdown or knockout of one or more additional immunosuppressive factors (e.g., one or more of the factors listed in Table 8). For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the cell lines within the composition may be further genetically modified to have a knockdown or knockout of the same additional immunosuppressive factor, of a different additional immunosuppressive factor for each cell line, or of some combination thereof.

In some embodiments, provided herein is a cancer vaccine composition comprising a therapeutically effective amount of cells from a cancer cell line wherein the cell line is modified to reduce production of SLAMF7, BTLA, EDNRB, TIGIT, KIR2DL1, KIR2DL2, KIR2DL3, TIM3 (HAVCR2), LAG3, ADORA2A and ARG1.

At least one of the cells within any of the vaccine compositions described herein may undergo one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) genetic modifications in order to achieve the partial or complete knockdown of immunosuppressive factor(s) described herein and/or the expression (or increased expression) of immunostimulatory factors described herein, TAAs, and/or neoantigens. In some embodiments, at least one cell line in the vaccine composition undergoes less than 5 (i.e., less than 4, less than 3, less than 2, 1, or 0) genetic modifications. In some embodiments, at least one cell in the vaccine composition undergoes no less than 5 genetic modifications.

Numerous methods of reducing or inhibiting expression of one or more immunosuppressive factors are known and available to those of ordinary skill in the art, embodiments of which are described herein.

Cancer cell lines are modified according to some embodiments to inhibit or reduce production of immunosuppressive factors. Provided herein are methods and techniques for selection of the appropriate technique(s) to be employed in order to inhibit production of an immunosuppressive factor and/or to reduce production of an immunosuppressive factor. Partial inhibition or reduction of the expression levels of an immunosuppressive factor may be accomplished using techniques known in the art.

In some embodiments, the cells of the cancer lines are genetically engineered in vitro using recombinant DNA techniques to introduce the genetic constructs into the cells. These DNA techniques include, but are not limited to, transduction (e.g., using viral vectors) or transfection procedures (e.g., using plasmids, cosmids, yeast artificial chromosomes (YACs), electroporation, liposomes). Any suitable method(s) known in the art to partially (e.g., reduce expression levels by at least 5, 10, 15, 20, 25, or 30%) or completely inhibit any immunosuppressive factor production by the cells can be employed.

In some embodiments, genome editing is used to inhibit or reduce production of an immunosuppressive factor by the cells in the vaccine. Non-limiting examples of genome editing techniques include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the CRISPR-Cas system. In certain embodiments, the reduction of gene expression and subsequently of biological active protein expression can be achieved by insertion/deletion of nucleotides via non-homologous end joining (NHEJ) or the insertion of appropriate donor cassettes via homology directed repair (HDR) that lead to premature stop codons and the expression of non-functional proteins or by insertion of nucleotides.

In some embodiments, spontaneous site-specific homologous recombination techniques that may or may not include the Cre-Lox and FLP-FRT recombination systems are used. In some embodiments, methods applying transposons that integrate appropriate donor cassettes into genomic DNA with higher frequency, but with little site/gene-specificity are used in combination with required selection and identification techniques. Non-limiting examples are the piggyBac and Sleeping Beauty transposon systems that use TTAA and TA nucleotide sequences for integration, respectively.

Furthermore, combinatorial approaches of gene editing methods consisting of meganucleases and transposons can be used.

In certain embodiments, techniques for inhibition or reduction of immunosuppressive factor expression may include using antisense or ribozyme approaches to reduce or inhibit translation of mRNA transcripts of an immunosuppressive factor; triple helix approaches to inhibit transcription of the gene of an immunosuppressive factor; or targeted homologous recombination.

Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to mRNA of an immunosuppressive factor. The antisense oligonucleotides bind to the complementary mRNA transcripts of an immunosuppressive factor and prevent translation. Absolute complementarity may be preferred but is not required. A sequence “complementary” to a portion of an RNA, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex. In the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may be tested, or triplex formation may be assayed. The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid. In some embodiments, oligonucleotides complementary to either the 5′ or 3-non-translated, non-coding regions of an immunosuppressive factor could be used in an antisense approach to inhibit translation of endogenous mRNA of an immunosuppressive factor. In some embodiments, inhibition or reduction of an immunosuppressive factor is carried out using an antisense oligonucleotide sequence within a short-hairpin RNA.

In some embodiments, lentivirus-mediated shRNA interference is used to silence the gene expressing the immunosuppressive factor. (See Wei et al., J. Immunother. 2012 35 (3)267-275 (2012), incorporated by reference herein.)

MicroRNAs (miRNA) are stably expressed RNAi hairpins that may also be used for knocking down gene expression. In some embodiments, ribozyme molecules-designed to catalytically cleave mRNA transcripts are used to prevent translation of an immunosuppressive factor mRNA and expression. In certain embodiments, ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs. In some embodiments, the use of hammerhead ribozymes that cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA are used. RNA endoribonucleases can also be used.

In some embodiments, endogenous gene expression of an immunosuppressive factor is reduced by inactivating or “knocking out” the gene or its promoter, for example, by using targeted homologous recombination. The percent reduction could, in some embodiments, be 100% (e.g., complete reduction). In other embodiments, the percent reduction is 90% or more. In some embodiments, endogenous gene expression is reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the promoter and/or enhancer genes of an immunosuppressive factor to form triple helical structures that prevent transcription of the immunosuppressive factor gene in target cells. In some embodiments, promoter activity is inhibited by a nuclease dead version of Cas9 (dCas9) and its fusions with KRAB, VP64 and p65 that cannot cleave target DNA. The dCas9 molecule retains the ability to bind to target DNA based on the targeting sequence. This targeting of dCas9 to transcriptional start sites is sufficient to reduce or knockdown transcription by blocking transcription initiation.

In some embodiments, the activity of an immunosuppressive factor is reduced using a “dominant negative” approach in which genetic constructs that encode defective immunosuppressive factors are used to diminish the immunosuppressive activity on neighboring cells.

In some embodiments, the administration of genetic constructs encoding soluble peptides, proteins, fusion proteins, or antibodies that bind to and “neutralize” intracellularly any other immunosuppressive factors are used. To this end, genetic constructs encoding peptides corresponding to domains of immunosuppressive factor receptors, deletion mutants of immunosuppressive factor receptors, or either of these immunosuppressive factor receptor domains or mutants fused to another polypeptide (e.g., an IgFc polypeptide) can be utilized. In some embodiments, genetic constructs encoding anti-idiotypic antibodies or Fab fragments of anti-idiotypic antibodies that mimic the immunosuppressive factor receptors and neutralize the immunosuppressive factor are used. Genetic constructs encoding these immunosuppressive factor receptor peptides, proteins, fusion proteins, anti-idiotypic antibodies or Fabs can be administered to neutralize the immunosuppressive factor.

Likewise, genetic constructs encoding antibodies that specifically recognize one or more epitopes of an immunosuppressive factor, or epitopes of conserved variants of an immunosuppressive factor, or peptide fragments of an immunosuppressive factor can also be used. Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)2 fragments, fragments produced by a Fab expression library, and epitope binding fragments of any of the above. Any technique(s) known in the art can be used to produce genetic constructs encoding suitable antibodies.

In some embodiments, the enzymes that cleave an immunosuppressive factor precursor to the active isoforms are inhibited to block activation of the immunosuppressive factor. Transcription or translation of these enzymes may be blocked by a means known in the art.

In further embodiments, pharmacological inhibitors can be used to reduce enzyme activities including, but not limited to COX-2 and IDO to reduce the amounts of certain immunosuppressive factors.

Tumor Associated Antigens (TAAs)

Vector-based and protein-based vaccine approaches are limited in the number of TAAs that can be targeted in a single formulation. In contrast, embodiments of the allogenic whole cell vaccine platform as described herein allow for the targeting of numerous, diverse TAAs. The breadth of responses can be expanded and/or optimized by selecting allogenic cell line(s) that express a range of TAAs and optionally genetically modifying the cell lines to express additional antigens, including neoantigens or nonsynonymous mutations (NSMs), of interest for a desired therapeutic target (e.g., cancer type).

As used herein, the term “TAA” refers to tumor-associated antigen(s) and can refer to “wildtype” antigens as naturally expressed from a tumor cell or can optionally refer to a mutant antigen, e.g., a design antigen or designed antigen or enhanced antigen or engineered antigen, comprising one or more mutations such as a neoepitope or one or more NSMs as described herein.

TAAs are proteins that can be expressed in normal tissue and tumor tissue, but the expression of the TAA protein is significantly higher in tumor tissue relative to healthy tissue. TAAs may include cancer testis antigens (CTs), which are important for embryonic development but restricted to expression in male germ cells in healthy adults. CTs are often expressed in tumor cells.

Neoantigens or neoepitopes are aberrantly mutated genes expressed in cancer cells. In many cases, a neoantigen can be considered a TAA because it is expressed by tumor tissue and not by normal tissue. Targeting neoepitopes has many advantages since these neoepitopes are truly tumor specific and not subject to central tolerance in thymus. A cancer vaccine encoding full length TAAs with neoepitopes arising from nonsynonymous mutations (NSMs) has potential to elicit a more potent immune response with improved breadth and magnitude.

As used herein, a nonsynonymous mutation (NSM) is a nucleotide mutation that alters the amino acid sequence of a protein. In some embodiments, a missense mutation is a change in one amino acid in a protein, arising from a point mutation in a single nucleotide. A missense mutation is a type of nonsynonymous substitution in a DNA sequence. Additional mutations are also contemplated, including but limited to truncations, frameshifts, or any other mutation that change the amino acid sequence to be different than the native antigen protein.

As described herein, in some embodiments, an antigen is designed by (i) referencing one or more publicly-available databases to identify NSMs in a selected TAA; (ii) identifying NSMs that occur in greater than 2 patients; (iii) introducing each NSM identified in step (ii) into the related TAA sequence; (iv) identifying HLA-A and HLA-B supertype-restricted MHC class I epitopes in the TAA that now includes the NSM; and and (v) including the NSMs that create new epitopes (SB and/or WB) or increases peptide-MHC affinity into a final TAA sequence. Exemplary NSMs predicted to create HLA-A and HLA-B supertype-restricted neoepitopes have been described in Example 40 of PCT/US2020/062840 (Pub. No. WO/2021/113328) and is incorporated by reference herein.

In some embodiments, an NSM identified in one patient tumor sample is included in the designed antigen (i.e., the mutant antigen arising from the introduction of the one or more NSMs). In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more NSMs are introduced into a TAA to generate the designed antigen. In some embodiments, target antigens could have a lower number NSMs and may need to use NSMs occurring only 1 time to reach the targeted homology to native antigen protein range (94-97%). In other embodiments, target antigens could have a high number of NSMs occurring at the 2 occurrence cut-off and may need to use NSMs occurring 3 times to reach the targeted homology to native antigen protein range (94-97%). Including a high number NSMs in the designed antigen would decrease the homology of the designed antigen to the native antigen below the target homology range (94-98%).

In some embodiments, 1, 2, 3, 4, 5 or 6 cell lines of a tumor cell vaccine according to the present disclosure comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more NSMs (and thus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more designed antigens) in at least one TAA.

In various embodiments, the sequence homology of the mutant (e.g., designed antigen) to the native full-length protein is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% over the full length of the antigen.

In some embodiments, the designed antigen is incorporated into a therapeutic allogenic whole cell cancer vaccine to induce antigen-specific immune responses to the designed TAAs and existing TAAs.

In some embodiments, the vaccine can be comprised of a therapeutically effective amount of at least one cancer cell line, wherein the cell line or the combination of the cell lines express at least one designed TAA. In other embodiments, the vaccine comprises a therapeutically effective amount of at least one cancer cell line, wherein the cell line or the combination of the cell lines expresses at least 2, 3, 4, 5, 6, 7, 8, 9 10 or more designed TAAs.

Provided herein are embodiments of vaccine compositions comprising a therapeutically effective amount of cells from at least one cancer cell line, wherein the at least one cancer cell line expresses (either natively, or is designed to express) one or more TAAs, neoantigens (including TAAs comprising one or more NSMs), CTs, and/or TAAs. In some embodiments, the cells are transduced with a recombinant lentivector encoding one or more TAAs, including TAAs comprising one or more NSMs, to be expressed by the cells in the vaccine composition.

In some embodiments, the TAAs, including TAAs comprising one or more NSMs or neoepitopes, and/or other antigens may endogenously be expressed on the cells selected for inclusion in the vaccine composition. In some embodiments, the cell lines may be modified (e.g., genetically modified) to express selected TAAs, including TAAs comprising one or more NSMs, and/or other antigens (e.g., CTs, TSAs, neoantigens).

Any of the tumor cell vaccine compositions described herein may present one or more TAAs, including TAAs comprising one or more NSMs or neoepitopes, and induce a broad antitumor response in the subject. Ensuring such a heterogeneous immune response may obviate some issues, such as antigen escape, that are commonly associated with certain cancer monotherapies.

According to various embodiments of the vaccine composition provided herein, at least one cell line of the vaccine composition may be modified to express one or more neoantigens, e.g., neoantigens implicated in lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), prostate cancer, glioblastoma, colorectal cancer, breast cancer including triple negative breast cancer (TNBC), bladder or urinary tract cancer, squamous cell head and neck cancer (SCCHN), liver hepatocellular (HCC) cancer, kidney or renal cell carcinoma (RCC) cancer, gastric or stomach cancer, ovarian cancer, esophageal cancer, testicular cancer, pancreatic cancer, central nervous system cancers, endometrial cancer, melanoma, and mesothelium cancer. In some embodiments, one or more of the cell lines expresses an unmutated portion of a neoantigen protein. In some embodiments, one or more of the cell lines expresses a mutated portion of a neoantigen protein.

In some embodiments, at least one of the cancer cells in any of the vaccine compositions described herein may naturally express, or be modified to express one or more TAAs, including TAAs comprising one or more NSMs, CTs, or TSAs/neoantigens. In certain embodiments, more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the cancer cell lines in the vaccine composition may express, or may be genetically modified to express, one or more of the TAAs, including TAAs comprising one or more NSMs, CTs, or TSAs/neoantigens. The TAAs, including TAAs comprising one or more NSMs, CTs, or TSAs/neoantigens expressed by the cell lines within the composition may all be the same, may all be different, or any combination thereof.

Because the vaccine compositions may contain multiple (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) cancer cell lines of different types and histology, a wide range and variety of TAAs, including TAAs comprising one or more NSMs, and/or neoantigens may be present in the composition (Table 9-25). The number of TAAs that can be targeted using a combination of cell lines (e.g., 5-cell line combination, 6-cell line combination, 7-cell line combination, 8-cell line combination, 9-cell line combination, or 10-cell line combination) and expression levels of the TAAs is higher for the cell line combination compared to individual cell lines in the combination.

In embodiments of the vaccine compositions provided herein, at least one (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the cancer cells in any of the vaccine compositions described herein may express, or be modified to express one or more TAAs, including TAAs comprising one or more NSMs or neoepitopes. The TAAs, including TAAs comprising one or more NSMs, expressed by the cells within the composition may all be the same, may all be different, or any combination thereof. Table 9 below lists exemplary non-small cell lung cancer TAAs, and exemplary subsets of lung cancer TAAs. In some embodiments, the TAAs are specific to NSCLC. In some embodiments, the TAAs are specific to GBM. In other embodiments, the TAAs are specific to prostate cancer.

In some embodiments, presented herein is a vaccine composition comprising a therapeutically effective amount of engineered cells from least one cancer cell line, wherein the cell lines or combination of cell lines express at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more of the TAAs in Tables 9-25. In other embodiments, the TAAs in Tables 9-25 are modified to include one or more NSM as described herein.

In some embodiments, a vaccine composition is provided comprising a therapeutically effective amount of engineered cells from at least one cancer cell line, wherein the cell lines express at least 2, 3, 4, 5, 6, 7, 8, 9, 10 of the TAAs in Tables 9-25 (or the TAAs in Tables 9-25 that have been modified to include one or more NSM). As provided herein, in various embodiments the cell lines express at least 2, 3, 4, 5, 6, 7, 8, 9, 10 of the TAAs in Tables 9-25 (or the TAAs in Tables 9-25 that have been modified to include one or more NSM) and are optionally modified to express or increase expression of one or more immunostimulatory factors of Table 6, and/or inhibit or decrease expression of one or more immunosuppressive factors in Table 8.

TABLE 9
Exemplary TAAs expressed in non-small cell lung cancer
TAA Name           NCBI Gene Symbol (Gene ID)
Survivin           BIRC5 (332)
CD44               CD44 (960)
CD44v6             CD44 (960)
CEA                CEACAM5 (1048)
CT83               CT83 (203413)
DEPDC1             DEPDC1 (55635)
DLL3               DLL3 (10683)
NYESO1             CTAG1 (1485)
BORIS              CTCFL (140690)
EGFR               EGFR (1956)
Her2               ERBB2 (2064)
PSMA               FOLH1 (2346)
KOC1               IGF2BP3 (10643)
VEGFR              KDR (3791) FLT1 (2321)
KIF20A             KIF20A (10112)
MPHOSPH1           KIF20B (9585)
KRAS               KRAS (3845)
LY6K               LY6K (54742)
MAGE-A1            MAGEA1 (4100)
MAGE-A3            MAGEA3 (4102)
MAGE-A4            MAGEA4 (4103)
MAGE-A6            MAGEA6 (4105)
Mesothelin         MSLN (10232)
MUC1               MUC1 (4582)
c-Myc              MYC (4609)
NUF2               NUF2 (83540)
PRAME              PRAME (23532)
CD133 (Prominin-1) PROM1 (8842)
PTK7               PTK7 (5754)
Securin            PTTG1 (9232)
STEAP1             STEAP1 (26872)
hTERT              TERT (7015)
p53                TP53 (7157)
5T4                TPBG (7162)
TTK (CT96)         TTK (7272)
Brachyury/TBXT     T (6862)
WT1                WT1 (7490
XAGE1B             XAGE1B (653067)

TABLE 10
Exemplary TAAs expressed in prostate cancer
TAA Name           NCBI Gene Symbol (Gene ID)
PAP                ACP3 (55)
Androgen Receptor  AR (367)
Survivin           BIRC5 (332)
NYESO1             CTAG1B (1485)
CXCL12             CXCL12 (6387)
CXCR4              CXCR4 (7852)
EGFR               EGFR (1956)
Her2               ERBB2 (2064)
PSMA               FOLH1 (2346)
GCNT1              GCNT1 (2650)
IDH1               IDH1 (3417)
FAP                FAP (2191)
c-KIT/CD117        KIT (3815)
PSA                KLK3 (354)
Galectin 8         LGALS8 (3964)
MAGE-A1            MAGEA1 (4100)
MAGE-A3            MAGEA3 (4102)
MAGE-A4            MAGEA4 (4103)
MAGE-C2            MAGEC2 (51438)
Midkine            MDK (4192)
MUC1               MUC1 (4582)
PDGF-B             PDGFB (5155)
PDGF-D             PDGFD (80310)
PDGFRβ             PDGFRB (5159)
PLAT (T-PA)        PLAT (5327)
uPA                PLAU (5328)
uPAR (CD87)        PLAUR (5329)
CD133 (Prominin-1) PROM1 (8842)
PSCA               PSCA (8000)
SART3              SART3 (9733)
Prostein           SLC45A3 (85414)
CD147              SLC7A11 (23657)
SSX2               SSX2 (6757)
STEAP1             STEAP1 (26872)
Brachyury/TBXT     T (6862)
hTERT              TERT (7015)
5T4                TPBG (7162)
VEGF-A             VEGFA (7422)

TABLE 11
Exemplary TAAs expressed in glioblastoma cancer
TAA Name         NCBI Gene Symbol (Gene ID)
AIM2             AIM2 (9447)
B4GALNT1         B4GALNT1 (2583)
Survivin         BIRC5 (4582)
Basigin (BSG)    BSG (682)
Cyclin B1        CCNB1 (891)
CDH5             CDH5 (1003)
GP39             CHI3L1 (1116)
Trp2             DOT (1638)
DLL3             DLL3 (10683)
DRD2             DRD2 (1813)
EGFRvIII         EGFR (1956)
Epha2            EPHA2 (1969)
Epha3            EPHA3 (2042)
Her2             ERBB2 (2064)
EZH2             EZH2 (2146)
PSMA             FOLH1 (2346)
FOSL1            FOSL1 (8061)
GSK3B            GSK3B (2932)
IDH1             IDH1 (3417)
IDH2             IDH2 (3418)
IL13RA2          IL13RA2 (3598)
IL4R             IL4R (3566)
LRP1             LRP1 (4035)
KOC1             IGF2BP3 (10643)
MAGE-A1          MAGEA1 (4100)
MAGE-A4          MAGEA4 (4103)
MUC1             MUC1 (4582)
MUL1             MUL1 (79594)
GP100 (PMEL)     PMEL (6490)
PRAME            PRAME (23532)
hCMVpp65*        ABQ23593 (UniProtKB - P06725 (PP65_HCMVA)
PROM1            PROM1 (8842)
PTHLH            PTHLH (4744)
SART1            SART1 (9092)
SART3            SART3 (9733)
CD147            SLC7A11 (23657)
SOX-2            SOX2 (6657)
SOX-11           SOX11 (6664)
STEAP1           STEAP1 (26872)
hTERT            TERT (7015)
Tenascin-C (TNC) TNC (3371)
TYR              TYR (7299)
Trp1 (TYRP1)     TYRP1 (7306)
WT1              WT1 (7490)
XPO1             XPO1 (7514)
pp65*            ABQ23593
*Viral antigen, no Gene ID is available. Accession number is used instead.

TABLE 12
Exemplary TAAs expressed in ovarian cancer
TAA Name                        NCBI Gene Symbol (Gene ID)
OY-TES-1                        ACRBP (84519)
A-Kinase Anchoring Protein 3    AKAP3 (10566)
Anti-Mullerian Hormone Receptor AMHR2 (269)
Axl Receptor Tyrosine Kinase    AXL (558)
Survivin                        BIRC5 (332)
Bruton's Tyrosine Kinase        BTK (695)
CD44                            CD44 (960)
Cell Cycle Checkpoint Kinase 1  CHEK1 (1111)
(CHK1)
Claudin 6                       CLDN6 ((074)
NY-ESO-1                        CTAG1B (1485)
LAGE1                           CTAG2 (30848)
BORIS                           CTCFL (140690)
Dickkopf-1                      DKK1 (22943)
DLL4                            DLL4 (54567)
Her2                            ERBB2 (2064)
HER3                            ERBB3 (2065)
FOLR1/FBP                       FOLR1 (2348)
GAGE1                           GAGE1 (2543)
GAGE2                           GAGE2A (729447)
IGFBP2                          IGFBP2 (3485)
FSHR                            FSHR (3969)
PLU-1                           KDM5B (10765)
Luteinizing Hormone Receptor    LHCGR (3973)
MAGE-A1                         MAGEA1 (4100)
MAGE-A10                        MAGEA10 (4109)
MAGE-A4                         MAGEA4 (4103)
MAGE-A9                         MAGEA9 (4108)
MAGE-C1                         MAGEC1 (9947)
Mesothelin                      MSLN (10232)
Muc1                            MUC1 (4582)
Muc16                           MUC16 (94025)
Glucocorticoid Receptor II      NR3C1 (2908)
PARP1                           PARP1 (142)
PIWIL1                          PIWIL1 (9271)
PIWIL2                          PIWIL2 (55124)
PIWIL3                          PIWIL3 (440822)
PIWIL4                          PIWIL4 (143689)
PRAME                           PRAME (23532)
SP17                            SPA17 (53340)
SPAG-9                          SPAG9 (9043)
STEAP1                          STEAP1 (26872)
hTERT                           TERT (7015)
WT1                             WT1 (7490)

TABLE 13
Exemplary TAAs expressed in colorectal cancer
TAA Name        NCBI Gene Symbol (Gene ID)
Survivin        BIRC5 (332)
B-RAF           BRAF (673)
CEA             CEACAM5 (1048)
βHCG            CGB3 (1082)
NYESO1          CTAG1B (1485)
EPCAM           EPCAM (4072)
EPH receptor A2 EPHA2 (1969)
Her2            ERBB2 (2064)
GUCY2C          GUCY2C (2984)
PSMA            FOLH1 (2346)
KRAS            KRAS (3845)
MAGE-A1         MAGEA1 (4100)
MAGE-A3         MAGEA3 (4102)
MAGE-A4         MAGEA4 (4103)
MAGE-A6         MAGEA6 (4105)
Mesothelin      MSLN (10232)
MUC1            MUC1 (4582)
PRAME           PRAME (23532)
CD133           PROM1 (8842)
RNF43           RNF43 (54894)
SART3           SART3 (9733)
STEAP1          STEAP1 (26872)
Brachyury/TBXT  T (6862)
TROP2           TACSTD2 (4070)
hTERT           TERT (7015)
TOMM34          TOMM34 (10953)
5T4             TPBG (7162)
WT1             WT1 (7490)

TABLE 14
Exemplary TAAs expressed in breast cancer
TAA Name             NCBI Gene Symbol (Gene ID)
Survivin             BIRC5 (332)
Cyclin B1            CCNB1 (891)
Cadherin-3           CDH3 (1001)
CEA                  CEACAM5 (1048)
CREB binding protein CREBBP (1387)
CS1                  CSH1 (1442)
CT83                 CT83 (203413)
NYESO1               CTAG1B (1485)
BORIS                CTCFL (140690)
Endoglin             ENG (2022)
PSMA                 FOLH1 (2346)
FOLR1α               FOLR1 (2348)
FOS like 1           FOSL1 (8061)
FOXM1                FOXM1 (2305)
GPNMB                GPNMB (10457)
MAGE A1              MAGEA1 (4100)
MAGE A3              MAGEA3 (4102)
MAGE A4              MAGEA4 (4103)
MAGE A6              MAGEA6 (4105)
Mesothelin           MSLN (10232)
MMP11                MMP11 (4320)
MUC1                 MUC1 (4582)
PRAME                PRAME (23532)
CD133                PROM1 (8842)
PTK7                 PTK7 (5754)
ROR1                 ROR1 (4919)
Mammaglobin A        SCGB2A2 (4250)
Syndecan-1           SDC1 (6382)
SOX2                 SOX2 (6657)
SPAG9                SPAG9 (9043)
STEAP1               STEAP1 (26872)
Brachyury/TBXT       T (6862)
TROP2                TACSTD2 (4070)
hTERT                TERT (7015)
WT1                  WT1 (7490)
YB-1                 YBX1 (4904)

TABLE 15
Exemplary TAAs expressed in bladder cancer
Androgen Receptor            AR (367)
ATG7                         ATG7 (10533)
AXL Receptor Tyrosine Kinase AXL (558)
Survivin                     BIRC5 (332)
BTK                          BTK (695)
CEACAM1                      CEACAM1 (634)
CEA                          CEACAM5 (1048)
βHCG                         CGB3 (1082)
NYESO1                       CTAG1B (1495)
LAGE1                        CTAG2 (30848)
DEPDC1                       DEPDC1 (55635)
EPH receptor B4              EPHB4 (2050)
HER2                         ERBB2 (2064)
FGFR3                        FGFR3 (2261)
VEGFR                        FLT3 (2322)
PSMA                         FOLH1 (2346)
FOLR1α (FBP)                 FOLR1 (2348)
IGF2BP3                      IGF2BP3 (10643)
MPHOSPH1                     KIF20B (9585)
LY6K                         LY6K (54742)
MAGEA1                       MAGEA1 (4100)
MAGEA3                       MAGEA3 (4102)
MAGEA6                       MAGEA6 (4105)
MAGEC2                       MAGEC2 (51438)
c-Met                        MET (4233)
MUC1                         MUC1 (4582)
Nectin-4                     NECTIN4 (81607)
NUF2                         NUF2 (83540)
RET                          RET (5979)
STEAP1                       STEAP1 (26872)
TDGF1 (Cripto1)              TDGF1 (6997)
hTERT                        TERT (7015)
TROP2                        TACSTD2 (4070)
WEE1                         WEE1 (7465)
WT1                          WT1 (7490)

TABLE 16
Exemplary TAAs expressed in head and/or neck cancer
TAA Name        NCBI Gene Symbol (Gene ID)
Survivin        BIRC5 (332)
BTK             BTK (695)
cyclin D1       CCND1 (595)
CDK4            CDK4 (1019)
CDK6            CDK6 (1021)
P16             CDKN2A (1029)
CEA             CEACAM5 (1048)
EGFR            EGFR (1956)
EPH receptor B4 EPHB4 (2050)
Her2            ERBB2 (2064)
HER3            ERBB3 (2065)
FGFR1           FGFR1 (2260)
FGFR2           FGFR2 (2263)
FGFR3           FGFR3 (2261)
PSMA            FOLH1 (2346)
IGF2BP3         IGF2BP3 (10643)
IMP3            IMP3 (55272)
MPHOSPH1        KIF20B (9585)
LY6K            LY6K (54742)
MAGE-A10        MAGEA10 (4109)
MAGE-A3         MAGEA3 (4102)
MAGE-A4         MAGE-A4 (4103)
MAGE-A6         MAGE-A6 (4105)
MUC1            MUC1 (4582)
NUF2            NUF2 (83540)
PRAME           PRAME (23532)
STEAP1          STEAP1 (26872)
Brachyury/TBXT  T (6862)
hTERT           TERT (7015)
p53             TP53 (7157)
HPV16 E6*       AVN72023
HPV16 E7*       AVN80203
HPV18 E6*       ALA62736
HPV18 E7*       ABP99745
*Viral antigen, no Gene ID is available; GenBank accession number is provided.

TABLE 17
Exemplary TAAs expressed in gastric cancer
TAA Name           NCBI Gene Symbol (Gene ID)
TEM-8 (ANTXR1)     ANTXR1 (84168)
Annexin A2 (ANXA2) ANXA2 (302)
Survivin           BIRC5 (332)
CCKBR              CCKBR (887)
Cadherin 17        CDH17 (1015)
CDKN2A             CDKN2A (1029)
CEA                CEACAM5 (1048)
Claudin 18         CLDN18 (51208)
CT83               CT83 (203413)
EPCAM              EPCAM (4072)
Her2               ERBB2 (2064)
Her3               ERBB3 (2065)
PSMA               FOLH1 (2346)
FOLR1              FOLR1 (2348)
FOXM1              FOXM1 (2305)
FUT3               FUT3 (2525)
Gastrin            GAST (2520)
KIF20A             KIF20A (10112)
LY6K               LY6K (54742)
MAGE-A1            MAGEA1 (4100)
MAGE-A3            MAGEA3 (4102)
MMP9               MMP9 (4318)
Mesothelin         MSLN (10232)
MUC1               MUC1 (4582)
MUC3A              MUC3A (4584)
PRAME              PRAME (23532)
PTPN11             PTPN11 (5781)
SART3              SART3 (9733)
SATB1              SATB1 (6304)
STEAP1             STEAP1 (26872)
hTERT              TERT (7015)
5T4 (TPBG)         TPBG (7162)
VEGFR1             FLT1 (2321)
WEE1               WEE1 (7465)
WT1                WT1 (7490)

TABLE 18
Exemplary TAAs expressed in liver cancer
TAA Name           NCBI Gene Symbol (Gene ID)
AKR1C3             AKR1C3 (8644)
MRP3 (ABCC3)       ABCC3 (8714)
AFP                AFP (174)
Annexin A2 (ANXA2) ANXA2 (302)
Survivin           BIRC5 (4582)
Basigin (BSG)      BSG (682)
CEA                CEACAM5 (1048)
NYESO1             CTAG1B (1485)
DKK-1              DKK1 (22943)
SART-2 (DSE)       DSE (29940)
EpCAM              EPCAM (4072)
Glypican-3         GPC3 (2719)
MAGE-A1            MAGEA1 (4100)
MAGE-A3            MAGEA3 (4102)
MAGE-A4            MAGEA4 (4103)
MAGE-A10           MAGEA10 (4109)
MAGE-C1            MAGEC1 (9947)
MAGE-C2            MAGEC2 (51438)
Midkine (MDK)      MDK (4192)
MUC-1              MUC1 (4582)
PRAME              PRAME (23532)
SALL-4             SALL4 (57167)
Spa17              SPA17 (53340)
SPHK2              SPHK2 (56848)
SSX-2              SSX2 (6757)
STAT3              STAT3 (6774)
hTERT              TERT (7015)
HCA661 (TFDP3)     TFDP3 (51270)
WT1                WT1 (7490)

TABLE 19
Exemplary TAAs expressed in esophageal cancer
TAA Name          NCBI Gene Symbol (Gene ID)
ABCA1             ABCA1 (19)
NYESO1            CTAG1B (1485)
LAGE1             CTAG2 (30848)
DKK1              DKK1 (22943)
EGFR              EGFR (1956)
EpCAM             EPCAM (4072)
Her2              ERBB2 (2065)
Her3              ERBB3 (2064)
FOLR1             FOLR1 (2348)
Gastrin (GAST)    GAST (2520)
IGF2BP3           IGF2BP3 (10643)
IMP3              IMP3 (55272)
LY6K              LY6K (54742)
MAGE-A1           MAGEA1 (4100)
MAGE-A3           MAGEA3 (4102)
MAGE-A4           MAGEA4 (4103)
MAGE-A11          MAGEA11 (4110)
Mesothelin (MSLN) MSLN (10232)
NUF2              NUF2 (83540)
PRAME             PRAME (23532)
PTPN11            PTPN11 (5781)
hTERT             TERT (7015)
TTK               TTK (7272)

TABLE 20
Exemplary TAAs expressed in kidney cancer
TAA Name                     NCBI Gene Symbol (Gene ID)
apolipoprotein L1            APOL1 (8542)
Axl Receptor Tyrosine Kinase AXL (558)
Survivin                     BIRC5 (332)
G250                         CA9 (768)
cyclin D1                    CCND1 (595)
CXCR4                        CXCR4 (7852)
EPH receptor B4              EPHB4 (2050)
FAP                          FAP (2191)
VEGFR                        FLT3 (2322)
GUCY2C                       GUCY2C (2984)
INTS1                        INTS1 (26173)
c-KIT/CD117                  KIT (3815)
c-Met                        MET (4233)
MMP7                         MMP7 (4316)
RAGE1                        MOK (5891)
Muc1                         MUC1 (4582)
PDGFRα                       PDGFRA (5156)
PDGFRβ                       PDGFRB (5159)
M2PK                         PKM (5315)
perilipin 2                  PLIN2 (123)
PRAME                        PRAME (23532)
PRUNE2                       PRUNE2 (158471)
RET                          RET (5979)
RGS5                         RGS5 (8490)
ROR2                         ROR2 (4920)
STEAP1                       STEAP1 (26872)
Tie-1                        TIE1 (7075)
5T4                          TPBG (7162)
gp75                         TYRP1 (7306)

TABLE 21
Exemplary TAAs expressed in pancreatic cancer
TAA Name                        NCBI Gene Symbol (Gene ID)
Survivin                        BIRC5 (332)
BTK                             BTK (695)
Connective Tissue Growth Factor CCN2 (1490)
CEA                             CEACAM5 (1048)
Claudin 18                      CLDN18 (51208)
NYESO1                          CTAG1B (1495)
CXCR4                           CXCR4 (7852)
EGFR                            EGFR (1956)
FAP                             FAP (2191)
PSMA                            FOLH1 (2346)
MAGE-A4                         MAGEA4 (4103)
Perlecan                        HSPG2 (3339)
Mesothelin                      MSLN (10232)
MUC1                            MUC1 (4582)
Muc16                           MUC16 (94025)
Mucin 5AC                       MUC5AC (4586)
CD73                            NT5E (4907)
G17 (gastrin1-17)               PBX2 (5089)
uPA                             PLAU (5328)
uPAR (CD87)                     PLAUR (5329)
PRAME                           PRAME (23532)
PSCA                            PSCA (8000)
Focal adhesion kinase           PTK2 (5747)
SSX2                            SSX2 (6757)
STEAP1                          STEAP1 (26872)
hTERT                           TERT (7015)
Neurotensin Receptor 1          TFIP11 (24144)
WT1                             WT1 (7490)

TABLE 22
Exemplary TAAs expressed in endometrial cancer
TAA Name    NCBI Gene Symbol (Gene ID)
OY-TES-1    ACRBP (84519)
ARMC3       ARMC3 (219681)
Survivin    BIRC5 (332)
BMI1        BMI1 (648)
BST2        BST2 (684)
BORIS       CTCFL (140690)
DKK1        DKK1 (22943)
DRD2        DRD2 (1813)
EpCam       EPCAM (4072)
EphA2       EphA2 (1969)
HER2/neu    ERBB2 (2064)
HER3        ERBB3 (2065
ESR2        ESR2 (2100)
MAGE-A3     MAGEA3 (4102)
MAGE-A4     MAGEA4 (4103)
MAGE-C1     MAGEC1 (9947)
MUC-1       MUC1 (4582)
MUC-16      MUC16 (94025)
SPA17       SPA17 (53340)
SSX-4       SSX4 (6757)
hTERT       TERT (7015)
HE4 (WFDC2) WFDC2 (10406)
WT1         WT1 (7490)
XPO1        XPO1 (7514)

TABLE 23
Exemplary TAAs expressed in skin cancer
TAA Name           NCBI Gene Symbol (Gene ID)
B4GALNT1           B4GALNT1 (2583)
Survivin           BIRC5 (332)
Endosialin (CD248) CD248 (57124)
CDKN2A             CDKN2A (1029)
CSAG2              CSAG2 (102423547)
CSPG4              CSPG4 (1464)
NYESO1             CTAG1B (1485)
Trp2 (DCT)         DCT (1638)
MAGE-A1            MAGEA1 (4100)
MAGE-A2            MAGEA2 (4101)
MAGE-A3            MAGEA3 (4102)
MAGE-A4            MAGEA4 (4103)
MAGE-A6            MAGEA6 (4105)
MAGE-A10           MAGEA10 (4109)
MITF               MITF (4286)
MART-1             MLANA (2315)
NFE2L2             NFE2L2 (4780)
PMEL               PMEL (6490)
PRAME              PRAME (23532)
NY-MEL-1           RAB38 (23682)
NEF                S100B (6285)
SEMA4D             SEMA4D (10507)
SSX2               SSX2 (6757)
SSX4               SSX4 (6759)
ST8SIA1            ST8SIA1 (6489)
hTERT              TERT (7015)
TYR                TYR (7299)
Trp1               TYRP1 (7306)

TABLE 24
Exemplary TAAs expressed in mesothelial cancer
TAA Name   NCBI Gene Symbol (Gene ID)
APEX1      APEX1 (328)
CHEK1      CHEK1 (1111)
NYESO1     CTAG1B (1485)
DHFR       DHFR (1719)
DKK3       DKK3 (27122)
EGFR       EGFR (1956)
ESR2       ESR2 (2100)
EZH1       EZH1 (2145)
EZH2       EZH2 (2146)
MAGE-A1    MAGEA1 (4100)
MAGE-A3    MAGEA3 (4102)
MAGE-A4    MAGEA4 (4103)
MCAM       MCAM (4162)
Mesothelin MSLN (10232)
MUC1       MUC1 (4582)
PTK2       PTK2 (5747)
SSX-2      SSX2 (6757)
STAT3      STAT3 (6774)
THBS2      THBS2 (7058)
5T4 (TPBG) TPBG (7162)
WT1        WT1 (7490)

TABLE 25
Exemplary TAAs expressed in small cell lung cancer
TAA Name     NCBI Gene Symbol (Gene ID)
AIM2         AIM2 (9447)
AKR1C3       AKR1C3 (8644)
ASCL1        ASCL1 (429)
B4GALNT1     B4GALNT1 (2583)
Survivin     BIRC5 (332)
Cyclin B1    CCNB1 (891)
CEA          CEACAM5 (1048)
CKB          CKB (1152)
DDC          DDC (1644)
DLL3         DLL3 (10863)
Enolase 2    ENO2 (2026)
Her2         ERBB2 (2064)
EZH2         EZH2 (2146)
Bombesin     GRP (2922)
KDM1A        KDM1A (23028)
MAGE-A1      MAGEA1 (4100)
MAGE-A3      MAGEA3 (4102)
MAGE-A4      MAGA4 (4103)
MAGE-A10     MAGEA10 (4109)
MDM2         MDM2 (4193)
MUC1         MUC1 (4582)
NCAM-1       NCAM1 (4684)
GP100        PMEL (6490)
SART-1       SART1 (9092)
SART-3       SART3 (9733)
SFRP1        SFRP1 (6422)
SOX-2        SOX2 (6657)
SSTR2        SSTR2 (6752)
Trp1 (TYRP1) TYRP1 (7306)

In some embodiments of the vaccine compositions provided herein, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the cell lines within the composition may be genetically modified to express or increase expression of the same immunostimulatory factor, TAA, including TAAs comprising one or more NSMs, and/or neoantigen; of a different immunostimulatory factor, TAA, and/or neoantigen; or some combination thereof. In some embodiments, the TAA sequence can be the native, endogenous, human TAA sequence. In some embodiments, the TAA sequence can be a genetically engineered sequence of the native endogenous, human TAA sequence. The genetically engineered sequence may be modified to increase expression of the TAA through codon optimization or the genetically engineered sequence may be modified to change the cellular location of the TAA (e.g., through mutation of protease cleavage sites).

Exemplary NCBI Gene IDs are presented in Table 25. As provided herein, these Gene IDs can be used to express (or overexpress) certain TAAs in one or more cell lines of the vaccine compositions of the disclosure.

In various embodiments, one or more of the cell lines in a composition described herein is modified to express mesothelin (MSLN), CT83 (kita-kyushu lung cancer antigen 1) TERT, PSMA, MAGEA1, EGFRvIII, hCMV pp65, TBXT, BORIS, FSHR, MAGEA10, MAGEC2, WT1, FBP, TDGF1, Claudin 18, LY6K, PRAME, HPV16/18 E6/E7, FAP, or mutated versions thereof (Table 26). The phrase “or mutated versions thereof” refers to sequences of the TAAs provided herein, that comprise one or more mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more substitution mutations), including neopepitopes or NSMs, as described herein. Thus, in various embodiments, one or more of the cell lines in a composition described herein is modified to express modMesothelin (modMSLN), modTERT, modPSMA, modMAGEA1, EGFRvIII, hCMV pp65, modTBXT, modBORIS, modFSHR, modMAGEA10, modMAGEC2, modWT1, modFBP, modTDGF1, modClaudin 18, modLY6K, modFAP, modPRAME, KRAS G12D mutation, KRAS G12V mutation, and/or HPV16/18 E6/E7. In other embodiments, the TAA or “mutated version thereof” may comprise fusions of 1, 2, or 3 or more of the TAAs or mutated versions provided herein. In some embodiments, the fusions comprise a native or wild-type sequence fused with a mutated TAA. In some embodiments, the individual TAAs in the fusion construct are separated by a cleavage site, such as a furin cleavage site. The present disclosure provides TAA fusion proteins such as, for example, modMAGEA1-EGFRvIII-pp65, modTBXT-modBORIS, modFSHR-modMAGEA10, modTBXT-modMAGEC2, modTBXT-modWT1, modTBXT-modWT1 (KRAS), modWT1-modFBP, modPSMA-modTDGF1, modWT1-modClaudin 18, modPSMA-modLY6K, modFAP-modClaudin 18, and modPRAME-modTBXT. Sequences for native TAAs can be readily obtained from the NCBI database (www.ncbi.nlm.nih.gov/protein). Sequences for some of the TAAs provided herein, mutated versions, and fusions thereof are provided in Table 26.

TABLE 26
Sequences of Exemplary Designed Antigens
TAA                       Sequence
modTBXT_modWT1_           agagtctgagctgtggctgcggttcaaagaactgaccaacgagatgatcgtgaccaagaacggcagacggatgttccccgtgct
(KRAS Mutations)(SEQ ID   gaaagtgaacgtgtccggactggaccccaacgccatgtacagctttctgctggacttcgtggtggccgacaaccacagatggaa
NO: 17)                   atacgtgaacggcgagtgggtgccaggcggaaaacctcaactgcaagcccctagctgcgtgtacattcaccctgacagcccca
                          atttcggcgcccactggatgaaggcccctgtgtccttcagcaaagtgaagctgaccaacaagctgaacggcggaggccagatca
                          tgctgaacagcctgcacaaatacgagcccagaatccacatcgtcagagtcggcggaccccagagaatgatcaccagccactg
                          cttccccgagacacagtttatcgccgtgaccgcctaccagaacgaggaaatcaccacactgaagatcaagtacaaccccttcgc
                          caaggccttcctggacgccaaagagggagcgaccacaaagagatgatcaaagagcccggcgacagccagcagccaggct
                          attctcaatggggatggctgctgccaggcaccagcacattgtgccctccagccaatcctcacagccagtttggaggcgccctgagc
                          ctgtctagcacccacagctacgacagataccccacactgcggagccacagaagcagcccctatccttctccttacgctcaccgga
                          acaacagccccacctacagcgataatagccccgcctgtctgagcatgctgcagtcccacgataactggtccagcctgagaatgc
                          ctgctcacccttccatgctgcccgtgtctcacaatgcctctccacctaccagcagctctcagtaccctagcctttggagcgtgtccaat
                          ggcgccgtgacactgggatctcaggcagccgctgtgtctaatggactgggagcccagttcttcagaggcagccctgctcactaca
                          cccctctgacacatcctgtgtctgcccctagcagcagggcttccctatgtataagggcgctgccgccgctaccgacatcgtggatt
                          ctcagtatgatgccgccgcacagggacacctgatcgcctcttggacacctgtgtctccaccttccatgagaggcagaaagggag
                          aagcgacttcctgctgctgcagaaccctgcctctacctgtgtgcctgaaccagcctctcagcacaccctgagatctggccctggatg
                          tctccagcagcctgaacagcagggcgttagagatcctggcggaatctgggccaaactgggagctgccgaagcctctgccgaatg
                          tctgcagggcagaagaagcagaggcgccagggatctgaacctcaccagatgggaagcgacgtgcacgacctgaatgctctg
                          ttgcctgccgtgccatctcttggcggaggcggaggatgtgctttgcctgtttctggtgctgcccagtgggctcccgtgctggattttgctc
                          ctcctggcgcttctgcctatggctctcttggaggacctgctcctccaccagctccacctccaccgccgcctccaccacctcacagcttt
                          atcaagcaagagccctcctggggcggagccgagcctcacgaaaaacagtgtctgagcgccttcaccgtgcactttttcggccagt
                          ttaccggcaccgtgggcgcctgtagatacggcccttttggaccaccaccacctagccaggcttctagcggacaggccagaatgttc
                          cccaacgctccttacctgcctagctgcctggaaagccagcctaccatcagaaaccagggcttcagcaccgtgaccttcgacggc
                          atgcctagctatggccacacaccatctcaccacgccgctcagttccccaatcacagcttcaagcacgaggaccctatgggccagc
                          agggatctctgggagagcagcagtatagcgtgccacctcctgtgtacggctgtcacacccctaccgatagctgcacaggcaatca
                          ggctctgctgctgaggatgcctttcagcagcgacaacctgtaccagatgacaagccagctggaatgcatgatttggaaccagatg
                          aacctgggcgccactctgaaaggcgtggccgctggatctagcagctccgtgaaatggacagccggccagagcaatcactccac
                          cggctacgagagcgacaatcacaccatgcctatcctgtgtggggcccagtaccggattcacacacacggcgtgttcaggggcatt
                          caggatgtgcgaagagtgcctggcgtggcccctacacttgtgggatctgccagcgaaaccagcgagaagcaccccttcatgtgc
                          gcctatccaggctgcaacaagcggtacttcaagctgagccacctgaagatgcacagccggaagcacacaggcgagaagctgt
                          accagtgcgacttcaaggactgcgagcggagattcagctgcagcgaccagctgaagagacaccagagaaggcacaccggc
                          gtgaagccctttcagtgcaagacctgccagcggaccttctcctggtccaaccacctgaaaacccacacaagaacccacaccgg
                          caagaccatcgagaagcccttcagctgtagatggcccagctgccagaagaagttcgcccggtctaacgagctggtgcatcacca
                          caacatgcaccagaggaacatgaccaaactgcagctggtgctgaggggaagaaagaggcggtccaccgagtacaagctggt
                          ggttgttggagccgatggcgtgggaaagagcgccctgacaattcagctgatccagaaccacttcgtgcgcggcagaaagagaa
                          gatctacagagtataagctcgtggtcgtgggcgctgtcggagtgggaaaatctgccctgaccatccaactcattcagaatcactttgt
                          gtgatga
modTBXT_modWT1__          MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTEHELRVGLEESELWLRFKELTNE
(KRAS Mutations)(SEQ ID   MIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVVADNHRWKYVNGEINVPGGKPQLQAPS
NO: 18)                   CVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMIT
                          SHCFPETQFIAVTAYQNEEITTLKIKYNPFAKAFLDAKERSDHKEMIKEPGDSQQPGYSQWG
                          WLLPGTSTLCPPANPHSQFGGALSLSSTHSYDRYPTLRSHRSSPYPSPYAHRNNSPTYSDN
                          SPACLSMLQSHDNWSSLRMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTLGSQAAA
                          VSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGFPMYKGAAAATDIVDSQYDAAAQGHLIASW
                          TPVSPPSMRGRKRRSDFLLLQNPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWA
                          KLGAAEASAECLQGRRSRGASGSEPHQMGSDVHDLNALLPAVPSLGGGGGCALPVSGAA
                          QWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEKQCLSAF
                          TVHFFGQFTGTVGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPTIRNQGFSTVT
                          FDGMPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGN
                          QALLLRMPFSSDNLYQMTSQLECMIWNQMNLGATLKGVAAGSSSSVKWTAGQSNHSTGYE
                          SDNHTMPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVGSASETSEKHPFMCAYPGCNKR
                          YFKLSHLKMHSRKHTGEKLYQCDFKDCERRFSCSDQLKRHQRRHTGVKPFQCKTCQRTFS
                          WSNHLKTHTRTHTGKTIEKPFSCRWPSCQKKFARSNELVHHHNMHQRNMTKLQLVLRGRK
                          RRSTEYKLVVVGADGVGKSALTIQLIQNHFVRGRKRRSTEYKLVVVGAVGVGKSALTIQLIQN
                          HFV
modBORIS (SEQ ID NO: 19)  atggccgctacagagattagcgtgctgagcgagcagttcaccaagatcaaagaactgaagctgatgctcgagaagggcctgaa
                          gaaagaagagaaggacggcgtctgccgcgagaagaaccacagaagcccatctgagctggaagcccagagaacctctggcg
                          ccttccaggacagcatcctggaagaggaagtggaactggttctggcccctctggaagagagcaagaagtacatcctgacactgc
                          agaccgtgcacttcacctctgaagccgtgcagctccaggacatgagcctgctgtctatccagcagcaagagggcgtgcaggttgt
                          ggttcagcaacctggacctggactgctgtggctgcaagagggacctagacagagcctgcagcagtgtgtggccatcagcatcca
                          gcaagagctgtactcccctcaagagatggaagtgctgcagtttcacgccctggaagaaaacgtgatggtggccatcgaggacag
                          caagctggctgtgtctctggccgaaaccaccggcctgatcaagctggaagaagaacaagagaagaatcagctgctcgccgaa
                          aagaccaaaaagcaactgttcttcgtggaaaccatgagcggcgacgagcggagcgacgaaatcgtgctgaccgtgtccaaca
                          gcaacgtcgaggaacaagaggaccagcctacagcctgtcaggccgatgccgagaaagccaagtttaccaagaaccagaga
                          aagaccaagggcgccaagggcaccttccactgcaacgtgtgcatgttcaccagcagccggatgagcagcttcaactgccacat
                          gaagacccacaccagcgagaagccccacctgtgccatctgtgcctgaaaaccttccggaccgtgactctgctgtggaactacgtg
                          aacacccacacaggcacccggccttacaagtgcaacgactgcaacatggccttcgtgaccagcggagaactcgtgcggcaca
                          gaagatacaagcacacccacgagaaacccttcaagtgcagcatgtgcaaatacgccagcatggaagcctccaagctgaagtg
                          tcacgtgcggagccatacaggcgagcaccctttccagtgctgccagtgtagctacgcctccagggacacctataagctgaagcg
                          gcacatgagaacccactctggggagaagccttacgagtgccacatctgccacaccagattcacccagagcggcaccatgaag
                          attcacatcctgcagaaacacggcaagaacgtgcccaagtaccagtgtcctcactgcgccaccattatcgccagaaagtccgac
                          ctgcgggtgcacatgaggaatctgcacgcctattctgccgccgagctgaaatgcagatactgcagcgccgtgttccacaagagat
                          acgccctgatccagcaccagaaaacccacaagaacgagaagcggtttaagtgcaagcactgctcctacgcctgcaagcaaga
                          gcgccacatgatcgcccacatccacacacacaccggcgaaaagcctttcacctgtctgagctgcaacaagtgcttccggcagaa
                          acagctgctgaacgcccacttcagaaagtaccacgacgccaacttcatccccaccgtgtacaagtgctccaagtgcggcaaggg
                          cttcagccggtggatcaatctgcaccggcacctggaaaagtgcgagtctggcgaagccaagtctgccgcctctggcaagggcag
                          aagaacccggaagagaaagcagaccattctgaaagaggccaccaagagccagaaagaagccgccaagcgctggaaaga
                          ggctgccaacggcgacgaagctgccgctgaagaagccagcacaacaaagggcgaacagttccccgaagagatgttccccgt
                          ggcctgcagagaaaccacagccagagtgaagcaagaggtggaccagggcgtcacatgcgagatgctgctgaataccatgga
                          caagtgatga
modBORIS (SEQ ID NO: 20)  MAATEISVLSEQFTKIKELKLMLEKGLKKEEKDGVCREKNHRSPSELEAQRTSGAFQDSILEE
                          EVELVLAPLEESKKYILTLQTVHFTSEAVQLQDMSLLSIQQQEGVQVVVQQPGPGLLWLQEG
                          PRQSLQQCVAISIQQELYSPQEMEVLQFHALEENVMVAIEDSKLAVSLAETTGLIKLEEEQEK
                          NQLLAEKTKKQLFFVETMSGDERSDEIVLTVSNSNVEEQEDQPTACQADAEKAKFTKNQRK
                          TKGAKGTFHCNVCMFTSSRMSSFNCHMKTHTSEKPHLCHLCLKTFRTVTLLWNYVNTHTGT
                          RPYKCNDCNMAFVTSGELVRHRRYKHTHEKPFKCSMCKYASMEASKLKCHVRSHTGEHPF
                          QCCQCSYASRDTYKLKRHMRTHSGEKPYECHICHTRFTQSGTMKIHILQKHGKNVPKYQCP
                          HCATIIARKSDLRVHMRNLHAYSAAELKCRYCSAVFHKRYALIQHQKTHKNEKRFKCKHCSY
                          ACKQERHMIAHIHTHTGEKPFTCLSCNKCFRQKQLLNAHFRKYHDANFIPTVYKCSKCGKGF
                          SRWINLHRHLEKCESGEAKSAASGKGRRTRKRKQTILKEATKSQKEAAKRWKEAANGDEAA
                          AEEASTTKGEQFPEEMFPVACRETTARVKQEVDQGVTCEMLLNTMDK
modMesothelin (SEQ ID NO: atggcattgcctacagctagacctctgctgggcagctgtggaacaccagctctgggaagcctgctgtttctgctgttcagcctcggat
21)                       gggtgcagccttctagaacactggccggcgagacaggacaagaagctgctcctcttgacggcgtgctggccaatcctcctaatat
                          cagctctctgagccccagacagctgctcggctttccttgtgccgaagtgtctggcctgagcaccgagagagtgtgggaacttgctgt
                          ggccctggctcagaaaaacgtgaagctgagcacagagcagctgagatgtctggcccaccagctgagtgaacctccagaggat
                          ctggatgccctgcctctggacctgctgctgttcctgaatcctgacgcctttagcggccctcaggcctgcaccagattcttcagcagaat
                          caccaaggccaatgtggatctgctgcccagaggcgcccctgagagacaaagacttctgcctgctgctctggcctgttggggcgtta
                          gaggatctctgctgtctgaggccgatgtgctggctcttggaggcctggcttgtaacctgcctggcagatttgtggccgagtctgctgag
                          gtgctgctgcctagactggtgtcctgtcctggacctctggatcaggaccagcaagaagccgctagagctgcacttcaaggcggcg
                          gacctccttatggacctcctctgacttggagcgtgtccaccatggacgctctgagaggactgctgcctgttctgggccagcctatcatc
                          cggtctatccctcagggaattgtggccgcttggcggcagagaagcttcagagatccctcttggagacagcccaagcagaccatcc
                          tgtggcctcggttcagatgggaagtcgagaaaaccgcctgtcctagcggcaagaaggccagagagatcgacgagagcctgatc
                          ttctacaagaagtgggaactcgaggcctgcgtggacgctgctctgctggctacacagatggacagagtgaacgctatccccttcac
                          ctatgagcagctggacgtgctgaagcacaagctggatgagctgtaccctcagggctaccccgagtctgtgattcagcacctgggct
                          acctgtttctgaagatgagccccgaggacatccggaagtggaacgtgaccagcctggaaaccctgaaggccctgctggaagtg
                          aacaagggccacgagatgtccccacaggctcctagaaggcctctgcctcaagtggccacactgatcgacagattcgtgaaagg
                          caggggccagctggacaaggacaccctggatacactgaccgccttctatcccggctatctgtgcagcctgtctcctgaggaactgt
                          cctctgtgcctcctagctctatttgggctgtgcggcctcaggacctggatacctgtgatcctagacagctggatgtcctgtatcctaagg
                          ctcggctggccttccagaacatgaacggcagcgagtacttcgtgaagatccagttcttccttggcggcgctcccaccgaggatctg
                          aaagctctgtcccagcagaatgtgtctatggacctggccacctttatgaagctgcggaccgatgctgtgctgcctctgacagtggcc
                          gaggtgcaaaaactgctgggccctcatgtggaaggactgaaggccgaagaacggcacagacccgtcagagactggattctga
                          gacagcggcaggacgacctggacacactggaacttggactgcaaggcggcatccccaatggctacctggtgctggatctgagc
                          gtgcaagaggccctctctggcacaccttgtttgctcggacctggaccagtgctgacagtgttggctctgctgctggcctctacactgg
                          cctgataa
modMesothelin (SEQ ID NO: MALPTARPLLGSCGTPALGSLLFLLFSLGINVQPSRTLAGETGQEAAPLDGVLANPPNISSLS
22)                       PRQLLGFPCAEVSGLSTERVWELAVALAQKNVKLSTEQLRCLAHQLSEPPEDLDALPLDLLL
                          FLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVLALG
                          GLACNLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPLTWSVSTMD
                          ALRGLLPVLGQPIIRSIPQGIVAAWRQRSFRDPSWRQPKQTILWPRFRWEVEKTACPSGKKA
                          REIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQH
                          LGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQL
                          DKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNM
                          NGSEYFVKIQFFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEG
                          LKAEERHRPVRDWILRQRQDDLDTLELGLQGGIPNGYLVLDLSVQEALSGTPCLLGPGPVLT
                          VLALLLASTLA
KRAS G12D mutation (SEQ   accgagtacaagctggtggttgttggagccgatggcgtgggaaagagcgccctgacaattcagctgatccagaaccacttcgtg
ID NO: 23)
KRAS G12D mutation (SEQ   TEYKLVVVGADGVGKSALTIQLIQNHFV
ID NO: 24)
KRAS G12V mutation (SEQ   acagagtataagctcgtggtcgtgggcgctgtcggagtgggaaaatctgccctgaccatccaactcattcagaatcactttgtg
ID NO: 25)
KRAS G12V mutation (SEQ   TEYKLVVVGAVGVGKSALTIQLIQNHFV
ID NO: 26)
modTERT (SEQ ID NO: 27)   atgcctagagcacctagatgtagagctgtgcggagcctgctgcggagccactatagagaagttctgcccctggccaccttcgtgcg
                          tagacttggacctcaaggatggcggctggtgcagagaggcgatcctgctgcttttagagccctggtggcccagtgtctcgtgtgcgtt
                          ccatgggatgctagacctccaccagctgctcccagcttcagacaggtgtcctgcctgaaagaactggtggccagagtgctgcagc
                          ggctgtgtgaaaggggcgccaaaaatgtgctggccttcggctttgccctgctggatgaagctagaggcggacctcctgaggccttt
                          acaacaagcgtgcggagctacctgcctaacaccgtgacagatgccctgagaggatctggcgcttggggactgctgctgagaag
                          agtgggagatgacgtgctggtgcatctgctggcccactgtgctctgtttgtgctggtggctcctagctgcgcctaccaagtttgcggcc
                          ctctgctgtatcagctgggcgctgctacacaggctagaccacctccacatgccagcggacctagaagaaggctgggctgcgaaa
                          gagcctggaaccactctgttagagaagccggcgtgccactgggattgcctgcacctggtgctcggagaagagatggcagcgcct
                          ctagatctctgcctctgcctaagaggcccagaagaggcgcagcacctgagcctgagagaacccctatcggccaaggatcttggg
                          cccatcctggcagaacaagaggccctagcgatagaggcttctgcgtggtgtctcctgccagacctgccgaggaagctacatctctt
                          gacggcgccctgagcggcacaagacactctcatccatctgtgggctgccagcaccatgccggacctccatctacaagcagacc
                          acctagaccttgggacaccccttgtcctccagtgtacgccgagacaaagcacttcctgtacagcagcggcgacaaagagcagct
                          gaggcctagcttcctgctgagctttctgaggccaagcctgacaggcgccagacggctgctggaaacaatcttcctgggcagcaga
                          ccctggatgcctggcacacttagaaggctgcctagactgccccagcggtactggcaaatgaggcccctgtttctggaactgctggg
                          caaccacgctcagtgcccttatggcgtgctgctgaaaacccactgtccactgagagccgtggttactccagctgctggcgtgtgtgc
                          cagagagaagccacagggatctgtggtggcccctgaggaagaggacaccgatcctagaaggctcgtgcagctgctgaggcag
                          catagctctccatggcaggtctacggattcgtgcgggcctgtctgcatagactggttccacctggactgtggggctccagacacaac
                          gagcggcggtttctgcggaacaccaagaagttcatcagcctgggaaagcacgccaagctgagcctgcaagagctgacctgga
                          agatgagcgtgtgggattgtgcttggctgcggagaagtcctggcgtgggatgtgttcctgccgccgaacacagactgcgggaaga
                          gatcctggccaagttcctgcactggctgatgtccgtgtacgtggtcgaactgctgcggtccctgttctgcgtgaccgagacaaccttc
                          cagaagaaccggctgttcttctaccggaagtccgtgtggtccaagctgcagagcatcggcatccggcagcatctgaagagagtg
                          cagctgagagagctgctcgaagccgaagttcggcagcacagaaaagccagactggccctgctgaccagcaggctgagattca
                          tccccaagcacgatggcctgcggcctattgtgaacatggactacgttgtgggcgccagaaccttccaccgggaaaagagagccg
                          agcggctgacctctagagtgaaggccctgtttagcgtgctgaactacgagcgggccagaaggccatctctgctgggagcctttgtg
                          ctcggcctggacgatattcatagagcctggcggacattcgtgctgagagtcagagcccaggatagccctcctgagctgtacttcgtg
                          aaggccgatgtgatgggcgcctacaacacaatccctcaggaccggctgaccgagatcattgccagcatcatcaagccccagaa
                          catgtactgtgtgcggagatacgccgtggtgcagaaagccacacatggccacgtgcgcaaggccttcaagagccatgtgtctacc
                          ctgaccgacctgcagccttacatgagacagttcgtggcctatctgcaagagacaagccctctgagggacgccgtgatcatcgaac
                          agagcagcagcctgaatgaggccagctccggcctgtttgacgtgttcctcagattcatgtgccaccacgccgtgcggatcagagg
                          caagagctacatccagtgccagggcattccacagggctccatcctgagcacactgctgtgcagcctgtgctacggcgacatgga
                          aaacaagctgttcgccggcattcggcgcgacggactgcttcttagactggtggacgacttcctgctcgtgacccctcatctgaccca
                          cgccaagacctttctgaaaacactcgtgcggggcgtgcccgagtatggctgtgtggtcaatctgagaaagaccgtggtcaacttcc
                          ccgtcgaggatgaagccctcggcggcacagcttttgtgcagatgcctgctcacggactgttcccttggtgctccctgctgctggacac
                          tagaaccctggaagtgcagagcgactacagcagctatgcccggacctctatcagagccagcctgaccttcaaccggggctttaa
                          ggccggcagaaacatgcggagaaagctgtttggagtgctgcggctgaagtgccacagcctgttcctcgacctgcaagtgaacag
                          cctgcagaccgtgtgcaccaatatctacaagattctgctgctgcaagcctaccggttccacgcctgtgttctgcagctgcccttccac
                          cagcaagtgtggaagaaccctacattcttcctgcggatcatcagcgacaccgccagcctgtgttacagcatcctgaaggccaaga
                          acgccggcatgtctctgggagctaaaggcgctgcaggacccctgccttttgaagctgttcagtggctgtgtcaccaggcctttctgct
                          gaagctgacccggcacagagtgacatatgtgcccctgctgggctccctgagaacagctcagatgcagctgtccagaaagctgcc
                          aggcacaaccctgacagccctggaagctgctgctaaccctgctctgcccagcgacttcaagaccatcctggactgatga
modTERT (SEQ ID NO: 28)   MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPW
                          DARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDEARGGPPEAFTTSVRS
                          YLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLAHCALFVLVAPSCAYQVCGPLLYQLGAAT
                          QARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRDGSASRSLPLPKRPRRG
                          AAPEPERTPIGQGSWAHPGRTRGPSDRGFCWSPARPAEEATSLDGALSGTRHSHPSVGC
                          QHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSFLRPSLTGARRLLE
                          TIFLGSRPWMPGTLRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAWTPAA
                          GVCAREKPQGSWAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLHRLVPPGLWGSRH
                          NERRFLRNTKKFISLGKHAKLSLQELTWKMSVWDCAWLRRSPGVGCVPAAEHRLREElLAK
                          FLHWLMSVYVVELLRSLFCVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELLEAEV
                          RQHRKARLALLTSRLRFIPKHDGLRPIVNMDYWGARTFHREKRAERLTSRVKALFSVLNYE
                          RARRPSLLGAFVLGLDDIHRAWRTFVLRVRAQDSPPELYFVKADVMGAYNTIPQDRLTEIIASI
                          IKPQNMYCVRRYAWQKATHGHVRKAFKSHVSTLTDLQPYMRQFVAYLQETSPLRDAVIIEQ
                          SSSLNEASSGLFDVFLRFMCHHAVRIRGKSYIQCQGIPQGSILSTLLCSLCYGDMENKLFAGI
                          RRDGLLLRLVDDFLLVTPHLTHAKTFLKTLVRGVPEYGCWNLRKTWNFPVEDEALGGTAF
                          VQMPAHGLFPWCSLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRL
                          KCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRIISDTASLCY
                          SILKAKNAGMSLGAKGAAGPLPFEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQMQLSR
                          KLPGTTLTALEAAANPALPSDFKTILD
modPSMA (SEQ ID NO: 29)   atgtggaatctgctgcacgagacagatagcgccgtggctaccgttagaaggcccagatggctttgtgctggcgctctggttctggct
                          ggcggcttttttctgctgggcttcctgttcggctggttcatcaagagcagcaacgaggccaccaacatcacccctaagcacaacatg
                          aaggcctttctggacgagctgaaggccgagaatatcaagaagttcctgtacaacttcacgcacatccctcacctggccggcaccg
                          agcagaattttcagctggccaagcagatccagagccagtggaaagagttcggcctggactctgtggaactggcccactacgatgt
                          gctgctgagctaccccaacaagacacaccccaactacatcagcatcatcaacgaggacggcaacgagatcttcaacaccagc
                          ctgttcgagcctccacctcctggctacgagaacgtgtccgatatcgtgcctccattcagcgctttcagcccacagcggatgcctgag
                          ggctacctggtgtacgtgaactacgccagaaccgaggacttcttcaagctggaatgggacatgaagatcagctgcagcggcaag
                          atcgtgatcgcccggtacagaaaggtgttccgcgagaacaaagtgaagaacgcccagctggcaggcgccaaaggcgtgatcc
                          tgtatagcgaccccgccgactattttgcccctggcgtgaagtcttaccccgacggctggaattttcctggcggcggagtgcagcggc
                          ggaacatccttaatcttaacggcgctggcgaccctctgacacctggctatcctgccaatgagtacgcctacagacacggaattgcc
                          gaggctgtgggcctgccttctattcctgtgcaccctgtgcggtactacgacgcccagaaactgctggaaaagatgggcggaagcg
                          cccctcctgactcttcttggagaggctctctgaaggtgccctacaatgtcggcccaggcttcaccggcaacttcagcacccagaaa
                          gtgaaaatgcacatccacagcaccaacgaagtgacccggatctacaacgtgatcggcacactgagaggcgccgtggaacccg
                          acaaatacgtgatcctcggcggccacagagacagctgggtgttcggaggaatcgaccctcaatctggcgccgctgtggtgtatga
                          gatcgtgcggtctttcggcaccctgaagaaagaaggatggcggcccagacggaccatcctgtttgcctcttgggacgccgagga
                          atttggcctgctgggatctacagagtgggccgaagagaacagcagactgctgcaagaaagaggcgtggcctacatcaacgccg
                          acagcagcatcgagggcaactacaccctgcggatcgattgcacccctctgatgtacagcctggtgcacaacctgaccaaagag
                          ctgaagtcccctgacgagggctttgagggcaagagcctgtacaagagctggaccaagaagtccccatctcctgagttcagcggc
                          atgcccagaatctctaagctggaaagcggcaacaacttcgaggtgttcttccagcggctgggaatcgcctctggaatcgccagat
                          acaccaagaactgggagacaaacaagttctccggctatcccctgtaccacagcgtgtacgagacatacgagctggtggaaaag
                          ttctacgaccccatgttcaagtaccacctgacagtggcccaagtgcgcggaggcatggtgttcgaactggccaatagcatcgtgct
                          gcccttcaactgcagagactacgccgtggtgctgcggaagtacgccgacaagatctacagcatcagcatgaagcacccgcaag
                          agatgaagacctacagcgtgtccttcgactccctgttcttcgccgtgaagaacttcaccaagatcgccagcaagttcagcgagcgg
                          ctgcaggacttcgacaagagcaaccctatcgtgctgaggatgatgaacgaccagctgatgttcctggaacgggccttcatcaacc
                          ctctgggactgcccgacagacccttctacaggcacgtgatctgtgcccctagcagccacaacaaatacgccggcgagagcttcc
                          ccggcatctacgatgccctgttcgacatcgagagcaacgtgaaccctagcaaggcctggggcgaagtgaagagacagatctac
                          gtggccgcattcacagtgcaggccgctgccgaaacactgtctgaggtggcctgatga
modPSMA (SEQ ID NO: 30)   MWNLLHETDSAVATVRRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFL
                          DELKAENIKKFLYNFTHIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHP
                          NYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQRMPEGYLVYVNYARTEDFFKLE
                          WDMKISCSGKIVIARYRKVFRENKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNFPG
                          GGVQRRNILNLNGAGDPLTPGYPANEYAYRHGIAEAVGLPSIPVHPVRYYDAQKLLEKMGG
                          SAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDKYVI
                          LGGHRDSWVFGGIDPQSGAAWYEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEW
                          AEENSRLLQERGVAYINADSSIEGNYTLRIDCTPLMYSLVHNLTKELKSPDEGFEGKSLYKSW
                          TKKSPSPEFSGMPRISKLESGNNFEVFFQRLGIASGIARYTKNWETNKFSGYPLYHSVYETY
                          ELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFNCRDYAWLRKYADKIYSISMKHPQE
                          MKTYSVSFDSLFFAVKNFTKIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFINPLGLPDR
                          PFYRHVICAPSSHNKYAGESFPGIYDALFDIESNVNPSKAWGEVKRQIYVAAFTVQAAAETLS
                          EVA
modMAGEA1-EGFRvIII-       atgtctctcgaacagagaagcctgcactgcaagcccgaggaagctctggaagctcagcaagaggctctgggccttgtgtgtgttc
pp65 (SEQ ID NO: 31)      aggccgctgccagcagcttttctcctctggtgctgggcacactggaagaggtgccaacagccggctctaccgatcctcctcaatctc
                          ctcaaggcgccagcgcctttcctaccaccatcaacttcacccggcagagacagcctagcgagggctctagctctcacgaggaaa
                          agggccctagcaccagctgcatcctggaaagcctgttccgggccgtgatcacaaagaaagtggccgacctcgtgggcttcctgct
                          gctgaagtacagagccagagaacccgtgaccaaggccgagatgctggaaagcgtgatcaagaactacaagcactgcttcagc
                          gagatcttcggcaaggccagcgagtctctgcagctcgtgtttggcatcgacgtgaaagaggccgatcctaccggccacagctacg
                          tgttcgtgacatgtctgggcctgagctacgatggcctgctgggcgacaatcagattatgctgaaaaccggcttcctgatcatcgtgct
                          ggtcatgatcgccatggaaggctctcacgcccctaaagaggaaatctgggaagaactgagcgtgatggaagtgtacgacggca
                          gagagcatagcgcctacggcgagcctagaaaactgctgacccaggacctggtgcaagagaagtacctcgagtacagacaggt
                          gcccgacagcgaccctgccagatacgaatttctgtggggccctagagcactggccgagacaagctatgtgaaggtgctggaata
                          cgtcatcaaggtgtccgccagagtgtgcttcttcttcccatctctgcgggaagccgctctgcgcgaagaggaagaaggcgtcagag
                          gccggaagagaagaagcctggaagagaaaaagggcaactacgtggtcaccgaccactgcagaggcagaaagcggagaa
                          gcgagtctagaggcagacggtgccctgagatgattagcgtgctgggccctatctctggccacgtgctgaaggccgtgttcagcag
                          aggcgatacacctgtgctgccccacgagacaagactgctgcagacaggcatccatgtgcgggtgtcacagccaagcctgatcct
                          ggtgtctcagtacacccctgacagcaccccttgtcacagaggcgacaaccagctccaggtgcagcacacctactttaccggcag
                          cgaggtggaaaacgtgtccgtgaacgtgcacaatcccaccggcagatccatctgtcccagccaagagcctatgagcatctacgt
                          gtacgccctgcctctgaagatgctgaacatccccagcatcaatgtgcatcactacccctctgccgccgagcggaaacacagacat
                          ctgcctgtggccgatgccgtgattcacgcctctggaaagcagatgtggcaggccagactgacagtgtccggactggcttggacca
                          gacagcagaaccagtggaaagaacccgacgtgtactacacctccgccttcgtgttccccacaaaggacgtggccctgagacac
                          gttgtgtgcgcccatgaactcgtgtgcagcatggaaaacacccgggccaccaagatgcaagtgatcggcgaccagtacgtgaa
                          ggtgtacctggaatccttctgcgaggacgtgccaagcggcaagctgttcatgcacgtgaccctgggctccgatgtggaagaggac
                          ctgaccatgaccagaaatccccagcctttcatgcggcctcacgagagaaatggcttcaccgtgctgtgccccaagaacatgatca
                          tcaagcccggcaagatcagccacatcatgctggatgtggccttcaccagccacgagcacttcggactgctgtgtcctaagagcat
                          ccccggcctgagcatcagcggcaacctgctgatgaatggccagcagatcttcctggaagtgcaggccattcgggaaaccgtgga
                          actgagacagtacgaccctgtggctgccctgttcttcttcgacatcgatctgctgctccagagaggccctcagtacagcgagcaccc
                          aacctttaccagccagtacagaatccagggcaagctggaatatcggcacacctgggatagacacgatgagggtgctgcacagg
                          gcgacgatgatgtgtggacaagcggcagcgatagcgacgaggaactggtcaccaccgagagaaagacccctagagttacag
                          gcggaggcgcaatggctggcgcttctacatctgccggacgcaagagaaagagcgcctcttctgccaccgcctgtacaagcggc
                          gtgatgacaagaggcaggctgaaagccgagagcacagtggcccctgaggaagatacagacgaggacagcgacaacgaga
                          ttcacaaccccgccgtgtttacctggcctccttggcaggctggcattctggctagaaacctggtgcctatggtggccacagtgcagg
                          gccagaacctgaagtaccaagagttcttctgggacgccaacgacatctaccggatcttcgccgaactggaaggcgtgtggcaac
                          cagccgctcagcccaaaagacgcagacacagacaggacgctctgcccggaccttgtattgccagcacacccaagaaacacc
                          ggggctgataa
modMAGEA1-EGFRvIII-       MSLEQRSLHCKPEEALEAQQEALGLVCVQAAASSFSPLVLGTLEEVPTAGSTDPPQSPQGA
pp65 (SEQ ID NO: 32)      SAFPTTINFTRQRQPSEGSSSHEEKGPSTSCILESLFRAVITKKVADLVGFLLLKYRAREPVTK
                          AEMLESVIKNYKHCFSEIFGKASESLQLVFGIDVKEADPTGHSYVFVTCLGLSYDGLLGDNQI
                          MLKTGFLIIVLVMIAMEGSHAPKEEIWEELSVMEVYDGREHSAYGEPRKLLTQDLVQEKYLEY
                          RQVPDSDPARYEFLWGPRALAETSYVKVLEYVIKVSARVCFFFPSLREAALREEEEGVRGRK
                          RRSLEEKKGNYVVTDHCRGRKRRSESRGRRCPEMISVLGPISGHVLKAVFSRGDTPVLPHE
                          TRLLQTGIHVRVSQPSLILVSQYTPDSTPCHRGDNQLQVQHTYFTGSEVENVSVNVHNPTG
                          RSICPSQEPMSIYVYALPLKMLNIPSINVHHYPSAAERKHRHLPVADAVIHASGKQMWQARLT
                          VSGLAWTRQQNQWKEPDVYYTSAFVFPTKDVALRHVVCAHELVCSMENTRATKMQVIGDQ
                          YVKVYLESFCEDVPSGKLFMHVTLGSDVEEDLTMTRNPQPFMRPHERNGFTVLCPKNMIIKP
                          GKISHIMLDVAFTSHEHFGLLCPKSIPGLSISGNLLMNGQQIFLEVQAIREIVELRQYDPVAAL
                          FFFDIDLLLQRGPQYSEHPTFTSQYRIQGKLEYRHTWDRHDEGAAQGDDDVWTSGSDSDE
                          ELVTTERKTPRVTGGGAMAGASTSAGRKRKSASSATACTSGVMTRGRLKAESTVAPEEDT
                          DEDSDNEIHNPAVFTWPPWQAGILARNLVPMVATVQGQNLKYQEFFWDANDIYRIFAELEG
                          VWQPAAQPKRRRHRQDALPGPCIASTPKKHRG
modTBXT-modBORIS (SEQ     atgtctagccctggaacagagtctgccggcaagagcctgcagtacagagtggaccatctgctgagcgccgtggaaaatgaactg
ID NO: 33)                caggccggaagcgagaagggcgatcctacagagcacgagctgagagtcggcctggaagagtctgagctgtggctgcggttca
                          aagaactgaccaacgagatgatcgtgaccaagaacggcagacggatgttccccgtgctgaaagtgaacgtgtccggactggac
                          cccaacgccatgtacagctttctgctggacttcgtggtggccgacaaccacagatggaaatacgtgaacggcgagtgggtgcca
                          ggcggaaaacctcaactgcaagcccctagctgcgtgtacattcaccctgacagccccaatttcggcgcccactggatgaaggcc
                          cctgtgtccttcagcaaagtgaagctgaccaacaagctgaacggcggaggccagatcatgctgaacagcctgcacaaatacga
                          gcccagaatccacatcgtcagagtcggcggaccccagagaatgatcaccagccactgcttccccgagacacagtttatcgccgt
                          gaccgcctaccagaacgaggaaatcaccacactgaagatcaagtacaaccccttcgccaaggccttcctggacgccaaagag
                          cggagcgaccacaaagagatgatcaaagagcccggcgacagccagcagccaggctattctcaatggggatggctgctgcca
                          ggcaccagcacattgtgccctccagccaatcctcacagccagtttggaggcgccctgagcctgtctagcacccacagctacgac
                          agataccccacactgcggagccacagaagcagcccctatccttctccttacgctcaccggaacaacagccccacctacagcgat
                          aatagccccgcctgtctgagcatgctgcagtcccacgataactggtccagcctgagaatgcctgctcacccttccatgctgcccgtg
                          tctcacaatgcctctccacctaccagcagctctcagtaccctagcctttggagcgtgtccaatggcgccgtgacactgggatctcag
                          gcagccgctgtgtctaatggactgggagcccagttcttcagaggcagccctgctcactacacccctctgacacatcctgtgtctgcc
                          cctagcagcamgcttccctatgtataagggcgctgccgccgctaccgacatcgtggattctcagtatgatgccgccgcacagg
                          gacacctgatcgcctcttggacacctgtgtctccaccttccatgagaggcagaaagagaagatccgccgccaccgagatcagcg
                          tgctgagcgagcagttcaccaagatcaaagaattgaagctgatgctcgagaaggggctgaagaaagaagagaaggacggcg
                          tctgccgcgagaagaatcacagaagccctagcgagctggaagcccagagaacatctggcgccttccaggacagcatcctgga
                          agaagaggtggaactggttctggcccctctggaagagagcaagaagtacatcctgacactgcagaccgtgcacttcacctctga
                          agccgtgcagctccaggacatgagcctgctgtctatccagcagcaagagggcgtgcaggttgtggttcagcaacctggacctgg
                          actgctctggctgcaagagggacctagacagtccctgcagcagtgtgtggccatcagcatccagcaagagctgtatagccctcaa
                          gagatggaagtgctgcagtttcacgccctcgaagagaacgtgatggtggccatcgaggacagcaagctggctgtgtctctggccg
                          aaacaaccggcctgatcaagctggaagaggaacaagagaagaaccagctgctggccgagaaaacaaaaaagcaactgttc
                          ttcgtggaaaccatgagcggcgacgagagaagcgacgagatcgtgctgacagtgtccaacagcaacgtggaagaacaagag
                          gaccagcctaccgcctgtcaggccgatgccgagaaagccaagtttaccaagaaccagagaaagaccaagggcgccaaggg
                          caccttccactgcaacgtgtgcatgttcaccagcagccggatgagcagcttcaactgccacatgaagacccacaccagcgagaa
                          gccccatctgtgtcacctgtgcctgaaaaccttccggacagtgacactgctgtggaactatgtgaacacccacacaggcacccgg
                          ccttacaagtgcaacgactgcaacatggccttcgtgaccagcggagaactcgtgcggcacagaagatacaagcacacccacg
                          agaaacccttcaagtgcagcatgtgcaaatacgcatccatggaagcctccaagctgaagtgccacgtgcgctctcacacaggcg
                          agcaccctttccagtgctgtcagtgtagctacgccagccgggacacctataagctgaagcggcacatgagaacccactctggcg
                          aaaagccctacgagtgccacatctgccacaccagattcacccagagcggcaccatgaagattcacatcctgcagaaacacggc
                          aagaacgtgcccaagtaccagtgtcctcactgcgccaccattatcgccagaaagtccgacctgcgggtgcacatgaggaatctg
                          cacgcctattctgccgccgagctgaaatgcagatactgcagcgccgtgttccacaagagatacgccctgatccagcaccagaaa
                          acccacaagaacgagaagcggtttaagtgcaagcactgcagctacgcctgcaagcaagagcgccacatgatcgcccacatcc
                          acacacacaccggggagaagccttttacctgcctgagctgcaacaagtgcttccggcagaaacagctgctcaacgcccacttca
                          gaaagtaccacgacgccaacttcatccccaccgtgtacaagtgctccaagtgcggcaagggcttcagccggtggatcaatctgc
                          accggcacctggaaaagtgcgagtctggcgaagccaagtctgccgcctctggcaagggcagaagaacccggaagagaaag
                          cagaccatcctgaaagaggccaccaagagccagaaagaagccgccaagcgctggaaagaggctgccaacggcgacgaa
                          gctgctgccgaagaagccagcacaacaaagggcgaacagttccccgaagagatgttccctgtggcctgcagagaaaccacag
                          ccagagtgaagcaagaggtcgaccagggcgtgacctgcgagatgctgctgaacaccatggacaagtgatga
modTBXT-modBORIS (SEQ     MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTEHELRVGLEESELWLRFKELTNE
ID NO: 34)                MIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVVADNHRWKYVNGEINVPGGKPQLQAPS
                          CVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMIT
                          SHCFPETQFIAVTAYQNEEITTLKIKYNPFAKAFLDAKERSDHKEMIKEPGDSQQPGYSQWG
                          WLLPGTSTLCPPANPHSQFGGALSLSSTHSYDRYPTLRSHRSSPYPSPYAHRNNSPTYSDN
                          SPACLSMLQSHDNWSSLRMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTLGSQAAA
                          VSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGFPMYKGAAAATDIVDSQYDAAAQGHLIASW
                          TPVSPPSMRGRKRRSAATEISVLSEQFTKIKELKLMLEKGLKKEEKDGVCREKNHRSPSELE
                          AQRTSGAFQDSILEEEVELVLAPLEESKKYILTLQTVHFTSEAVQLQDMSLLSIQQQEGVQVV
                          VQQPGPGLLWLQEGPRQSLQQCVAISIQQELYSPQEMEVLQFHALEENVMVAIEDSKLAVSL
                          AETTGLIKLEEEQEKNQLLAEKTKKQLFFVETMSGDERSDEIVLTVSNSNVEEQEDQPTACQ
                          ADAEKAKFTKNQRKTKGAKGTFHCNVCMFTSSRMSSFNCHMKTHTSEKPHLCHLCLKTFRT
                          VTLLWNYVNTHTGTRPYKCNDCNMAFVTSGELVRHRRYKHTHEKPFKCSMCKYASMEASK
                          LKCHVRSHTGEHPFQCCQCSYASRDTYKLKRHMRTHSGEKPYECHICHTRFTQSGTMKIHI
                          LQKHGKNVPKYQCPHCATIIARKSDLRVHMRNLHAYSAAELKCRYCSAVFHKRYALIQHQKT
                          HKNEKRFKCKHCSYACKQERHMIAHIHTHTGEKPFTCLSCNKCFRQKQLLNAHFRKYHDAN
                          FIPTVYKCSKCGKGFSRWINLHRHLEKCESGEAKSAASGKGRRTRKRKQTILKEATKSQKEA
                          AKRWKEAANGDEAAAEEASTTKGEQFPEEMFPVACRETTARVKQEVDQGVTCEMLLNTMD
                          K
modTBXT-modMAGEC2         atggctctgctgctggtttctctgctggccctgctgtctctcggctctggatgtcaccacagaatctgccactgcagcaaccgggtgttc
(SEQ ID NO: 35)           ctgtgccagaaaagcaaagtgaccgagatcctgagcgacctgcagcggaatgccatcgagctgagattcgtgctgaccaagct
                          gcaagtgatccagaagggcgccttcagcggcttcggcgacctggaaaagatcgagatcagccagaacaacgtgctggaagtg
                          atcgaggcccacgtgttcagcaacctgcctaagctgcacgagatcagaatcgagaaggccaacaacctgctgtacatcaaccc
                          cgaggccttccagaacttccccaacctgcagtacctgctgatctccaacaccggcatcaaacatctgcccgacgtgcacaagatc
                          cacagcctgcagaaggtgctgctggacatccaggacaacatcaacatccacacaatcgagcggaactacttcctgggcctgag
                          cttcgagagcgtgatcctgtggctgaacaagaacggcatccaagagatccacaactgcgccttcaatggcacccagctggacga
                          gctgaacctgtccgacaacaacaatctggaagaactgcccaacgacgtgttccacagagccagcggacctgtgatcctggacat
                          cagcagaaccagaatccactctctgcccagctacggcctggaaaacctgaagaagctgcgggccagaagcacctacaatctg
                          aaaaagctgcctacgctggaaaccctggtggccctgatggaagccagcctgacataccctagccactgctgcgcctttgccaact
                          ggcggagacagatctctgagctgcaccccatctgcaacaagagcatcctgcggcaagaggtggactacatgacacaggccag
                          aggccagagattcagcctggccgaggataacgagagcagctacagcagaggcttcgacatgacctacaccgagttcgactac
                          gacctgtgcaacaaggtggtggacgtgacatgcagccccaagcctgatgccttcaatccctgcgaggacatcatgggctacaac
                          atcctgagagtgctgatctggttcatcagcatcctggccatcaccgagaacatcatcgtgctggtcatcctgaccaccagccagtac
                          aagctgaccgtgcctatgttcctgatgtgcaacctggccttcgccgatctgtgcatcggcatctacctgctgctgatcgccagcgtgg
                          acattcacaccaagagccagtaccacaactacgccatcgactggcagacaggcgccggatgtgatgccgccggattctttacag
                          tgttcgccagcgagctgtccgtgtacaccctgacagctatcaccctggaacggtggcacaccatcacacacgctatgcagctgga
                          ctgcaaagtgcacctgagacacagcgcctccgtgatggttatgggctggatcttcgccttcgctgccgctctgttccccatctttggcat
                          cagctcctacatgaaggtgtccatctatctgcccatggacatcgacagccctctgagccagctgtacgtgatgagtctgctggtgctg
                          aatgtgctggcctttgtggtcatctgcggctgctacatctatatctacctgacagtgcggaaccccaacatcgtgtccagctccagcg
                          acacccggatcgctaagagaatggccatgctgatcttcaccgactttctgtgcatggcccctatcagcctgttcgccattagcgctag
                          cctgaaggtgcccctgatcaccgtgtccaaggccaagattctgctggtcctgttctaccccatcaacagctgcgccaatcctttcctgt
                          acgccatcttcaccaagaacttcaggcggaacttcttcatcctgctgagcaagcggggctgttacaagatgcaggcccagatctac
                          cggaccgagacactgtccaccgtgcacaacacacaccccagaaacggccactgtagcagcgcccctagagtgacaaatggct
                          ccacctacatcctggtgccactgagccatctggcccagaacagaggccggaagagaagaagccccagggctcccaagagac
                          agagatgcatgcccgaagaggacctgcagagccagagcgaaacacagggactcgaaggtgctcaggctcctctggccgtgg
                          aagaagatgccagcagctctaccagcacctccagcagcttccctagcagctttccattcagctcctctagctctagcagcagctgtt
                          accctctgatccccagcacacccgagaaggtgttcgccgacgacgagacacctaatccactgcagtctgcccagatcgcctgca
                          gcagtacactggtggttgctagcctgcctctggaccagtctgatgagggaagcagcagccagaaagaggaaagccctagcaca
                          ctccaggtgctgcccgatagcgagagcctgcctagaagcgagatctacaagaaaatgaccgacctggtgcagttcctcctgttca
                          agtaccagatgaaggaacccatcaccaaggccgaaatcctggaaagcgtgatcagaaactacgaggaccactttccactgctg
                          ttcagcgaggccagcgagtgcatgctgctcgtgtttagcatcgacgtgaagaaggtggaccccaccggccacagctttgtgctggt
                          tacaagcctgggactgacctacgacggcatgctgtccgatgtgcagagcatgcctaagaccggcatcctgatcctgattctgagca
                          tcgtgttcatcgagggctactgcacccctgaggaagtgatttgggaagccctgaacatgatgggcctgtacgatggcatggaacac
                          ctgatctacggcgagcccagaaaactgctgacccaggactgggtgcaagagaactacctggaataccggcagatgcccggca
                          gcgatcctgccagatatgagtttctgtggggccctagagcacatgccgagatccggaagatgagcctgctgaagttcctggccaa
                          agtgaacggcagcgacccaatcagcttcccactttggtacgaagaggccctgaaggacgaggaagagagagcccaggatag
                          aatcgccaccaccgacgacacaacagccatggcctctgcctcttctagcgccaccggcagctttagctaccccgagtgataa
modTBXT-modMAGEC2         MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTEHELRVGLEESELWLRFKELTNE
(SEQ ID NO: 36)           MIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFWADNHRWKYVNGEINVPGGKPQLQAPS
                          CVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMIT
                          SHCFPETQFIAVTAYQNEEITTLKIKYNPFAKAFLDAKERSDHKEMIKEPGDSQQPGYSQWG
                          WLLPGTSTLCPPANPHSQFGGALSLSSTHSYDRYPTLRSHRSSPYPSPYAHRNNSPTYSDN
                          SPACLSMLQSHDNWSSLRMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTLGSQAAA
                          VSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGFPMYKGAAAATDIVDSQYDAAAQGHLIASW
                          TPVSPPSMRGRKRRSPPVPGVPFRNVDNDSLTSVELEDWVDAQHPTDEEEEEASSASSTL
                          YLVFSPSSFSTSSSLILGGPEEEEVPSGVIPNLTESIPSSPPQGPPQGPSQSPLSSCCSSFLW
                          SSFSEESSSQKGEDTGTCQGLPDSESSFTYTLDEKVAKLVEFLLLKYEAEEPVTEAEMLMIVI
                          KYKDYFPVILKRAREFMELLFGLALIEVGPDHFCVFANTVGLTDEGSDDEGMPENSLLIIILSVI
                          FIKGNCASEEVIWEVLNAVGVYAGREHFVYGKPRELLTNVWVQGHYLEYWEVPHSSPLYYE
                          FLWGPRAHSESIKKKVLEFLAKLNNTVPSFFPSWYKDALKDVEERVQATIDTADDATVMASE
                          SLSVMSSNVSFSE

In some embodiments, provided herein is a vaccine composition comprising a therapeutically effective amount of cells from at least two cancer cell lines, wherein each cell line or a combination of the cell lines expresses at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the TAAs of Tables 25. In other embodiments, the TAAs in Tables 25 are modified to include one or more NSMs as described herein. In some embodiments, at least one cell line is modified to increase production of at least 1, 2, or 3 immunostimulatory factors, e.g., immunostimulatory factors from Table 6. In some embodiments, a vaccine composition is provided comprising a therapeutically effective amount of the cells from at least one cancer cell line, wherein each cell line or combination of cell lines is modified to reduce at least 1, 2, or 3 immunosuppressive factors, e.g., immunosuppressive factors from Table 8. In some embodiments, a vaccine composition is provided comprising two cocktails, wherein each cocktail comprises three cell lines modified to express 1, 2, or 3 immunostimulatory factors and to inhibit or reduce expression of 1, 2, or 3 immunosuppressive factors, and wherein each cell line expresses at least 10 TAAs or TAAs comprising one or more NSMs.

Methods and assays for determining the presence or expression level of a TAA in a cell line according to the disclosure or in a tumor from a subject are known in the art. By way of example, Warburg-Christian method, Lowry Assay, Bradford Assay, spectrometry methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), immunoblotting and antibody-based techniques such as western blot, ELISA, immunoelectrophoresis, protein immunoprecipitation, flow cytometry, and protein immunostaining are all contemplated by the present disclosure.

The antigen repertoire displayed by a patient's tumor can be evaluated in some embodiments in a biopsy specimen using next generation sequencing and antibody-based approaches. Similarly, in some embodiments, the antigen repertoire of potential metastatic lesions can be evaluated using the same techniques to determine antigens expressed by circulating tumor cells (CTCs). Assessment of antigen expression in tumor biopsies and CTCs can be representative of a subset of antigens expressed. In some embodiments, a subset of the antigens expressed by a patient's primary tumor and/or CTCs are identified and, as described herein, informs the selection of cell lines to be included in the vaccine composition in order to provide the best possible match to the antigens expressed in a patient's tumor and/or metastatic lesions.

Embodiments of the present disclosure provides compositions of cell lines that (i) are modified as described herein and (ii) express a sufficient number and amount of TAAs such that, when administered to a patient afflicted with a cancer, cancers, or cancerous tumor(s), a TAA-specific immune response is generated.

Methods of Stimulating an Immune Response and Methods of Treatment

The vaccine compositions described herein may be administered to a subject in need thereof. Provided herein are methods for inducing an immune response in a subject, which involve administering to a subject an immunologically effective amount of the genetically modified cells. Also provided are methods for preventing or treating a tumor in a subject by administering an anti-tumor effective amount of the vaccine compositions described herein. Such compositions and methods may be effective to prolong the survival of the subject.

According to various embodiments, administration of any one of the vaccine compositions provided herein can increase pro-inflammatory cytokine production (e.g., IFNγ secretion) by leukocytes. In some embodiments, administration of any one of the vaccine compositions provided herein can increase pro-inflammatory cytokine production (e.g., IFNγ secretion) by leukocytes by at least 1.5-fold, 1.6-fold, 1.75-fold, 2-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold or more. In other embodiments, the IFNγ production is increased by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25-fold or higher compared to unmodified cancer cell lines. Assays for determining the amount of cytokine production are well-known in the art and described herein. Without being bound to any theory or mechanism, the increase in pro-inflammatory cytokine production (e.g., IFNγ secretion) by leukocytes is a result of either indirect or direct interaction with the vaccine composition.

In some embodiments, administration of any one of the vaccine compositions provided herein comprising one or more modified cell lines as described herein can increase the uptake of cells of the vaccine composition by phagocytic cells, e.g., by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold or more, as compared to a composition that does not comprise modified cells.

In some embodiments, the vaccine composition is provided to a subject by an intradermal injection. Without being bound to any theory or mechanism, the intradermal injection, in at least some embodiments, generates a localized inflammatory response recruiting immune cells to the injection site. Following administration of the vaccine, antigen presenting cells (APCs) in the skin, such as Langerhans cells (LCs) and dermal dendritic cells (DCs), uptake the vaccine cell line components by phagocytosis and then migrate through the dermis to the draining lymph node. At the draining lymph node, DCs or LCs that have phagocytized the vaccine cell line components are expected to prime naïve T cells and B cells. Priming of naïve T and B cells is expected to initiate an adaptive immune response to tumor associated antigens (TAAs) expressed by the vaccine cell line components. Certain TAAs expressed by the vaccine cell line components are also expressed by the patient's tumor. Expansion of antigen specific T cells at the draining lymph node and trafficking of these T cells to the tumor microenvironment (TME) is expected to generate a vaccine-induced anti-tumor response.

According to various embodiments, immunogenicity of the allogenic vaccine composition can be further enhanced through genetic modifications that reduce expression of immunosuppressive factors while increasing the expression or secretion of immunostimulatory signals. Modulation of these factors aims to enhance the uptake vaccine cell line components by LCs and DCs in the dermis, trafficking of DCs and LCs to the draining lymph node, T cell and B cell priming in the draining lymph node, and, thereby resulting in more potent anti-tumor responses.

In some embodiments, the breadth of TAAs targeted in the vaccine composition can be increased through the inclusion of multiple cell lines. For example, different histological subsets within a certain tumor type tend to express different TAA subsets. As a further example, in NSCLC, adenocarcinomas, and squamous cell carcinomas express different antigens. The magnitude and breadth of the adaptive immune response induced by the vaccine composition can, according to some embodiments of the disclosure, be enhanced through the inclusion of additional cell lines expressing the same or different immunostimulatory factors. For example, expression of an immunostimulatory factor, such as IL-12, by one cell line within a cocktail of three cell lines can act locally to enhance the immune responses to all cell lines delivered into the same site. The expression of an immunostimulatory factor by more than one cell line within a cocktail, such as GM-CSF, can increase the amount of the immunostimulatory factor in the injection site, thereby enhancing the immune responses induced to all components of the cocktail. The degree of HLA mismatch present within a vaccine cocktail may further enhance the immune responses induced by that cocktail.

As described herein, in various embodiments, a method of stimulating an immune response specific to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more TAAs in a subject is provided comprising administering a therapeutically effective amount of a vaccine composition comprising modified cancer cell lines.

An “immune response” is a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus, such as a cell or antigen (e.g., formulated as an antigenic composition or a vaccine). An immune response can be a B cell response, which results in the production of specific antibodies, such as antigen specific neutralizing antibodies. An immune response can also be a T cell response, such as a CD4+ response or a CD8+ response. B cell and T cell responses are aspects of a “cellular” immune response. An immune response can also be a “humoral” immune response, which is mediated by antibodies. In some cases, the response is specific for a particular antigen (that is, an “antigen specific response”), such as one or more TAAs, and this specificity can include the production of antigen specific antibodies and/or production of a cytokine such as interferon gamma which is a key cytokine involved in the generation of a Th₁ T cell response and measurable by ELISpot and flow cytometry.

Vaccine efficacy can be tested by measuring the T cell response CD4+ and CD8+ after immunization, using flow cytometry (FACS) analysis, ELISpot assay, or other method known in the art. Exposure of a subject to an immunogenic stimulus, such as a cell or antigen (e.g., formulated as an antigenic composition or vaccine), elicits a primary immune response specific for the stimulus, that is, the exposure “primes” the immune response. A subsequent exposure, e.g., by immunization, to the stimulus can increase or “boost” the magnitude (or duration, or both) of the specific immune response. Thus, “boosting” a preexisting immune response by administering an antigenic composition increases the magnitude of an antigen (or cell) specific response, (e.g., by increasing antibody titer and/or affinity, by increasing the frequency of antigen specific B or T cells, by inducing maturation effector function, or a combination thereof).

The immune responses that are monitored/assayed or stimulated by the methods described herein include, but not limited to: (a) antigen specific or vaccine specific IgG antibodies; (b) changes in serum cytokine levels that may include and is not limited to: IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17A, IL-20, IL-22, TNFα, IFNγ, TGFβ, CCL5, CXCL10; (c) IFNγ responses determined by ELISpot for CD4 and CD8 T cell vaccine and antigen specific responses; (d) changes in IFNγ responses to TAA or vaccine cell components; (e) increased T cell production of intracellular cytokines in response to antigen stimulation: IFNγ, TNFα, and IL-2 and indicators of cytolytic potential: Granzyme A, Granzyme B, Perforin, and CD107a; (f) decreased levels of regulatory T cells (Tregs), mononuclear monocyte derived suppressor cells (M-MDSCs), and polymorphonuclear derived suppressor cells (PMN-MDSCs); (g) decreased levels of circulating tumor cells (CTCs); (h) neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR); (i) changes in immune infiltrate in the TME; and (j) dendritic cell maturation.

Assays for determining the immune responses are described herein and well known in the art. DC maturation can be assessed, for example, by assaying for the presence of DC maturation markers such as CD80, CD83, CD86, and MHC II. (See Dudek, A., et al., Front. Immunol., 4:438 (2013)). Antigen specific or vaccine specific IgG antibodies can be assessed by ELISA or flow cytometry. Serum cytokine levels can be measured using a multiplex approach such as Luminex or Meso Scale Discovery Electrochemiluminescence (MSD). T cell activation and changes in lymphocyte populations can be measured by flow cytometry. CTCs can be measured in PBMCs using a RT-PCR based approach. The NLR and PLR ratios can be determined using standard complete blood count (CBC) chemistry panels. Changes in immune infiltrate in the TME can be assessed by flow cytometry, tumor biopsy and next-generation sequencing (NGS), or positron emission tomography (PET) scan of a subject.

Given the overlap in TAA expression between cancers and tumors of different types, the present disclosure provides, in certain embodiments, compositions that can treat multiple different cancers. For example, one vaccine composition comprising two cocktails of three cell lines each may be administered to a subject suffering from two or more types of cancers and said vaccine composition is effective at treating both, additional or all types of cancers. In exemplary embodiments, and in consideration of the TAA expression profile, the same vaccine composition comprising modified cancer cell lines is used to treat prostate cancer and testicular cancer, gastric and esophageal cancer, or endometrial, ovarian, and breast cancer in the same patient (or different patients). TAA overlap can also occur within subsets of hot tumors or cold tumors. For example, TAA overlap occurs in GBM and SCLC, both considered cold tumors. Exemplary TAAs included in embodiments of the vaccine composition include GP100, MAGE-A1, MAGE-A4, MAGE-A10, Sart-1, Sart-3, Trp-1, and Sox2. In some embodiments, cell lines included in the vaccine composition can be selected from two tumor types of similar immune landscape to treat one or both of the tumor types in the same individual.

As used herein, changes in or “increased production” of, for example a cytokine such as IFNγ, refers to a change or increase above a control or baseline level of production/secretion/expression and that is indicative of an immunostimulatory response to an antigen or vaccine component.

Combination Treatments and Regimens

Formulations, Adjuvants, and Additional Therapeutic Agents

The compositions described herein may be formulated as pharmaceutical compositions. The term “pharmaceutically acceptable” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each component must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It must also be suitable for use in contact with tissue, organs or other human component without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. (See Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 5th Edition; Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, Fla., 2004)).

Embodiments of the pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration (i.e., parenteral, intravenous, intra-arterial, intradermal, subcutaneous, oral, inhalation, transdermal, topical, intratumoral, transmucosal, intraperitoneal or intra-pleural, and/or rectal administration). Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; dimethyl sulfoxide (DMSO); antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or one or more vials comprising glass or polymer (e.g., polypropylene). The term “vial” as used herein means any kind of vessel, container, tube, bottle, or the like that is adapted to store embodiments of the vaccine composition as described herein.

In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. The term “carrier” as used herein encompasses diluents, excipients, adjuvants, and combinations thereof. Pharmaceutically acceptable carriers are well known in the art (See Remington: The Science and Practice of Pharmacy, 21st Edition). Exemplary “diluents” include sterile liquids such as sterile water, saline solutions, and buffers (e.g., phosphate, tris, borate, succinate, or histidine). Exemplary “excipients” are inert substances that may enhance vaccine stability and include but are not limited to polymers (e.g., polyethylene glycol), carbohydrates (e.g., starch, glucose, lactose, sucrose, or cellulose), and alcohols (e.g., glycerol, sorbitol, or xylitol).

In various embodiments, the vaccine compositions and cell line components thereof are sterile and fluid to the extent that the compositions and/or cell line components can be loaded into one or more syringes. In various embodiments, the compositions are stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. In some embodiments, the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, by the use of surfactants, and by other means known to one of skill in the art. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, it may be desirable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, and/or sodium chloride in the composition. In some embodiments, prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

In some embodiments, sterile injectable solutions can be prepared by incorporating the active compound(s) in the required amount(s) in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. In certain embodiments, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, embodiments of methods of preparation include vacuum drying and freeze-drying that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The innate immune system comprises cells that provide defense in a non-specific manner to infection by other organisms. Innate immunity in a subject is an immediate defense, but it is not long-lasting or protective against future challenges. Immune system cells that generally have a role in innate immunity are phagocytic, such as macrophages and dendritic cells. The innate immune system interacts with the adaptive (also called acquired) immune system in a variety of ways.

In some embodiments, the vaccine compositions alone activate an immune response (i.e., an innate immune response, an adaptive immune response, and/or other immune response). In some embodiments, one or more adjuvants are optionally included in the vaccine composition or are administered concurrently or strategically in relation to the vaccine composition, to provide an agent(s) that supports activation of innate immunity in order to enhance the effectiveness of the vaccine composition. An “adjuvant” as used herein is an “agent” or substance incorporated into the vaccine composition or administered simultaneously or at a selected time point or manner relative to the administration of the vaccine composition. In some embodiments, the adjuvant is a small molecule, chemical composition, or therapeutic protein such as a cytokine or checkpoint inhibitor. A variety of mechanisms have been proposed to explain how different agents function (e.g., antigen depots, activators of dendritic cells, macrophages). An agent may act to enhance an acquired immune response in various ways and many types of agents can activate innate immunity. Organisms, like bacteria and viruses, can activate innate immunity, as can components of organisms, chemicals such as 2′-5′ oligo A, bacterial endotoxins, RNA duplexes, single stranded RNA and other compositions. Many of the agents act through a family of molecules referred to herein as “toll-like receptors” (TLRs). Engaging a TLR can also lead to production of cytokines and chemokines and activation and maturation of dendritic cells, components involved in development of acquired immunity. The TLR family can respond to a variety of agents, including lipoprotein, peptidoglycan, flagellin, imidazoquinolines, CpG DNA, lipopolysaccharide and double stranded RNA. These types of agents are sometimes called pathogen (or microbe)-associated molecular patterns. In some embodiments, the adjuvant is a TLR4 agonist.

One adjuvant that in some embodiments may be used in the vaccine compositions is a monoacid lipid A (MALA) type molecule. An exemplary MALA is MPL® adjuvant as described in, e.g., Ulrich J. T. and Myers, K. R., Chapter 21 in Vaccine Design, the Subunit and Adjuvant Approach, Powell, M. F. and Newman, M. J., eds. Plenum Press, NY (1995).

In other embodiments, the adjuvant may be “alum”, where this term refers to aluminum salts, such as aluminum phosphate and aluminum hydroxide.

In some embodiments, the adjuvant may be an emulsion having vaccine adjuvant properties. Such emulsions include oil-in-water emulsions. Incomplete Freund's adjuvant (IFA) is one such adjuvant. Another suitable oil-in-water emulsion is MF-59™ adjuvant which contains squalene, polyoxyethylene sorbitan monooleate (also known as Tween® 80 surfactant) and sorbitan trioleate. Other suitable emulsion adjuvants are Montanide™ adjuvants (Seppic Inc., Fairfield N.J.) including Montanide™ ISA 50V which is a mineral oil-based adjuvant, Montanide™ ISA 206, and Montanide™ IMS 1312. While mineral oil may be present in the adjuvant, in one embodiment, the oil component(s) of the compositions of the present disclosure are all metabolizable oils.

In some embodiments, the adjuvant may be AS02™ adjuvant or AS04™ adjuvant. AS02™ adjuvant is an oil-in-water emulsion that contains both MPL™ adjuvant and QS-21™ adjuvant (a saponin adjuvant discussed elsewhere herein). AS04™ adjuvant contains MPL™ adjuvant and alum. The adjuvant may be Matrix-M™ adjuvant. The adjuvant may be a saponin such as those derived from the bark of the Quillaja saponaria tree species, or a modified saponin, see, e.g., U.S. Pat. Nos. 5,057,540; 5,273,965; 5,352,449; 5,443,829; and 5,560,398. The product QS-21™ adjuvant sold by Antigenics, Inc. (Lexington, Mass.) is an exemplary saponin-containing co-adjuvant that may be used with embodiments of the composition described herein. In other embodiments, the adjuvant may be one or a combination of agents from the ISCOM™ family of adjuvants, originally developed by Iscotec (Sweden) and typically formed from saponins derived from Quillaja saponaria or synthetic analogs, cholesterol, and phospholipid, all formed into a honeycomb-like structure.

In some embodiments, the adjuvant or agent may be a cytokine that functions as an adjuvant, see, e.g., Lin R. et al. Clin. Infec. Dis. 21(6):1439-1449 (1995); Taylor, C. E., Infect. Immun. 63(9):3241-3244 (1995); and Egilmez, N. K., Chap. 14 in Vaccine Adjuvants and Delivery Systems, John Wiley & Sons, Inc. (2007). In various embodiments, the cytokine may be, e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF); see, e.g., Change D. Z. et al. Hematology 9(3):207-215 (2004), Dranoff, G. Immunol. Rev. 188:147-154 (2002), and U.S. Pat. No. 5,679,356; or an interferon, such as a type I interferon, e.g., interferon-α (IFN-α) or interferon-β (IFN-β), or a type II interferon, e.g., interferon-γ (IFNγ), see, e.g., Boehm, U. et al. Ann. Rev. Immunol. 15:749-795 (1997); and Theofilopoulos, A. N. et al. Ann. Rev. Immunol. 23:307-336 (2005); an interleukin, specifically including interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-2 (IL-2); see, e.g., Nelson, B. H., J. Immunol. 172(7): 3983-3988 (2004); interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-12 (IL-12); see, e.g., Portielje, J. E., et al., Cancer Immunol. Immunother. 52(3): 133-144 (2003) and Trinchieri. G. Nat. Rev. Immunol. 3(2):133-146 (2003); interleukin-15 (11-15), interleukin-18 (IL-18); fetal liver tyrosine kinase 3 ligand (Flt3L), or tumor necrosis factor α (TNFα).

In some embodiments, the adjuvant may be unmethylated CpG dinucleotides, optionally conjugated to the antigens described herein.

Examples of immunopotentiators that may be used in the practice of the compositions and methods described herein as adjuvants include: MPL™; MDP and derivatives; oligonucleotides; double-stranded RNA; alternative pathogen-associated molecular patterns (PAMPS); saponins; small-molecule immune potentiators (SMIPs); cytokines; and chemokines.

When two or more adjuvants or agents are utilized in combination, the relative amounts of the multiple adjuvants may be selected to achieve the desired performance properties for the composition which contains the adjuvants, relative to the antigen alone. For example, an adjuvant combination may be selected to enhance the antibody response of the antigen, and/or to enhance the subject's innate immune system response. Activating the innate immune system results in the production of chemokines and cytokines, which in turn may activate an adaptive (acquired) immune response. An important consequence of activating the adaptive immune response is the formation of memory immune cells so that when the host re-encounters the antigen, the immune response occurs quicker and generally with better quality. In some embodiments, the adjuvant(s) may be pre-formulated prior to their combination with the compositions described herein.

Embodiments of the vaccine compositions described herein may be administered simultaneously with, prior to, or after administration of one or more other adjuvants or agents, including therapeutic agents. In certain embodiments, such agents may be accepted in the art as a standard treatment or prevention for a particular cancer. Exemplary agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatories, immune checkpoint inhibitors, chemotherapeutics, radiotherapeutics, or other active and ancillary agents. In other embodiments, the agent is one or more isolated TAA as described herein.

In some embodiments, a vaccine composition provided herein is administered to a subject that has not previously received certain treatment or treatments for cancer or other disease or disorder. As used herein, the phrase “wherein the subject refrains from treatment with other vaccines or therapeutic agents” refers to a subject that has not received a cancer treatment or other treatment or procedure prior to receiving a vaccine of the present disclosure. In some embodiments, the subject refrains from receiving one or more therapeutic vaccines (e.g., flu vaccine, covid-19 vaccine such as AZD1222, BNT162b2, mRNA-1273, and the like) prior to the administration of the therapeutic vaccine as described in various embodiments herein. In some embodiments, the subject refrains from receiving one or more antibiotics prior to the administration of the therapeutic vaccine as described in various embodiments herein. “Immune tolerance” is a state of unresponsiveness of the immune system to substances, antigens, or tissues that have the potential to induce an immune response. The vaccine compositions of the present disclosure, in certain embodiments, are administered to avoid the induction of immune tolerance or to reverse immune tolerance.

In various embodiments, the vaccine composition is administered in combination with one or more active agents used in the treatment of cancer, including one or more chemotherapeutic agents. Examples of such active agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and paclitaxel protein-bound particles (ABRAXANE®) and doxetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine, docetaxel, platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoid or retinoic acid or retinoic acid derivative such as all-trans retinoic acid (ATRA), VESANOID® (tretinoin), ACCUTANE® (isotretinoin, 9-cis-retinoid, 13-cis-retinoic acid), vitamin A acid) TARGRETIN™ (bexarotene), PANRETIN™ (alitretinoin); and ONTAK™ (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Further cancer active agents include sorafenib and other protein kinase inhibitors such as afatinib, axitinib, bevacizumab, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, trastuzumab, vandetanib, vemurafenib, and sunitinib; sirolimus (rapamycin), everolimus and other mTOR inhibitors.

In further embodiments, the vaccine composition is administered in combination with a TLR4 agonist, TLR8 agonist, or TLR9 agonist. Such an agonist may be selected from peptidoglycan, polyl:C, CpG, 3M003, flagellin, and Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a (LeIF).

In some embodiments, the vaccine composition is administered in combination with a cytokine as described herein. In some embodiments, the compositions disclosed herein may be administered in conjunction with molecules targeting one or more of the following: Adhesion: MAdCAM1, ICAM1, VCAM1, CD103; Inhibitory Mediators: IDO, TDO; MDSCs/Tregs: NOS1, arginase, CSFR1, FOXP3, cyclophosphamide, PI3Kgamma, PI3Kdelta, tasquinimod; Immunosuppression: TGFβ, IL-10; Priming and Presenting: BATF3, XCR1/XCL1, STING, INFalpha; Apoptotic Recycling: IL-6, surviving, IAP, mTOR, MCL1, PI3K; T-Cell Trafficking: CXCL9/10/11, CXCL1/13, CCL2/5, anti-LIGHT, anti-CCR5; Oncogenic Activation: WNT-beta-cat, MEK, PPARgamma, FGFR3, TKIs, MET; Epigenetic Reprogramming: HDAC, HMA, BET; Angiogenesis immune modulation: VEGF (alpha, beta, gamma); Hypoxia: HIF1alpha, adenosine, anit-ADORA2A, anti-CD73, and anti-CD39.

In certain embodiments, the compositions disclosed herein may be administered in conjunction with a histone deacetylase (HDAC) inhibitor. HDAC inhibitors include hydroxamates, cyclic peptides, aliphatic acids and benzamides. Illustrative HDAC inhibitors contemplated for use herein include, but are not limited to, Suberoylanilide hydroxamic acid (SAHANorinostat/Zolinza), Trichostatin A (TSA), PXD-101, Depsipeptide (FK228/romidepsin/ISTODAX®), panobinostat (LBH589), MS-275, Mocetinostat (MGCD0103), ACY-738, TMP195, Tucidinostat, valproic acid, sodium phenylbutyrate, 5-aza-2′-deoxycytidine (decitabine). See e.g., Kim and Bae, Am J Transl Res 2011; 3(2):166-179; Odunsi et al., Cancer Immunol Res. 2014 Jan. 1; 2(1): 37-49. Other HDAC inhibitors include Vorinostat (SAHA, MK0683), Entinostat (MS-275), Panobinostat (LBH589), Trichostatin A (TSA), Mocetinostat (MGCD0103), ACY-738, Tucidinostat (Chidamide), TMP195, Citarinostat (ACY-241), Belinostat (PXD101), Romidepsin (FK228, Depsipeptide), MC1568, Tubastatin A HCl, Givinostat (ITF2357), Dacinostat (LAQ824), CUDC-101, Quisinostat (JNJ-26481585) 2HCI, Pracinostat (SB939), PCI-34051, Droxinostat, Abexinostat (PCI-24781), RGFP966, AR-42, Ricolinostat (ACY-1215), Valproic acid sodium salt (Sodium valproate), Tacedinaline (CI994), CUDC-907, Sodium butyrate, Curcumin, M344, Tubacin, RG2833 (RGFP109), Resminostat, Divalproex Sodium, Scriptaid, and Tubastatin A.

In certain embodiments, the vaccine composition is administered in combination with chloroquine, a lysosomotropic agent that prevents endosomal acidification and which inhibits autophagy induced by tumor cells to survive accelerated cell growth and nutrient deprivation. More generally, the compositions comprising heterozygous viral vectors as described herein may be administered in combination with active agents that act as autophagy inhibitors, radiosensitizers or chemosensitizers, such as chloroquine, misonidazole, metronidazole, and hypoxic cytotoxins, such as tirapazamine. In this regard, such combinations of a heterozygous viral vector with chloroquine or other radio or chemo sensitizer, or autophagy inhibitor, can be used in further combination with other cancer active agents or with radiation therapy or surgery.

In other embodiments, the vaccine composition is administered in combination with one or more small molecule drugs that are known to result in killing of tumor cells with concomitant activation of immune responses, termed “immunogenic cell death”, such as cyclophosphamide, doxorubicin, oxaliplatin and mitoxantrone. Furthermore, combinations with drugs known to enhance the immunogenicity of tumor cells such as patupilone (epothilone B), epidermal-growth factor receptor (EGFR)-targeting monoclonal antibody 7A7.27, histone deacetylase inhibitors (e.g., vorinostat, romidepsin, panobinostat, belinostat, and entinostat), the n3-polyunsaturated fatty acid docosahexaenoic acid, furthermore proteasome inhibitors (e.g., bortezomib), shikonin (the major constituent of the root of Lithospermum erythrorhizon) and oncolytic viruses, such as TVec (talimogene laherparepvec). In some embodiments, the compositions comprising heterozygous viral vectors as described herein may be administered in combination with epigenetic therapies, such as DNA methyltransferase inhibitors (e.g., decitabine, 5-aza-2′-deoxycytidine) which may be administered locally or systemically.

In other embodiments, the vaccine composition is administered in combination with one or more antibodies that increase ADCC uptake of tumor by DCs. Thus, embodiments of the present disclosure contemplate combining cancer vaccine compositions with any molecule that induces or enhances the ingestion of a tumor cell or its fragments by an antigen presenting cell and subsequent presentation of tumor antigens to the immune system. These molecules include agents that induce receptor binding (e.g., Fc or mannose receptors) and transport into the antigen presenting cell such as antibodies, antibody-like molecules, multi-specific multivalent molecules and polymers. Such molecules may either be administered intratumorally with the composition comprising heterozygous viral vector or administered by a different route. For example, a composition comprising heterozygous viral vector as described herein may be administered intratumorally in conjunction with intratumoral injection of rituximab, cetuximab, trastuzumab, Campath, panitumumab, ofatumumab, brentuximab, pertuzumab, Ado-trastuzumab emtansine, Obinutuzumab, anti-HER1, -HER2, or -HER3 antibodies (e.g., MEHD7945A; MM-111; MM-151; MM-121; AMG888), anti-EGFR antibodies (e.g., nimotuzumab, ABT-806), or other like antibodies. Any multivalent scaffold that is capable of engaging Fc receptors and other receptors that can induce internalization may be used in the combination therapies described herein (e.g., peptides and/or proteins capable of binding targets that are linked to Fc fragments or polymers capable of engaging receptors).

In certain embodiments, the vaccine composition may be further combined with an inhibitor of ALK, PARP, VEGFRs, EGFR, FGFR1-3, HIF1a, PDGFR1-2, c-Met, c-KIT, Her2, Her3, AR, PR, RET, EPHB4, STAT3, Ras, HDAC1-11, mTOR, and/or CXCR4.

In certain embodiments, a cancer vaccine composition may be further combined with an antibody that promotes a co-stimulatory signal (e.g., by blocking inhibitory pathways), such as anti-CTLA-4, or that activates co-stimulatory pathways such as an anti-CD40, anti-CD28, anti-ICOS, anti-OX40, anti-CD27, anti-ICOS, anti-CD127, anti-GITR, IL-2, IL-7, IL-15, IL-21, GM-CSF, IL-12, and INFα.

Retinoic Acid

In certain embodiments, a retinoid, retinoic acid or retinoic acid derivative such as all-trans retinoic acid (ATRA), VESANOID® (tretinoin), ACCUTANE® (isotretinoin, 9-cis-retinoid, 13-cis-retinoic acid, vitamin A acid), TARGRETIN™ (bexarotene), PANRETIN™ (alitretinoin), and ONTAK™ (denileukin diftitox) is administered in combination with the vaccine compositions described herein.

Various studies, including clinical trials, have looked at the use of retinoic acid in the treatment of cancers, including glioblastoma. (See, e.g., Penas-Prado M, et al., Neuro Oncol., 2014, 17(2):266-273; Butowski N, et al., Int J Radiat Oncol Biol Phys., 2005, 61(5):1454-1459; Jaeckle K A, et al., J Clin Oncol., 2003, 21(12): 2305-2311; Yung W K, et al., Clin Cancer Res., 1996, 2(12):1931-1935; and SJ, Levin V A, et al., Neuro Oncol., 2004, 6(3):253-258.) Embodiments of the present disclosure provide concomitant use of ATRA and/or related retinoids in combination with allogeneic tumor cell vaccines to improve immune response and efficacy by altering the tumor microenvironment. In some embodiments, ATRA is administered at a dose of 25-100 mg per square meter of body surface area per day. In various embodiments, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145 or 150 mg per square meter of body surface area per day is administered. In one embodiment, ATRA is administered orally and is optionally administered in accordance with the dosing frequency of other concomitant anti-tumor agents as described herein. In one embodiment, ATRA is administered twice in one day. PK studies of ATRA have demonstrated that the drug auto-catalyzes and serum levels decrease with continuous dosing. Thus, in certain embodiments, the ATRA dosing schedule includes one or two weeks on and one or two weeks off.

In one exemplary embodiment, in combination with allogeneic tumor cell vaccines described herein, ATRA is administered at doses of 25-100 mg per square meter per day in two divided doses for 7 continuous days, followed by 7 days without administration of ATRA, followed by the same cycle of 7 days on and 7 days off for as long as the vaccine therapy is being administered. In another embodiment, ATRA is administered at the same time as cyclophosphamide as described herein.

In some embodiments, ATRA is administered in combination with a vaccine composition as described herein for the treatment of cancer including, but not limited to, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), prostate cancer, glioblastoma, colorectal cancer, breast cancer including triple negative breast cancer (TNBC), bladder or urinary tract cancer, squamous cell head and neck cancer (SCCHN), liver hepatocellular (HCC) cancer, kidney or renal cell carcinoma (RCC) cancer, gastric or stomach cancer, ovarian cancer, esophageal cancer, testicular cancer, pancreatic cancer, central nervous system cancers, endometrial cancer, melanoma, and mesothelium cancer.

Checkpoint Inhibitors

In certain embodiments, a checkpoint inhibitor molecule is administered in combination with the vaccine compositions described herein. Immune checkpoints refer to a variety of inhibitory pathways of the immune system that are crucial for maintaining self-tolerance and for modulating the duration and amplitude of an immune responses. Tumors use certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumor antigens. (See Pardoll, 2012 Nature 12:252; Chen and Mellman Immunity 39:1 (2013)). Immune checkpoint inhibitors include any agent that blocks or inhibits in a statistically significant manner, the inhibitory pathways of the immune system. Such inhibitors may include antibodies, or antigen binding fragments thereof, that bind to and block or inhibit immune checkpoint receptors or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Illustrative immune checkpoint molecules that may be targeted for blocking or inhibition include, but are not limited to, CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, BTLA, SIGLEC9, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, γδ, and memory CD8+ (αβ) T cells), CD160 (also referred to as BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies, or antigen binding fragments thereof, or other binding proteins, that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, BTLA, SIGLEC9, 2B4, CD160, and CGEN-15049.

Illustrative immune checkpoint inhibitors include anti-PD1, anti-PDL1, and anti-PDL2 agents such as A167, AB122, ABBV-181, ADG-104, AK-103, AK-105, AK-106, AGEN2034, AM0001, AMG-404, ANB-030, APL-502, APL-501, zimberelimab, atezolizumab, AVA-040, AVA-040-100, avelumab, balstilimab, BAT-1306, BCD-135, BGB-A333, BI-754091, budigalimab, camrelizumab, CB-201, CBT-502, CCX-4503, cemiplimab, cosibelimab, cetrelimab, CS-1001, CS-1003, CX-072, CX-188, dostarlimab, durvalumab, envafolimab, sugemalimab, HBM9167, F-520, FAZ-053, genolimzumab, GLS-010, GS-4224, hAB21, HLX-10, HLX-20, HS-636, HX-008, IMC-001, IMM-25, INCB-86550, JS-003, JTX-4014, JYO-34, KL-A167, LBL-006, Iodapolimab, LP-002, LVGN-3616, LYN-00102, LMZ-009, MAX-10181, MEDI-0680, MGA-012 (Retifanlimab), MSB-2311, nivolumab, pembrolizumab, prolgolimab, prololimab, sansalimab, SCT-110A, SG-001, SHR-1316, sintilimab, spartalizumab, RG6084, RG6139, RG6279, CA-170, CA-327, STI-3031, toleracyte, toca 521, Sym-021, TG-1501, tislelizumab, toripalimab, TT-01, ZKAB-001, and the anti-PD-1 antibodies capable of blocking interaction with its ligands PD-L1 and PD-L2 described in WO/2017/124050.

Illustrative multi-specific immune checkpoint inhibitors, where at least one target is anti-PD1, anti-PDL1, or anti-PDL2, include ABP-160 (CD47×PD-L1), AK-104 (PD-1×CTLA-4), AK-112 (PD-1×VEGF), ALPN-202 (PD-L1×CTLA-4×CD28), AP-201 (PD-L1×OX-40), AP-505 (PD-L1×VEGF), AVA-0017 (PD-L1×LAG-3), AVA-0021 (PD-L1×LAG-3), AUPM-170 (PD-L1×VISTA), BCD-217 (PD-1×CTLA-4), BH-2950 (PD-1×HER2), BH-2996h (PD-1×PD-L1), BH-29xx (PD-L1×CD47), bintrafusp alfa (PD-L1×TGFβ), CB-213 (PD-1×LAG-3), CDX-527 (CD27×PD-L1), CS-4100 (PD-1×PD-L1), DB-001 (PD-L1×HER2), DB-002 (PD-L1×CTLA-4), DSP-105 (PD-1×4-1BBL), DSP-106, (PD-1×CD70), FS-118 (LAG-3×PD-L1), FS-222 (CD137/4-1BB×PD-L1), GEN-1046 (PD-L1×CD137/4-1BB), IBI-318 (PD-1×PD-L1), IBI-322 (PD-L1×CD-47), KD-033 (PD-L1×IL-15), KN-046 (PD-L1×CTLA-4), KY-1043 (PD-L1×IL-2), LY-3434172 (PD-1×PD-L1), MCLA-145 (PD-L1×CD137), MEDI-5752 (PD-1×CTLA-4), MGD-013 (PD-1×LAG-3), MGD-019 (PD-1×CTLA-4), ND-021 (PD-L1×4-1BB×HSA), OSE-279 (PD-1×PD-L1), PRS-332 (PD-1×HER2), PRS-344 (PD-L1×CD137), PSB-205 (PD-1×CTLA-4), R-7015 (PD-L1×TGFβ), RO-7121661 (PD-1×TIM-3), RO-7247669 (PD-1×LAG-3), SHR-1701 (PD-L1×TGFβ2), SL-279252 (PD-1×OX40L), TSR-075 (PD-1×LAG-3), XmAb-20717 (CTLA-4×PD-1), XmAb-23104 (PD-1×ICOS), and Y-111 (PD-L1×CD-3).

Additional illustrative immune checkpoint inhibitors include anti-CTLA4 agents such as: ADG-116, AGEN-2041, BA-3071, BCD-145, BJ-003, BMS-986218, BMS-986249, BPI-002, CBT-509, CG-0161, Olipass-1, HBM-4003, HLX-09, IBI-310, ipilimumab, JS-007, KN-044, MK-1308, ONC-392, REGN-4659, RP-2, tremelimumab, and zalifrelimab. Additional illustrative multi-specific immune checkpoint inhibitors, where at least one target is anti-CTLA4, include: AK-104 (PD-1×CTLA-4), ALPN-202 (PD-L1×CTLA-4×CD28), ATOR-1015 (CTLA-4×OX40), ATOR-1144 (CTLA-4×GITR), BCD-217 (PD-1×CTLA-4), DB-002 (PD-L1×CTLA-4), FPT-155 (CD28×CTLA-4), KN-046 (PD-L1×CTLA-4),), MEDI-5752 (PD-1×CTLA-4), MGD-019 (PD-1×CTLA-4), PSB-205 (PD-1×CTLA-4), XmAb-20717 (CTLA-4×PD-1), and XmAb-22841 (CTLA-4×LAG-3). Additional illustrative immune checkpoint inhibitors include anti-LAG3 agents such as BI-754111, BJ-007, eftilagimod alfa, GSK-2831781, HLX-26, IBI-110, IMP-701, IMP-761, INCAGN-2385, LBL-007, MK-4280, REGN-3767, relatlimab, Sym-022, TJ-A3, and TSR-033. Additional illustrative multi-specific immune checkpoint inhibitors, where at least one target is anti-LAG3, include: CB-213 (PD-1×LAG-3), FS-118 (LAG-3×PD-L1), MGD-013 (PD-1×LAG-3), AVA-0017 (PD-L1×LAG-3), AVA-0021 (PD-L1×LAG-3), RO-7247669 (PD-1×LAG-3), TSR-075 (PD-1×LAG-3), and XmAb-22841 (CTLA-4×LAG-3). Additional illustrative immune checkpoint inhibitors include anti-TIGIT agents such as AB-154, ASP8374, BGB-A1217, BMS-986207, CASC-674, COM-902, EOS-884448, HLX-53, IBI-939, JS-006, MK-7684, NB-6253, RXI-804, tiragolumab, and YH-29143. Additional illustrative multi-specific immune checkpoint inhibitors, where at least one target is anti-TIGIT are contemplated. Additional illustrative immune checkpoint inhibitors include anti-TIM3 agents such as: BGB-A425, BMS-986258, ES-001, HLX-52, INCAGN-2390, LBL-003, LY-3321367, MBG-453, SHR-1702, Sym-023, and TSR-022. Additional illustrative multi-specific immune checkpoint inhibitors, where at least one target is anti-TIM3, include: AUPM-327 (PD-L1×TIM-3), and RO-7121661 (PD-1×TIM-3). Additional illustrative immune checkpoint inhibitors include anti-VISTA agents such as: HMBD-002, and PMC-309. Additional illustrative multi-specific immune checkpoint inhibitors, where at least one target is anti-VISTA, include CA-170 (PD-L1×VISTA). Additional illustrative immune checkpoint inhibitors include anti-BTLA agents such as: JS-004. Additional illustrative multi-specific immune checkpoint inhibitors, where at least one target is anti-BTLA are contemplated. Illustrative stimulatory immune checkpoints include anti-OX40 agents such as ABBV-368, GSK-3174998, HLX-51, IBI-101, INBRX-106, INCAGN-1949, INV-531, JNJ-6892, and KHK-4083. Additional illustrative multi-specific stimulatory immune checkpoints, where at least one target is anti-OX40, include AP-201 (PD-L1×OX-40), APVO-603 (CD138/4-1BB×OX-40), ATOR-1015 (CTLA-4×OX-40), and FS-120 (OX40×CD137/4-1BB). Additional illustrative stimulatory immune checkpoints include anti-GITR agents such as BMS-986256, CK-302, GWN-323, INCAGN-1876, MK-4166, PTZ-522, and TRX-518. Additional illustrative multi-specific stimulatory immune checkpoints, where at least one target is anti-GITR, include ATOR-1144 (CTLA-4×GITR). Additional illustrative stimulatory immune checkpoints include anti-CD137/4-1BB agents such a: ADG-106, AGEN-2373, AP-116, ATOR-1017, BCY-3814, CTX-471, EU-101, LB-001, LVGN-6051, RTX-4-1BBL, SCB-333, urelumab, utomilumab, and WTiNT. Additional illustrative multi-specific stimulatory immune checkpoints, where at least one target is anti-CD137/4-1BB, include ALG.APV-527 (CD137/4-1BB×5T4), APVO-603 (CD137/4-1BB×OX40), BT-7480 (Nectin-4×CD137/4-1BB), CB-307 (CD137/4-1BB×PSMA), CUE-201 (CD80×CD137/4-1BB), DSP-105 (PD-1×CD137/4-1BB), FS-120 (Ox40×CD137/4-1BB), FS-222 (PD-L1×CD137/4-1BB), GEN-1042 (CD40×CD137/4-1BB), GEN-1046 (PD-L1×CD137/4-1BB), INBRX-105 (PD-L1×CD137/4-1BB), MCLA-145 (PD-L1×CD137/4-1BB), MP-0310 (CD137/4-1BB×FAP), ND-021 (PD-L1×CD137/4-1BB×HSA), PRS-343 (CD137/4-1BB×HER2), PRS-342 (CD137/4-1BB×GPC3), PRS-344 (CD137/4-1BB×PD-L1), RG-7827 (FAP×4-1BBL), and RO-7227166 (CD-19×4-1BBL).

Additional illustrative stimulatory immune checkpoints include anti-ICOS agents such as BMS-986226, GSK-3359609, KY-1044, and vopratelimab. Additional illustrative multi-specific stimulatory immune checkpoints, where at least one target is anti-ICOS, include XmAb-23104 (PD-1×ICOS). Additional illustrative stimulatory immune checkpoints include anti-CD127 agents such as MD-707 and OSE-703. Additional illustrative multi-specific stimulatory immune checkpoints, where at least one target is anti-CD127 are contemplated. Additional illustrative stimulatory immune checkpoints include anti-CD40 agents such as ABBV-428, ABBV-927, APG-1233, APX-005M, BI-655064, bleselumab, CD-40GEX, CDX-1140, LVGN-7408, MEDI-5083, mitazalimab, and selicrelumab. Additional Illustrative multi-specific stimulatory immune checkpoints, where at least one target is anti-CD40, include GEN-1042 (CD40×CD137/4-1BB). Additional illustrative stimulatory immune checkpoints include anti-CD28 agents such as FR-104 and theralizumab. Additional illustrative multi-specific stimulatory immune checkpoints, where at least one target is anti-CD28, include ALPN-101 (CD28×ICOS), ALPN-202 (PD-L1×CD28), CUE-201 (CD80×CD137/4-1BB), FPT-155 (CD28×CTLA-4), and REGN-5678 (PSMA×CD28). Additional illustrative stimulatory immune checkpoints include anti-CD27 agents such as: HLX-59 and varlilumab. Additional illustrative multi-specific stimulatory immune checkpoints, where at least one target is anti-CD27, include DSP-160 (PD-L1×CD27/CD70) and CDX-256 (PD-L1×CD27). Additional illustrative stimulatory immune checkpoints include anti-IL-2 agents such as ALKS-4230, BNT-151, CUE-103, NL-201, and THOR-707. Additional illustrative multi-specific stimulatory immune checkpoints, where at least one target is anti-IL-2, include CUE-102 (IL-2×WT1). Additional illustrative stimulatory immune checkpoints include anti-IL-7 agents such as BNT-152. Additional illustrative multi-specific stimulatory immune checkpoints, where at least one target is anti-IL-7 are contemplated. Additional illustrative stimulatory immune checkpoints include anti-IL-12 agents such as AK-101, M-9241, and ustekinumab. Additional illustrative multi-specific stimulatory immune checkpoints, where at least one target is antilL-12 are contemplated.

As described herein, the present disclosure provides methods of administering vaccine compositions, cyclophosphamide, checkpoint inhibitors, retinoids (e.g., ATRA), and/or other therapeutic agents such as Treg inhibitors. Treg inhibitors are known in the art and include, for example, bempegaldesleukin, fludarabine, gemcitabine, mitoxantrone, Cyclosporine A, tacrolimus, paclitaxel, imatinib, dasatinib, bevacizumab, idelalisib, anti-CD25, anti-folate receptor 4, anti-CTLA4, anti-GITR, anti-OX40, anti-CCR4, anti-CCR5, anti-CCR8, or TLR8 ligands.

Dosing

A “dose” or “unit dose” as used herein refers to one or more vaccine compositions that comprise therapeutically effective amounts of one more cell lines. A dose can be a single vaccine composition, two separate vaccine compositions, or two separate vaccine compositions plus one or more compositions comprising one or more therapeutic agents described herein. When in separate compositions, the two or more compositions of the “dose” are meant to be administered “concurrently”. In some embodiments, the two or more compositions are administered at different sites on the subject (e.g., arm, thigh, or back). As used herein, “concurrent” administration of two compositions or therapeutic agents indicates that within about 30 minutes of administration of a first composition or therapeutic agent, the second composition or therapeutic agent is administered. In cases where more than two compositions and/or therapeutic agents are administered concurrently, each composition or agent is administered within 30 minutes, wherein timing of such administration begins with the administration of the first composition or agent and ends with the beginning of administration of the last composition or agent. In some cases, concurrent administration can be completed (i.e., administration of the last composition or agent begins) within about 30 minutes, or within 15 minutes, or within 10 minutes, or within 5 minutes of start of administration of first composition or agent. Administration of a second (or multiple) therapeutic agents or compositions “prior to” or “subsequent to” administration of a first composition means that the administration of the first composition and another therapeutic agent is separated by at least 30 minutes, e.g., at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, or at least 48 hours.

The amount (e.g., number) of cells from the various individual cell lines in the vaccine compositions can be equal (as defined herein), approximately (as defined herein) equal, or different. In various embodiments, each cell line of a vaccine composition is present in an approximately equal amount. In other embodiments, 2 or 3 cell lines of one vaccine composition are present in approximately equal amounts and 2 or 3 different cell lines of a second composition are present in approximately equal amounts.

In some embodiments, the number of cells from each cell line (in the case where multiple cell lines are administered), is approximately 5.0×10⁵, 1.0×10⁶, 2.0×10⁶, 3.0×10⁶, 4.0×10⁶, 5.0×10⁶, 6.0×10⁶, 7.0×10⁶, 8×10⁶, 9.0×10⁶, 1.0×10⁷, 2.0×10⁷, 3.0×10⁷, 4.0×10⁷, 5.0×10⁷, 6.0×10⁷, 8.0×10⁷, 9.0×10⁷, 1.0×10⁸, 2.0×10⁸, 3.0×10⁸, 4.0×10⁸ or 5.0×10⁸ cells. In one embodiment, approximately 10 million (e.g., 1.0×10⁷) cells from one cell line are contemplated. In another embodiment, where 6 separate cell lines are administered, approximately 10 million cells from each cell line, or 60 million (e.g., 6.0×10⁷) total cells are contemplated.

The total number of cells administered in a vaccine composition, e.g., per administration site, can range from 1.0×10⁶ to 3.0×10⁸. For example, in some embodiments, 2.0×10⁶, 3.0×10⁶, 4.0×10⁶, 5.0×10⁶, 6.0×10⁶, 7.0×10⁶, 8×10⁶, 9.0×10⁶, 1.0×10⁷, 2.0×10⁷, 3.0×10⁷, 4.0×10⁷, 5.0×10⁷, 6.0×10⁷, 8.0×10⁷, 9.0×10⁷, 1.0×10⁸, 2.0×10⁸, or 3.0×10⁸ cells are administered.

As described herein, the number of cell lines contained with each administration of a cocktail or vaccine composition can range from 1 to 10 cell lines. In some embodiments, the number of cells from each cell line are not equal, and different ratios of cell lines are included in the cocktail or vaccine composition. For example, if one cocktail contains 5.0×10⁷ total cells from 3 different cell lines, there could be 3.33×10⁷ cells of one cell line and 8.33×10⁶ of the remaining 2 cell lines.

The vaccine compositions and compositions comprising additional therapeutic agents (e.g., chemotherapeutic agents, checkpoint inhibitors, and the like) may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial and sublingual injection or infusion techniques. Also envisioned are embodiments where the vaccine compositions and compositions comprising additional therapeutic agents (e.g., chemotherapeutic agents, checkpoint inhibitors, and the like) are administered intranodally or intratumorally.

In some embodiments, the vaccine compositions are administered intradermally. In related embodiments, the intradermal injection involves injecting the cocktail or vaccine composition at an angle of administration of 5 to 15 degrees.

The injections (e.g., intradermal or subcutaneous injections), can be provided at a single site (e.g. arm, thigh or back), or at multiple sites (e.g. arms and thighs). In some embodiments, the vaccine composition is administered concurrently at two sites, where each site receives a vaccine composition comprising a different composition (e.g., cocktail). For example, in some embodiments, the subject receives a composition comprising three cell lines in the arm, and three different, or partially overlapping cell lines in the thigh. In some embodiments, the subject receives a composition comprising one or more cell lines concurrently in each arm and in each thigh.

In some embodiments, the subject receives multiple doses of the cocktail or vaccine composition and the doses are administered at different sites on the subject to avoid potential antigen competition at certain (e.g., draining) lymph nodes. In some embodiments, the multiple doses are administered by alternating administration sites (e.g., left arm and right arm, or left thigh and right thigh) on the subject between doses. In some embodiments, the multiple doses are administered as follows: a first dose is administered in one arm, and second dose is administered in the other arm; subsequent doses, if administered, continue to alternate in this manner. In some embodiments, the multiple doses are administered as follows: a first dose is administered in one thigh, and second dose is administered in the other thigh; subsequent doses, if administered, continue to alternate in this manner. In some embodiments, the multiple doses are administered as follows: a first dose is administered in one thigh, and second dose is administered in one arm; subsequent doses if administered can alternate in any combination that is safe and efficacious for the subject. In some embodiments, the multiple doses are administered as follows: a first dose is administered in one thigh and one arm, and second dose is administered in the other arm and the other thigh; subsequent doses if administered can alternate in any combination that is safe and efficacious for the subject.

In some embodiments, the subject receives, via intradermal injection, a vaccine composition comprising a total of six cell lines (e.g., NCI-H460, NCI-H520, DMS 53, LK-2, NCI-H23, and A549 or other 6-cell line combinations described herein) in one, two or more separate cocktails, each cocktail comprising one or a mixture two or more of the 6-cell lines. In some embodiments, the subject receives, via intradermal injection, a vaccine composition comprising a mixture of three cell lines (e.g., three of NCI-H460, NCI-H520, DMS 53, LK-2, NCI-H23, and A549 or three cell lines from other 6-cell line combinations described herein). In some embodiments, the subject receives, via intradermal injection to the arm (e.g., upper arm), a vaccine composition comprising a mixture of three cell lines, comprising NCI-H460, NCI-H520, and A549; and the subject concurrently receives, via intradermal injection to the leg (e.g., thigh), a vaccine composition comprising a mixture of three cell lines, comprising DMS 53, LK-2, and NCI-H23.

Where an additional therapeutic agent is administered, the doses or multiple doses may be administered via the same or different route as the vaccine composition(s). By way of example, a composition comprising a checkpoint inhibitor is administered in some embodiments via intravenous injection, and the vaccine composition is administered via intradermal injection. In some embodiments, cyclophosphamide is administered orally, and the vaccine composition is administered intradermally. In other embodiments, ATRA is administered orally, and the vaccine composition is administered intradermally.

Regimens

The vaccine compositions according to the disclosure may be administered at various administration sites on a subject, at various times, and in various amounts. The efficacy of a tumor cell vaccine may be impacted if the subject's immune system is in a state that is amenable to the activation of antitumor immune responses. For example, the vaccine efficacy may be impacted if the subject is undergoing or has received radiation therapy, chemotherapy or other prior treatments. In some embodiments, therapeutic efficacy will require inhibition of immunosuppressive elements of the immune system and fully functional activation and effector elements. In addition to the immunosuppressive factors described herein, other elements that suppress antitumor immunity include, but are not limited to, T regulatory cells (Tregs) and checkpoint molecules such as CTLA-4, PD-1 and PD-L1.

In some embodiments, timing of the administration of the vaccine relative to previous chemotherapy and radiation therapy cycles is set in order to maximize the immune permissive state of the subject's immune system prior to vaccine administration. The present disclosure provides methods for conditioning the immune system with one or low dose administrations of a chemotherapeutic agent such as cyclophosphamide prior to vaccination to increase efficacy of whole cell tumor vaccines. In some embodiments, metronomic chemotherapy (e.g., frequent, low dose administration of chemotherapy drugs with no prolonged drug-free break) is used to condition the immune system. In some embodiments, metronomic chemotherapy allows for a low level of the drug to persist in the blood, without the complications of toxicity and side effects often seen at higher doses. By way of example, administering cyclophosphamide to condition the immune system includes, in some embodiments, administration of the drug at a time before the receipt of a vaccine dose (e.g., 15 days to 1 hour prior to administration of a vaccine composition) in order to maintain the ratio of effector T cells to regulatory T cells at a level less than 1.

In some embodiments, a chemotherapy regimen (e.g., myeloablative chemotherapy, cyclophosphamide, and/or fludarabine regimen) may be administered before some, or all of the administrations of the vaccine composition(s) provided herein. Cyclophosphamide (CYTOXAN™, NEOSAR™) is a well-known cancer medication that interferes with the growth and spread of cancer cells in the body. Cyclophosphamide may be administered as a pill (oral), liquid, or via intravenous injection. Numerous studies have shown that cyclophosphamide can enhance the efficacy of vaccines. (See, e.g., Machiels et al., Cancer Res., 61:3689, 2001; Greten, T. F., et al., J. Immunother., 2010, 33:211; Ghiringhelli et al., Cancer Immunol. Immunother., 56:641, 2007; Ge et al., Cancer Immunol. Immunother., 61:353, 2011; Laheru et al., Clin. Cancer Res., 14:1455, 2008; and Borch et al., Oncolmmunol, e1207842, 2016). “Low dose” cyclophosphamide as described herein, in some embodiments, is effective in depleting Tregs, attenuating Treg activity, and enhancing effector T cell functions. In some embodiments, intravenous low dose administration of cyclophosphamide includes 40-50 mg/kg in divided doses over 2-5 days. Other low dose regimens include 1-15 mg/kg every 7-10 days or 3-5 mg/kg twice weekly. Low dose oral administration, in accordance with some embodiments of the present disclosure, includes 1-5 mg/kg per day for both initial and maintenance dosing. Dosage forms for the oral tablet are 25 mg and 50 mg. In some embodiments, cyclophosphamide is administered as an oral 50 mg tablet for the 7 days leading up to the first and optionally each subsequent doses of the vaccine compositions described herein.

In some embodiments, cyclophosphamide is administered as an oral 50 mg tablet on each of the 7 days leading up to the first, and optionally on each of the 7 days preceding each subsequent dose(s) of the vaccine compositions. In another embodiment, the patient takes or receives an oral dose of 25 mg of cyclophosphamide twice daily, with one dose being the morning upon rising and the second dose being at night before bed, 7 days prior to each administration of a cancer vaccine cocktail or unit dose. In certain embodiments, the vaccine compositions are administered intradermally multiple times over a period of years. In some embodiments, a checkpoint inhibitor is administered every two weeks or every three weeks following administration of the vaccine composition(s).

In another embodiment, the patient receives a single intravenous dose of cyclophosphamide of 200, 250, 300, 500 or 600 mg/m² at least one day prior to the administration of a cancer vaccine cocktail or unit dose of the vaccine composition. In another embodiment, the patient receives an intravenous dose of cyclophosphamide of 200, 250, 300, 500 or 600 mg/m² at least one day prior to the administration vaccine dose number 4, 8, 12 of a cancer vaccine cocktail or unit dose. In another embodiment, the patient receives a single dose of cyclophosphamide at 1000 mg/kg as an intravenous injection at least one hour prior to the administration of a cancer vaccine cocktail or unit dose. In some embodiments, an oral high dose of 200 mg/kg or an IV high dose of 500-1000 mg/m² of cyclophosphamide is administered.

The administration of cyclophosphamide can be via any of the following: oral (e.g., as a capsule, powder for solution, or a tablet); intravenous (e.g., administered through a vein (IV) by injection or infusion); intramuscular (e.g., via an injection into a muscle (IM)); intraperitoneal (e.g., via an injection into the abdominal lining (IP)); and intrapleural (e.g., via an injection into the lining of the lung).

In some embodiments, immunotherapy checkpoint inhibitors (e.g., anti-CTLA4, anti-PD-1 antibodies such as pembrolizumab, and nivolumab, anti-PDL1 such as durvalumab) may be administered before, concurrently, or after the vaccine composition. In certain embodiments, pembrolizumab is administered 2 mg/kg every 3 weeks as an intravenous infusion over 60 minutes. In some embodiments, pembrolizumab is administered 200 mg every 3 weeks as an intravenous infusion over 30 minutes. In some embodiments pembrolizumab is administered 400 mg every 6 weeks as an intravenous infusion over 30 minutes. In some embodiments, durvalumab is administered 10 mg/kg every two weeks. In some embodiments, nivolumab is administered 240 mg every 2 weeks (or 480 mg every 4 weeks). In some embodiments, nivolumab is administered 1 mg/kg followed by ipilimumab on the same day, every 3 weeks for 4 doses, then 240 mg every 2 weeks (or 480 mg every 4 weeks). In some embodiments, nivolumab is administered 3 mg/kg followed by ipilimumab 1 mg/kg on the same day every 3 weeks for 4 doses, then 240 mg every 2 weeks (or 480 mg every 4 weeks). In some embodiments, nivolumab is administered or 3 mg/kg every 2 weeks.

In some embodiments, durvalumab or pembrolizumab is administered every 2, 3, 4, 5, 6, 7 or 8 weeks for up to 8 administrations and then reduced to every 6, 7, 8, 9, 10, 11 or 12 weeks as appropriate.

In other embodiments, the present disclosure provides that PD-1 and PD-L1 inhibitors are administered with a fixed dosing regimen (i.e., not weight-based). In non-limiting examples, a PD-1 inhibitor is administered weekly or at weeks 2, 3, 4, 6 and 8 in an amount between 100-1200 mg. In non-limiting examples, a PD-L1 inhibitor is administered weekly or at weeks 2, 3, 4, 6 and 8 in an mount between 250-2000 mg.

In some embodiments, a vaccine composition or compositions as described herein is administered concurrently or in combination with a PD-1 inhibitor dosed either Q1W, Q2W, Q3W, Q4W, Q6W, or Q8W, between 100 mg and 1500 mg fixed or 0.5 mg/kg and 15 mg/kg based on weight. In another embodiment, a vaccine composition or compositions as described herein is administered concurrently in combination with PD-L1 inhibitor dosed either Q2W, Q3W, or Q4W between 250 mg and 2000 mg fixed or 2 mg/kg and 30 mg/kg based on weight. In other embodiments, the aforementioned regimen is administered but the compositions are administered in short succession or series such that the patient receives the vaccine composition or compositions and the checkpoint inhibitor during the same visit.

The plant Cannabis sativa L. has been used as an herbal remedy for centuries and is an important source of phytocannabinoids. The endocannabinoid system (ECS) consists of receptors, endogenous ligands (endocannabinoids) and metabolizing enzymes, and plays a role in different physiological and pathological processes. Phytocannabinoids and synthetic cannabinoids can interact with the components of ECS or other cellular pathways and thus may affect the development or progression of diseases, including cancer. In cancer patients, cannabinoids can be used as a part of palliative care to alleviate pain, relieve nausea and stimulate appetite. In addition, numerous cell culture and animal studies have demonstrated antitumor effects of cannabinoids in various cancer types. (For a review, see Daris, B., et al., Bosn. J. Basic. Med. Sci., 19(1):14-23 (2019).) Phytocannabinoids are a group of C21 terpenophenolic compounds predominately produced by the plants from the genus Cannabis. There are several different cannabinoids and related breakdown products. Among these are tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), cannabichromene (CBC), Δ8-THC, cannabidiolic acid (CBDA), cannabidivarin (CBDV), and cannabigerol (CBG).

In certain embodiments of the present disclosure, use of all phytocannabinoids is stopped prior to or concurrent with the administration of a Treg cell inhibitor such as cyclophosphamide, and/or is otherwise stopped prior to or concurrent with the administration of a vaccine composition according to the present disclosure. In some embodiments, where multiple administrations of cyclophosphamide or vaccine compositions occur, the cessation optionally occurs prior to or concurrent with each administration. In certain embodiments, use of phytocannabinoids is not resumed until a period of time after the administration of the vaccine composition(s). For example, abstaining from cannabinoid administration for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days prior to administration and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after administration of cyclophosphamide or a vaccine dose is contemplated.

In some embodiments, patients will receive the first dose of the vaccine within 6-12 weeks after completion of chemotherapy. High dose chemotherapy used in cancer treatment ablates proliferating cells and depletes immune cell subsets. Upon completion of chemotherapy, the immune system will begin to reconstitute. The time span for T cells to recur is roughly 2-3 weeks. Because T cells are an immunological cell subset targeted for activation, in some embodiments, the cancer vaccine is administered within a window where there are sufficient T cells to prime, yet the subject remains lymphopenic. This environment, in which there are less cells occupying the niche will allow the primed T cells to rapidly divide, undergoing “homeostatic proliferation” in response to increased availability of cytokines (e.g., IL7 and IL15). Thus, by dosing the vaccine at this window, the potential efficacy of embodiments of the cancer vaccine platform as described herein is maximized to allow for the priming of antigen specific T cells and expansion of the vaccine associated T cell response.

Methods of Selecting Cell Lines and Preparing Vaccines

Cell Line Selection

For a given cancer or in instances where a patient is suffering from more than one cancer, a cell line or combination of cell lines is identified for inclusion in a vaccine composition based on several criteria. In some embodiments, selection of cell lines is performed stepwise as provided below. Not all cancer indications will require all of the selection steps and/or criteria.

Step 1. Cell lines for each indication are selected based on the availability of RNA-seq data such as for example in the Cancer Cell Line Encyclopedia (CCLE) database. RNA-seq data allows for the identification of candidate cell lines that have the potential to display the greatest breadth of antigens specific to a cancer indication of interest and informs on the potential expression of immunosuppressive factors by the cell lines. If the availability of RNA-seq data in the CCLE is limited, RNA-seq data may be sourced from the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) database or other sources known in the art. In some embodiments, potential expression of a protein of interest (e.g., a TAA) based on RNA-seq data is considered “positive” when the RNA-seq value is >0.

Step 2. For all indications, cell lines derived from metastatic sites are prioritized to diversify antigenic breadth and to more effectively target later-stage disease in patients with metastases. Cell lines derived from primary tumors are included in some embodiments to further diversify breadth of the vaccine composition. The location of the metastases from which the cell line are derived is also considered in some embodiments. For example, in some embodiments, cell lines can be selected that are derived from lymph node, ascites, and liver metastatic sites instead of all three cell lines derived from liver metastatic sites.

Step 3. Cell lines are selected to cover a broad range of classifications of cancer types. For example, tubular adenocarcinoma is a commonly diagnosed classification of gastric cancer. Thus, numerous cell lines may be chosen matching this classification. For indications where primary tumor sites vary, cell lines can be selected to meet this diversity. For example, for small cell carcinoma of the head and neck (SCCHN), cell lines were chosen, in some embodiments, to cover tumors originating from the oral cavity, buccal mucosa, and tongue. These selection criteria enable targeting a heterogeneous population of patient tumor types. In some embodiments, cell lines are selected to encompass an ethnically diverse population to generate a cell line candidate pool derived from diverse histological and ethnical backgrounds.

Step 4. In some embodiments, cell lines are selected based on additional factors. For example, in metastatic colorectal cancer (mCRC), cell lines reported as both microsatellite instable high (MSI-H) and microsatellite stable (MSS) may be included. As another example, for indications that are viral driven, cell lines encoding viral genomes may be excluded for safety and/or manufacturing complexity concerns.

Step 5. In some embodiments, cell lines are selected to cover a varying degree of genetic complexity in driver mutations or indication-associated mutations. Heterogeneity of cell line mutations can expand the antigen repertoire to target a larger population within patients with one or more tumor types. By way of example, breast cancer cell lines can be diversified on deletion status of Her2, progesterone receptor, and estrogen receptor such that the final unit dose includes triple negative, double negative, single negative, and wild type combinations. Each cancer type has a complex genomic landscape and, as a result, cell lines are selected for similar gene mutations for specific indications. For example, melanoma tumors most frequently harbor alterations in BRAF, CDKN2A, NRAS and TP53, therefore selected melanoma cell lines, in some embodiments, contain genetic alterations in one or more of these genes.

Step 6. In some embodiments, cell lines are further narrowed based on the TAA, TSA, and/or cancer/testis antigen expression based on RNA-seq data. An antigen or collection of antigens associated with a particular tumor or tumors is identified using search approaches evident to persons skilled in the art (See, e.g., such as www.ncbi.nlm.nih.gov/pubmed/, and clinicaltrials.gov). In some embodiments, antigens can be included if associated with a positive clinical outcome or identified as highly expressed by the specific tumor or tumor types while expressed at lower levels in normal tissues.

Step 7. After Steps 1 through 6 are completed, in some embodiments, the list of remaining cell line candidates are consolidated based on cell culture properties and considerations such as doubling time, adherence, size, and serum requirements. For example, cell lines with a doubling time of less than 80 hours or cell lines requiring media serum (FBS, FCS)<10% can be selected. In some embodiments, adherent or suspension cell lines that do not form aggregates can be selected to ensure proper cell count and viability.

Step 8. In some embodiments, cell lines are selected based on the expression of immunosuppressive factors (e.g., based on RNA-seq data sourced from CCLE or EMBL as described in Step 1).

In some embodiments, a biopsy of a patient's tumor and subsequent TAA expression profile of the biopsied sample will assist in the selection of cell lines. Embodiments of the present disclosure therefore provide a method of preparing a vaccine composition comprising the steps of determining the TAA expression profile of the subject's tumor; selecting cancer cell lines; modifying cancer cell lines; and irradiating cell lines prior to administration to prevent proliferation after administration to patients.

Preparing Vaccine Compositions

In certain embodiments, after expansion in manufacturing, all of the cells in a modified cell line are irradiated, suspended, and cryopreserved. In some embodiments, cells are irradiated 10,000 cGy. According to some embodiments, cells are irradiated at 7,000 to 15,000 cGy. According to some embodiments, cells are irradiated at 7,000 to 15,000 cGy.

In certain embodiments, each vial contains a volume of 120±10 μL (1.2×10⁷ cells). In some embodiments, the total volume injected per site is 300 μL or less. In some embodiments, the total volume injected per site is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 μL. Where, for example, the total volume injected is 300 μL, the present disclosure provides, in some embodiments that 3×100 μL volumes, or 2×150 μL, are injected, for a total of 300 μL.

In some embodiments, the vials of the component cell lines are stored in the liquid nitrogen vapor phase until ready for injection. In some embodiments, each of the component cell lines are packaged in separate vials.

As described herein, prior to administration, in some embodiments the contents of two vials are removed by needle and syringe and are injected into a third vial for mixing. In some embodiments, this mixing is repeated for each cocktail. In other embodiments, the contents of six vials are divided into two groups—A and B, where the contents of three vials are combined or mixed, optionally into a new vial (A), and the contents of the remaining three vials are combined or mixed, optionally into a new vial (B).

In certain embodiments, the cells will be irradiated prior to cryopreservation to prevent proliferation after administration to patients. In some embodiments, cells are irradiated at 7,000 to 15,000 cGy in order to render the cells proliferation incompetent.

In some embodiments, cell lines are grown separately and in the same growth culture media. In some embodiments, cell lines are grown separately and in different cell growth culture media.

Xeno-Free Conversion of Whole Tumor Cell Vaccine Component Cell Lines

Analysis of antibody responses in subjects treated with a whole tumor cell vaccine has suggested a negative correlation between survival and the development of IgG antibody responses to the bovine α-Gal antigen. (See Xia et al., Cell Chem Biol 23(12):1515-1525 (2016)). This is significant because most whole tumor cell vaccines are comprised of tumor cell lines that have been expanded and cryopreserved in media containing fetal bovine serum (FBS), which contains the bovine α-Gal antigen.

In some embodiments, to prevent the immune response to foreign antigens that are present in FBS, the cell lines disclosed herein are adapted to xeno-free media composed of growth factors and supplements essential for cell growth that are from human source, prior to large scale cGMP manufacturing.

By way of example and as described herein, cell line DMS 53 (e.g., DMS 53 which has been modified in vitro to (i) express GM-CSF (SEQ ID NO: 8), IL-12 (SEQ ID NO: 10), membrane-bound CD40L (SEQ ID NO: 3), TGFβ1 shRNA (SEQ ID NO: 54), TGFβ2 shRNA (SEQ ID NO: 55); and (ii) decrease expression of CD276 using a zinc-finger nuclease targeting CD276 (SEQ ID NO: 57) has been adapted to xeno-free media. In some embodiments, the expression of the surface protein mCD40L, GM-CSF, and/or IL-12 are each or independently expressed at levels equal to or greater than the expression levels observed when DMS 53 is cultured in FBS media (i.e., “baseline expression level”). In one embodiment, expression of the surface protein mCD40L and reduction of CD276 expression are comparable to pre-adapted cells. In another embodiment, cells secrete undetectable levels of TGFβ1 and TGFβ2 as determined by ELISA and as described in Example 4. In another embodiment, cells express approximately 77 ng/10⁶/24 hours of GM-CSF and 86 ng/10⁶/24 hours of IL-12.

In some embodiments, the transgene expression is approximately 1, 1.2, 1.5, 1.6, 2.0, 2.5, 3, 3.5, 4, 4.5, or 5-fold greater in the xeno-free media compared baseline expression level. In some embodiments, IL-12 is expressed at approximately 50, 60, 70, 80, 90, 100, or 150 ng/10⁶/24 hours. In some embodiments, GM-CSF is expressed at approximately 50, 60, 70, 80, 90, 100, or 150 ng/10⁶/24 hours.

In some embodiments, the doubling time of DMS 53 in xeno-free media is less than or equal to approximately 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 hours or more. In one embodiment, the doubling time of DMS 53 in xeno-free media is between approximately 75-125 hours, or between approximately 88 to 105 hours. In other embodiments, the doubling time of DMS 53 is less than approximately 250 hours or less than approximately 206 hours.

As described herein at, for example, Example 4, modified DMS 53 was observed to generate robust antigen specific IFNγ responses. In some embodiments, antigen specific IFNγ responses are maintained following adaptation to xeno-free media.

As used herein, the terms “adapting” and “converting” or “conversion” are used interchangeably to refer to transferring/changing cells to a different media as will be appreciated by those of skill in the art. The xeno-free media formulation chosen can be, in some embodiments, the same across all cell lines or, in other embodiments, can be different for different cell lines. In some embodiments, the media composition will not contain any non-human materials and can include human source proteins as a replacement for FBS alone, or a combination of human source proteins and human source recombinant cytokines and growth factors (e.g., EGF). Additionally, the xeno-free media compositions can, in some embodiments, also contain additional supplements (e.g., amino acids, energy sources) that enhance the growth of the tumor cell lines. The xeno-free media formulation will be selected for its ability to maintain cell line morphology and doubling time no greater than twice the doubling time in FBS and the ability to maintain expression of transgenes comparable to that in FBS.

A number of procedures may be instituted to minimize the possibility of inducing IgG, IgA, IgE, IgM and IgD antibodies to bovine antigens. These include but are not limited to: cell lines adapted to growth in xeno-free media; cell lines grown in FBS and placed in xeno-free media for a period of time (e.g., at least three days) prior to harvest; cell lines grown in FBS and washed in xeno-free media prior to harvest and cryopreservation; cell lines cryopreserved in media containing Buminate (a USP-grade pharmaceutical human serum albumin) as a substitute for FBS; and/or cell lines cryopreserved in a medial formulation that is xeno-free, and animal-component free (e.g., CryoStor). In some embodiments, implementation of one or more of these procedures may reduce the risk of inducing anti-bovine antibodies by removing the bovine antigens from the vaccine compositions.

According to one embodiment, the vaccine compositions described herein do not comprise non-human materials. In some embodiments, the cell lines described herein are formulated in xeno-free media. Use of xeno-free media avoids the use of immunodominant xenogeneic antigens and potential zoonotic organisms, such as the BSE prion. By way of example, following gene modification, the cell lines are transitioned to xeno-free media and are expanded to generate seed banks. The seed banks are cryopreserved and stored in vapor-phase in a liquid nitrogen cryogenic freezer.

In Vitro Assays

The ability of allogeneic whole cell cancer vaccines such as those described herein, to elicit anti-tumor immune responses, and to demonstrate that modifications to the vaccine cell lines enhance vaccine-associated immune responses, can be modelled with in vitro assays. Without being bound by any theory, the genetic modifications made to the vaccine cell line components augment adaptive immune responses through enhancing dendritic cell (DC) function in the vaccine microenvironment. The potential effects of expression of TAAs, immunosuppressive factors, and/or immunostimulatory factors can be modelled in vitro, for example, using flow cytometry-based assays and the IFNγ ELISpot assay.

In some embodiments, to model the effects of modifications to the vaccine cell line components in vitro, DCs are derived from monocytes isolated from healthy donor peripheral blood mononuclear cells (PBMCs) and used in downstream assays to characterize immune responses in the presence or absence of one or more immunostimulatory or immunosuppressive factors. The vaccine cell line components are phagocytized by donor-derived immature DCs during co-culture with the unmodified parental vaccine cell line (control) or the modified vaccine cell line components. The effect of modified vaccine cell line components on DC maturation, and thereby subsequent T cell priming, can be evaluated using flow cytometry to detect changes in markers of DC maturation such as CD40, CD83, CD86, and HLA-DR. Alternatively, the immature DCs are matured after co-culture with the vaccine cell line components, the mature DCs are magnetically separated from the vaccine cell line components, and then co-cultured with autologous CD14-PBMCs for 6 days to mimic in vivo presentation and stimulation of T cells. IFNγ production, a measurement of T cell stimulatory activity, is measured in the IFNγ ELISpot assay or the proliferation and characterization of immune cell subsets is evaluated by flow cytometry. In the IFNγ ELISpot assay, PBMCs are stimulated with autologous DCs loaded with the unmodified parental vaccine cell line components to assess potential responses against unmodified tumor cells in vivo.

The IFNγ ELISpot assay can be used to evaluate the potential of the allogenic vaccine to drive immune responses to clinically relevant TAAs expressed by the vaccine cell lines. To assess TAA-specific responses in the IFNγ ELISpot assay, following co-culture with DCs, the PBMCs are stimulated with peptide pools comprising known diverse MHC-I epitopes for TAAs of interest. In various embodiments, the vaccine composition may comprise 3 cell lines that induce IFNγ responses to at least 3, 4, 5, 6, 7, 8, 9, 10, or 11 non-viral antigens, or at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the antigens evaluated for an IFNγ response. In some embodiments, the vaccine composition may be a unit dose of 6 cell lines that induce IFNγ responses to at least 5, 6, 7, 8, 9, 10 or 11 non-viral antigens, or at least 60%, 70%, 80%, 90%, or 100% of the antigens evaluated for an IFNγ response.

In Vivo Mouse Models

Induction of antigen specific T cells by the allogenic whole cell vaccine can be modeled in vivo using mouse tumor challenge models. The vaccines provided in embodiments herein may not be administered directly to mouse tumor model due to the diverse xenogeneic homology of TAAs between mouse and human. However, a murine homolog of the vaccines can be generated using mouse tumor cell lines. Some examples of additional immune readouts in a mouse model are: characterization of humoral immune responses specific to the vaccine or TAAs, boosting of cellular immune responses with subsequent immunizations, characterization of DC trafficking and DC subsets at draining lymph nodes, evaluation of cellular and humoral memory responses, reduction of tumor burden, and determining vaccine-associated immunological changes in the TME, such as the ratio of tumor infiltrating lymphocytes (TILs) to Tregs. Standard immunological methods such as ELISA, IFNγ ELISpot, and flow cytometry will be used.

Kits

The vaccine compositions described herein may be used in the manufacture of a medicament, for example, a medicament for treating or prolonging the survival of a subject with cancer, e.g., lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), prostate cancer, glioblastoma, colorectal cancer, breast cancer including triple negative breast cancer (TNBC), bladder or urinary tract cancer, squamous cell head and neck cancer (SCCHN), liver hepatocellular (HCC) cancer, kidney or renal cell carcinoma (RCC) cancer, gastric or stomach cancer, ovarian cancer, esophageal cancer, testicular cancer, pancreatic cancer, central nervous system cancers, endometrial cancer, melanoma, and mesothelium cancer.

Also provided are kits for treating or prolonging the survival of a subject with cancer containing any of the vaccine compositions described herein, optionally along with a syringe, needle, and/or instructions for use. Articles of manufacture are also provided, which include at least one vessel or vial containing any of the vaccine compositions described herein and instructions for use to treat or prolong the survival of a subject with cancer. Any of the vaccine compositions described herein can be included in a kit comprising a container, pack, or dispenser together with instructions for administration.

In some embodiments, provided herein is a kit comprising at least two vials, each vial comprising a vaccine composition (e.g., cocktail A and cocktail B), wherein each vial comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more cell lines, wherein the cell lines are modified to inhibit or reduce production of one or more immunosuppressive factors, and/or express or increase expression of one or more immunostimulatory factors, and/or express a heterogeneity of tumor associated antigens, or neoantigens.

By way of example, a kit comprising 6 separate vials is provided, wherein each vial comprises one of the following cell lines: NCI-H460, NCI-H520, DMS 53, LK-2, NCI-H23, and A549. As another example, a kit comprising 6 separate vials is provided, wherein each vial comprises one of the following cell lines: DMS 53, DBTRG-05MG, LN-229, SF-126, GB-1, and KNS-60. As another example, a kit comprising 6 separate vials is provided, wherein each vial comprises one of the following cell lines: DMS53, PC3, NEC8, NTERA-2cl-D1, DU-145, and LNCAP. As another example, a kit comprising 6 separate vials is provided, wherein each vial comprises one of the following cell lines: DMS 53, HCT-15, HuTu80, LS411N, HCT-116 and RKO. As another example, a kit comprising 6 separate vials is provided, wherein each vial comprises one of the following cell lines: DMS 53, OVTOKO, MCAS, TOV-112D, TOV-21G, and ES-2. As another example, a kit comprising 6 separate vials is provided, wherein each vial comprises one of the following cell lines: DMS 53, HSC-4, HO-1-N-1, DETROIT 562, KON, and OSC-20. As another example, a kit comprising 6 separate vials is provided, wherein each vial comprises one of the following cell lines: DMS 53, J82, HT-1376, TCCSUP, SCaBER, and UM-UC-3. As another example, a kit comprising 6 separate vials is provided, wherein each vial comprises one of the following cell lines: DMS 53, MKN-1, MKN-45, MKN-74, OCUM-1, and Fu97. As another example, a kit comprising 6 separate vials is provided, wherein each vial comprises one of the following cell lines: DMS 53, AU565, CAMA-1, HS-578T, MCF-7, and T-47D. As another example, a kit comprising 6 separate vials is provided, wherein each vial comprises one of the following cell lines: DMS 53, PANC-1, KP-3, KP-4, SUIT-2, and PSN1.

In some embodiments, provided herein is a kit comprising at least two vials, each vial comprising a vaccine composition (e.g., cocktail A and cocktail B), wherein each vial comprises at least three cell lines, wherein the cell lines are modified to reduce production or expression of one or more immunosuppressive factors, and/or modified to increase expression of one or more immunostimulatory factors, and/or express a heterogeneity of tumor associated antigens, or neoantigens. The two vials in these embodiments together are a unit dose. Each unit dose can have from about 5×10⁶ to about 5×10⁷ cells per vial, e.g., from about 5×10⁶ to about 3×10⁷ cells per vial.

In some embodiments, provided herein is a kit comprising at least six vials, each vial comprising a vaccine composition, wherein each vaccine composition comprises one cell line, wherein the cell line is modified to inhibit or reduce production of one or more immunosuppressive factors, and/or modified to express or increase expression of one or more immunostimulatory factors, and/or expresses a heterogeneity of tumor associated antigens, or neoantigens. Each of the at least six vials in the embodiments provided herein can be a unit dose of the vaccine composition. Each unit dose can have from about 2×10⁶ to about 50×10⁶ cells per vial, e.g., from about 2×10⁶ to about 10×10⁶ cells per vial.

In some embodiments, provided herein is a kit comprising separate vials, each vial comprising a vaccine composition, wherein each vaccine composition comprises one cell line, wherein the cell line is modified to inhibit or reduce production of one or more immunosuppressive factors, and/or modified to express or increase expression of one or more immunostimulatory factors, and/or expresses, a heterogeneity of tumor associated antigens, or neoantigens. Each of the vials in the embodiments provided herein can be a unit dose of the vaccine composition. Each unit dose can have from about 2×10⁶ to about 50×10⁶ cells per vial, e.g., from about 2×10⁶ to about 10×10⁶ cells per vial.

In one exemplary embodiment, a kit is provide comprising two cocktails of 3 cell lines each (i.e., total of 6 cell lines in 2 different vaccine compositions) as follows: 8×10⁶ cells per cell line; 2.4×10⁷ cells per injection; and 4.8×10⁷ cells total dose. In another exemplary embodiment, 1×10⁷ cells per cell line; 3.0×10⁷ cells per injection; and 6.0×10⁷ cells total dose is provided. In some embodiments, a vial of any of the kits disclosed herein contains about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mL of a vaccine composition of the disclosure. In some embodiments, the concentration of cells in a vial is about 5×10⁷ cells/mL to about 5×10⁸/cells mL.

The kits as described herein can further comprise needles, syringes, and other accessories for administration.

Described herein and in the co-filed sequence listing are various polynucleotide and polypeptide sequences. If there are discrepancies, the sequences provided in the text control.

EXAMPLES

International patent application number PCT/US2020/062840 (Pub. No. WO/2021/113328) describes numerous methods and materials related to modified, whole cell cancer vaccines, which are incorporated by reference herein in their entirety. In some embodiments, the present disclosure including the following Examples provide additional and/or alternative cancer cell and cell line modifications.

Example 28 of PCT/US2020/062840 (Pub. No. WO/2021/113328) demonstrates that the reduction of TGFβ1, TGFβ2, and CD276 expression with concurrent overexpression of GM-CSF, CD40L, and IL-12 in of the NSCLC vaccine comprising two cocktails, each cocktail composed of three cell line components, a total of 6 component cell lines, significantly increases the antigenic breadth and magnitude of cellular immune responses compared to belagenpumatucel-L.

Cancer immunotherapy through induction of anti-tumor cellular immunity has become a promising approach targeting cancer. Many therapeutic cancer vaccine platforms are targeting tumor associated antigens (TAAs) that are overexpressed in tumor cells, however, a cancer vaccine using these antigens must be potent enough to break tolerance. The cancer vaccines described in various embodiments herein are designed with the capacity to elicit broad and robust cellular responses against tumors. Neoepitopes are non-self epitopes generated from somatic mutations arising during tumor growth. Tumor types with higher mutational burden are correlated with durable clinical benefit in response to checkpoint inhibitor therapies. Targeting neoepitopes has many advantages because these neoepitopes are truly tumor specific and not subject to central tolerance in the thymus. A cancer vaccine encoding full length TAAs with neoepitopes arising from nonsynonymous mutations (NSMs) has potential to elicit a more potent immune response with improved breadth and magnitude. Example 40 of PCT/US2020/062840 (Pub. No. WO/2021/113328) describes improving breadth and magnitude of vaccine-induced cellular immune responses by introducing non-synonymous mutations (NSM) into prioritized full-length tumor associated antigens (TAAs).

Example 1: Driver Mutation Identification and Design Process

Based on the number of alleles harboring a mutation and the fraction of tumor cells with the mutation, mutations can be classified as clonal (truncal mutations, present in all tumor cells sequenced) and subclonal (shared and private mutations, present in a subset of regions or cells within a single biopsy). Unlike the majority of neoepitopes that are private mutations and not found in more than one patient, driver mutations in known driver genes typically occur early in cancer evolution and are found in all or a subset of tumor cells across patients. Driver mutations show a tendency to be clonal and give a fitness advantage to the tumor cells that carry them and are crucial for the tumors transformation, growth and survival. In various embodiments, the present disclosure provides methods for selecting and targeting driver mutations as an effective strategy to overcome intra- and inter-tumor neoantigen heterogeneity and tumor escape. Inclusion of a pool of driver mutations that occur at high frequency in a vaccine can promote potent anti-tumor immune responses.

The following Example provides the process for identifying and selecting driver mutations for inclusion in a cancer vaccine according to the present disclosure. This process was followed for the Examples described herein.

Identification of Frequently Mutated Oncogenes for Each Indication

Oncogenes have the potential to initiate and maintain cancer phenotype and are often mutated in tumor cells. Missense driver mutations represent a greater fraction of the total mutations in oncogenes, and these driver mutations are implicated in oncogenesis by deregulating the control of normal cell proliferation, differentiation, and death, leading to growth advantage for the malignant clone.

To identify frequently mutated oncogenes for each indication, the dataset of “curated set of non-redundant studies” specific for each indication was first selected and explored from the publicly available database cBioPortal. Then a complete list of mutated genes was downloaded from the indication-specific dataset, and the cancer genes (oncogenes) were sorted out from the list and ranked by the percentage of samples with one or more mutations (mutation frequency). Any oncogenes with greater than 5% mutation frequency were selected for driver mutation identification and selection.

Identification of Driver Mutations in Selected Oncogenes

Once the oncogenes were selected, the non-redundant data set was queried with the HUGO Gene Nomenclature Committee gene symbol for the oncogene of interest. Missense mutations occurring in the target oncogene were downloaded and sorted by frequency of occurrence. Missense mutations occurring in the same amino acid position in 0.5% of profiled patient samples in each selected oncogene were included as driver mutations for further prioritization.

Prioritization and Selection of Identified Driver Mutations

Previous studies have shown that long peptide-based vaccine could potentially include MHC class I and II epitopes, thus eliciting robust cytotoxic and T helper cell responses. Therefore, a long peptide sequence containing a given driver mutation that is 28-35 amino acid in length was generated for CD4 and CD8 epitope analysis. A respective driver mutation was placed in the middle of a 28-35-mer peptide and flanked by roughly 15 aa on either side taken from the respective non-mutated, adjacent, natural human protein backbone. When two (or more) driver mutations occur within 9 amino acids of a protein sequence, a long peptide sequence containing two (or more) driver mutations was also generated for CD4 and CD8 epitope analysis so long as there were at least 8 amino acids before and after each driver mutation.

These driver mutation-containing long peptide sequences were first evaluated based on the number of CD8 epitopes introduced by inclusion of a driver mutation using the publicly available NetMHCpan 4.0 (http://www.cbs.dtu.dk/services/NetMHCpan-4.0/) database. Then the selected driver mutations from the CD8 epitope analysis were further prioritized based on the number of CD4 epitopes introduced by inclusion of a driver mutation using the publicly available NetMHCIIpan 4.0 (http://www.cbs.dtu.dk/services/NetMHCIIpan/) database. The final list of driver mutations was generated based on the collective info on CD4 and CD8 epitope analysis and frequencies of these driver mutations.

For the CD8 epitope prediction, the HLA class I supertypes included are HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*26:01, HLA-B*07:02, HLA-B*08:01, HLA-B*27:05, HLA-B*39:01, HLA-B*40:01, HLA-B*58:01, and HLA-B*15:01 (Table 1-1). The threshold for strong binder was set at the recommended threshold of 0.5, which means any peptides with predicted % rank lower than 0.5 will be annotated as strong binders. The threshold for weak binder was set at the recommended 2.0, which means any peptides with predicted % rank lower than 2.0 but higher than 0.5 would be annotated as weak binders. Only epitopes that contain the driver mutation are included in the analysis.

TABLE 1-1
HLA Class I supertypes used to predict CD8 epitopes
Supertype Representative
A01       HLA-A*01:01
A02       HLA-A*02:01
A03       HLA-A*03:01
A24       HLA-A*24:02
A26       HLA-A*26:01
B07       HLA-B*07:02
B08       HLA-B*08:01
B27       HLA-B*27:05
B39       HLA-B*39:01
B44       HLA-B*40:01
B58       HLA-B*58:01
B62       HLA-B*15:01

For the CD4 epitope prediction, forty-six HLA Class II alleles are included and shown in Table 1-2. The threshold for strong binder was set at the recommended threshold of 2, which means any peptides with predicted % rank lower than 2 will be annotated as strong binders. The threshold for weak binder was set at the recommended 10, which means any peptides with predicted % rank lower than 10 but higher than 2 will be annotated as weak binders. For each driver mutation-containing sequence, all strong or weak binder CD4 epitopes that are 13, 14, 15, 16 and 17 amino acids in length were analyzed and recorded, respectively. Only epitopes that contain the driver mutation are included in the analysis. The highest number of CD4 epitopes for an allele predicted for 13, 14, 15, 16 or 17 amino acid epitopes was used for further analysis. The maximum number of strong or weak binders for each Class II allele was determined and the sum of the total predicted epitopes for each locus DRB1, DRB 3/4/5, DQA1/DQB1 and DPB1 were recorded. The total number of CD4 epitopes is the sum of the number of epitopes in each locus (DRB1+DRB 3/4/5+DQA1/DQB1+DPB1).

TABLE 1-2
HLA Class II alleles used to predict CD4 epitopes
DRB1	DRB3/4/5	DQA1/DQB1	DPB1
DRB1*0101	DRB3*0101	DQA1*0501/DQB1*0201	DPA1*0201/DPB1*0101
DRB1*0301	DRB3*0202	DQA1*0201/DQB1*0201	DPA1*0103/DPB1*0201
DRB1*0302	DRB3*0301	DQA1*0501/DQB1*0301	DPA1*0103/DPB1*0401
DRB1*0401	DRB4*0101	DQA1*0301/DQB1*0302	DPA1*0103/DPB1*0402
DRB1*0402	DRB5*0101	DQA1*0401/DQB1*0402	DPA1*0202/DPB1*0501
DRB1*0403	DRB5*0102	DQA1*0101/DQB1*0501	DPA1*0201/DPB1*1401
DRB1*0404		DQA1*0102/DQB1*0502
DRB1*0405		DQA1*0102/DQB1*0602
DRB1*0407
DRB1*0411
DRB1*0701
DRB1*0802
DRB1*0901
DRB1*1101
DRB1*1102
DRB1*1103
DRB1*1104
DRB1*1201
DRB1*1301
DRB1*1302
DRB1*1303
DRB1*1304
DRB1*1401
DRB1*1402
DRB1*1501
DRB1*1601

The general criteria of driver mutation down selection are:

1. If there is only one driver mutation at certain position, this driver mutation will be selected if inclusion of this mutation results in >1 CD8 epitope. Driver mutations that introduce zero CD8 epitope will be excluded.

2. If there are more than one driver mutation at the same position, the driver mutation that introduces greater number of CD8 epitopes will be selected.

3. If two driver mutations at the same position introduce the same number of CD8 epitopes, the mutation with higher frequency will be selected.

4. If two driver mutations at the same position have the similar number of CD8 epitopes and similar frequencies, the mutation with greater number of CD4 epitopes will be selected.

5. When two driver mutations occur within 9 amino acids of a protein sequence, each driver mutation was evaluated alone and combined.

Patient Sample Coverage by Selected Driver Mutations

After driver mutations were prioritized and selected for each indication, the sequences encoding these driver mutations were assembled, separated by furin cleavage site to generate construct inserts. Each insert could potentially include up to 20 driver mutation-containing sequences. Once construct inserts were assembled, the analysis of patient sample coverage by each insert was performed. Briefly, the dataset of “curated set of non-redundant studies” specific for each indication was queried with the HUGO Gene Nomenclature Committee gene symbol for the oncogenes with identified driver mutations. Expression data was downloaded and Patient Samples that were “not profiled” for the oncogene containing the driver mutation were omitted. If a Patient ID was associated with more than one sample from different anatomical sites, for example from the primary tumor and a metastatic site, expression for both samples was retained in the final data set. The remaining samples was used to calculate the frequency of a driver mutation. The patient sample coverage by each insert was calculated based on the collective information of the total number of samples with one selected driver mutation, the total number of samples with >2 driver mutations from same antigen and the total number of samples with >2 driver mutations from different antigens.

Example 2: Glioblastoma Multiforme (GBM) Driver Mutation Identification, Selection and Design

Example 2 describes the process for identification, selection, and design of driver mutations expressed by GBM patient tumors and that expression of these driver mutations by GBM vaccine component cell lines can generate a GBM anti-tumor response in an HLA diverse population.

Example 29 of WO/2021/113328 first described a GBM vaccine that included two cocktails, each including three modified cell lines as follows. Cocktail A: (a) LN-229 is modified to (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ1 and CD276; and (iii) express modPSMA; (b) GB-1 is modified to (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression of TGFβ1 and CD276; (c) SF-126 is modified to (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) express modTERT; and Cocktail B: (a) DMS 53 is modified to (i) increase expression of GM-CSF and membrane bound CD40L; and (ii) decrease expression of TGFβ2 and CD276; (b) DBTRG-05MG is modified to (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression of TGFβ1 and CD276; and (c) KNS 60 is modified to (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ1, TGFβ2, and CD276; and (iii) express modMAGEA1, EGFRvIII, and hCMV pp65.

As described herein, driver mutations have now been identified and included in LN-229 and GB-1 of the GBM vaccine and potent immune responses have been detected.

Identification of Frequently Mutated Oncogenes in GBM

Table 2-1 below shows the selected oncogenes that exhibit greater than 5% mutation frequency (percentage of samples with one or more mutations) in 429 glioblastoma profiled patient samples.

TABLE 2-1
Oncogenes in GBM with greater than 5% mutation frequency
		Number of samples		Percentage of samples
	Total number	with one or more	Profiled	with one or more	Is Cancer Gene
Gene	of mutations	mutations	Samples	mutations	(source: OncoKB)
PTEN	144	139	429	32.40%	Yes
TP53	152	128	429	29.80%	Yes
EGFR	118	95	429	22.10%	Yss
NF1	68	49	429	11.40%	Yes
PIK3CA	46	41	429	9.60%	Yss
PIK3R1	41	39	429	9.10%	Yss
RB1	40	39	429	9.10%	Yss
ATRX	48	38	429	8.90%	Yes
PCLO	36	29	429	6.80%	Yes

Identification of Driver Mutations in Selected GBM Oncogenes

The GBM driver mutations in PTEN, TP53, EGFR, PIK3CA and PIK3R1 occurring in ≥0.5% of profiled patient samples (Frequency) are listed in Table 2-2. Among all GBM oncogenes listed in Table 2-1 above, there are no missense mutations occurring in ≥0.5% of profiled patient samples in NF1, RB1, ATRX, IDH1 and PCLO.

TABLE 2-2
Identified driver mutations in selected GBM oncogenes
	Driver	Number of samples	Total number of	Fre-
Gene	Mutation	with mutation	samples	quency
PTEN	R130Q	3	429	0.7%
	G132D	4	429	0.9%
	R173H	6	429	1.4%
TP53	R158H	3	429	0.7%
	H179R	3	429	0.7%
	V216M	3	429	0.7%
	C275Y	3	429	0.7%
	R175H	8	429	1.9%
	G245S	4	429	0.9%
	R273C	4	429	0.9%
	R273H	4	429	0.9%
	Y220C	6	429	1.4%
	R248W	5	429	1.2%
	R282W	5	429	1.2%
	R248Q	8	429	1.9%
EGFR	G63R	3	429	0.7%
	R252C	3	429	0.7%
	T263P	3	429	0.7%
	H304Y	3	429	0.7%
	S645C	3	429	0.7%
	R108K	4	429	0.9%
	A289D	5	429	1.2%
	V774M	5	429	1.2%
	R222C	6	429	1.4%
	A289T	6	429	1.4%
	G598V	15	429	3.5%
	A289V	17	429	4.0%
PIK3CA	E545K	3	429	0.7%
	M1043V	3	429	0.7%
	H1047R	4	429	0.9%
PIK3R1	G376R	6	429	1.4%

Prioritization and Selection of Identified GBM Driver Mutations

The results of the completed CD4 and CD8 epitope analysis, the total number of HLA-A and HLA-B supertype-restricted 9-mer CD8 epitopes, the total number of CD4 epitopes and frequency (%) for each mutation are shown in Table 2-3. Twenty-two GBM driver mutations encoded by 17 peptide sequences were selected and included as vaccine targets.

TABLE 2-3
Prioritization and selection of identified GBM driver mutations
		Number of		Number of	Included as
		total CD8	Frequency	total CD4	a vaccine
	Driver	epitopes	(%)	epitopes	target?
Gene	mutations	(SB + WB)	(n = 429)	(SB + WB)	Yes (Y) or No (N)
PTEN	R130Q	3	0.7	0	N
	G132D	3	0.9	11	N
	R130Q G132D	3	1.6	23	Y
	R173H	8	1.4	0	Y
TP53	R158H	6	0.7	0	Y
	R175H	2	1.9	0	N
	H179R	0	0.7	8	N
	R175H H179R	1	2.6	17	Y
	V216M	7	0.7	3	Y
	Y220C	2	1.4	0	N
	V216M Y220C	6	2.1	0	N
	G245S	3	0.9	0	N
	R248Q	0	1.9	0	N
	R248W	3	1.2	15	N
	G245S R248W	3	2.1	28	Y
	R273C	1	0.9	0	N
	R273H	1	0.9	0	N
	C275Y	1	0.7	49	N
	R273C C275Y	1	1.6	11	N
	R273H C275Y	1	1.6	97	Y
	R282W	0	1.2	14	N
EGFR	G63R	4	0.7	8	Y
	R108K	4	0.9	0	Y
	R222C	0	1.4	0	N
	R252C	1	0.7	0	Y
	T263P	0	0.7	0	N
	A289D	1	1.2	11	Y
	A289T	1	1.4	0	N
	A289V	1	4	7	N
	H304Y	1	0.7	49	Y
	G598V	1	3.5	7	Y
	S645C	2	0.7	0	Y
	V774M	3	1.2	3	Y
PIK3R1	G376R	3	1.4	8	Y
PIK3CA	E545K	0	0.7	0	N
	M1043V	1	0.7	7	N
	H1047R	2	0.9	12	N
	M1043 H1047R	2	1.6	46	Y

The total number of CD8 epitopes for each HLA-A and HLA-B supertype introduced by 22 selected GBM driver mutations, encoded by 17 peptide sequences, is shown in Table 2-4.

TABLE 2-4
CD8 epitopes introduced by 22 selected GBM driver
mutations encoded by 17 peptide sequences
				Total
		HLA-A	HLA-B	number
		Supertypes	Supertypes	of CD8
Gene	Mutations	(n = 5)	(n = 7)	epitopes
PTEN	R130Q G132D	3	0	3
	R173H	4	4	8
TP53	R158H	2	4	6
	R175H H179R	0	1	1
	V216M	1	6	7
	G245S R248W	1	2	3
	R273H C275Y	0	1	1
EGFR	G63R	2	2	4
	R108K	2	2	4
	R252C	1	0	1
	A289D	1	0	1
	H304Y	1	0	1
	G598V	0	1	1
	S645C	0	2	2
	V774M	0	3	3
PIK3R1	G376R	1	2	3
PIK3CA	M1043V H1047R	0	2	2

The total number of CD4 epitopes for Class II locus DRB1, DRB 3/4/5, DQA1/DQB1 and DPB1 introduced by 22 selected GBM driver mutations, encoded by 17 peptide sequences, is shown in Table 2-5.

TABLE 2-5
CD4 epitopes introduced by 22 selected GBM driver
mutations encoded by 17 peptide sequences
		DRB1	DRB3/4/5	DQA1 DQB1	DPB1	Total number of
Gene	Mutations	n = 26	n = 6	n = 8	n = 6	CD4 epitopes
PTEN	R130Q G132D	5	3	10	5	23
	R173H	0	0	0	0	0
TP53	R158H	0	0	0	0	0
	R175H H179R	0	0	0	17	17
	V216M	0	0	0	3	3
	G245S R248W	10	8	1	9	28
	R273H C275Y	38	14	4	41	97
EGFR	G63R	0	0	0	8	8
	R108K	0	0	0	0	0
	R252C	0	0	0	0	0
	A289D	2	3	6	0	11
	H304Y	18	11	1	19	49
	G598V	0	0	0	7	7
	S645C	0	0	0	0	0
	V774M	0	0	0	3	3
PIK3R1	G376R	0	0	0	8	8
PIK3CA	M1043V H1047R	25	0	0	21	46

GBM Patient Sample Coverage by Selected Driver Mutations

As shown in Table 2-6, the 22 selected GBM driver mutations were assembled into two construct inserts.

TABLE 2-6
Generation of two constructs encoding 22 selected GBM driver mutations
						Total CD4
Construct	Gene	Mutations	Frequency	Total CD8	Total CD4	and CD8
GBM	EGFR	G598V	2.4%	1	7	8
construct 1	TP53	R175H H179R	1.8%	1	17	18
insert	TP53	G245S R248W	1.4%	3	28	31
	PIK3CA	M1043V H1047R	1.1%	2	46	48
	PTEN	R130Q G132D	1.1%	3	23	26
	TP53	R273H C275Y	1.1%	1	97	98
	PIK3R1	G376R	1.0%	3	8	11
	PTEN	R173H	0.5%	8	0	8
	TP53	V216M	0.5%	7	3	10
	TP53	R158H	0.5%	6	0	6
GBM	EGFR	A289D	0.8%	1	11	12
construct 2	EGFR	V774M	0.8%	3	3	6
insert	EGFR	R108K	0.6%	4	0	4
	EGFR	S645C	0.5%	2	0	2
	EGFR	R252C	0.5%	1	0	1
	EGFR	H304Y	0.5%	1	49	50
	EGFR	G63R	0.5%	4	8	12

Once two construct inserts were assembled, analysis of GBM patient sample coverage by each insert was performed. The results indicated GBM patient sample coverage by the Construct 1 insert was 11.1% (Table 2-7). GBM patient sample coverage by the Construct 2 insert was 3% (Table 2-8). In total, GBM patient sample coverage by both Construct 1 and 2 inserts was 14.3% (Table 2-9).

TABLE 2-7
GBM patient sample coverage by Construct 1
		Total number of	Total Sample
Coverage Construct 1 Insert	Driver Mutation Target Gene	Samples with	(n = 624)
Sample Description	PTEN	TP53	EGFR	PIK3R1	PIK3CA	Driver Mutations	Coverage
# of samples with one DM	10	30	13	6	6	65	10.4%
# of samples with ≥2 DMs from same antigen	0	0	1	0	0	1	0.2%
# of samples with ≥2 DMs from different antigens						3	0.5%
					Total	69	11.1%

TABLE 2-8
GBM patient sample coverage by Construct 2
			Total
		Total number of	Sample
Coverage Construct 2 Insert	Driver Mutation Target Gene	Samples with	(n = 624)
Sample Description	PTEN	TP53	EGFR	PIK3R1	PIK3CA	Driver Mutations	Coverage
# of samples with one DM	0	0	17	0	0	17	2.7%
# of samples with ≥2 DMs from same antigen	0	0	2	0	0	2	0.3%
# of samples with ≥2 DMs from different antigens						0	0.0%
						19	3.0%

TABLE 2-9
GBM patient sample coverage by Constructs 1 and 2
			Total
Coverage All Driver Mutations		Total number of	Sample
(Construct 1 & 2 Inserts)	Driver Mutation Target Gene	Samples with	(n = 624)
Sample Description	PTEN	TP53	EGFR	PIK3R1	PIK3CA	Driver Mutations	Coverage
# of samples with one DM	10	30	30	5	5	83	13.3%
# of samples with ≥2 DMs from same antigen	0	0	3	0	0	3	0.5%
# of samples with ≥2 DMs from different antigens						3	0.5%
						89	14.3%

Oncogene Sequences and Insert Sequences of GBM Driver Mutation Construct 1 and 2

Native DNA and protein sequences of oncogenes with the selected driver mutations are included in Table 2-10. DNA and protein sequences of GBM Construct 1 and GBM Construct 2 inserts encoding selected driver mutations are also included in Table 2-10.

The Construct 1 (SEQ ID NO: 48 and SEQ ID NO: 49) insert gene encodes 374 amino acids containing the driver mutation sequences identified from PTEN (SEQ ID NO: 39), TP53 (SEQ ID NO: 41), EGFR (SEQ ID NO: 43), PIK3R1 (SEQ ID NO: 45) and PIK3CA (SEQ ID NO: 47) that were separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37). The Construct 2 (SEQ ID NO: 50 and SEQ ID NO: 51) insert gene encodes 260 amino acids containing the driver mutation sequences identified from EGFR (SEQ ID NO: 43) that were separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37).

TABLE 2-10
Native oncogene sequences and driver mutation insert sequences for GBM Construct 1 and GBM
construct 2
PTEN        DNA Sequence
(SEQ ID NO: 1 ATGACAGCCA TCATCAAAGA GATCGTTAGC AGAAACAAAA GGAGATATCA AGAGGATGGA
38)         61 TTCGACTTAG ACTTGACCTA TATTTATCCA AACATTATTG CTATGGGATT TCCTGCAGAA
            121 AGACTTGAAG GCGTATACAG GAACAATATT GATGATGTAG TAAGGTTTTT GGATTCAAAG
            181 CATAAAAACC ATTACAAGAT ATACAATCTT TGTGCTGAAA GACATTATGA CACCGCCAAA
            241 TTTAATTGCA GAGTTGCACA ATATCCTTTT GAAGACCATA ACCCACCACA GCTAGAACTT
            301 ATCAAACCCT TTTGTGAAGA TCTTGACCAA TGGCTAAGTG AAGATGACAA TCATGTTGCA
            361 GCAATTCACT GTAAAGCTGG AAAGGGACGA ACTGGTGTAA TGATATGTGC ATATTTATTA
            421 CATCGGGGCA AATTTTTAAA GGCACAAGAG GCCCTAGATT TCTATGGGGA AGTAAGGACC
            481 AGAGACAAAA AGGGAGTAAC TATTCCCAGT CAGAGGCGCT ATGTGTATTA TTATAGCTAC
            541 CTGTTAAAGA ATCATCTGGA TTATAGACCA GTGGCACTGT TGTTTCACAA GATGATGTTT
            601 GAAACTATTC CAATGTTCAG TGGCGGAACT TGCAATCCTC AGTTTGTGGT CTGCCAGCTA
            661 AAGGTGAAGA TATATTCCTC CAATTCAGGA CCCACACGAC GGGAAGACAA GTTCATGTAC
            721 TTTGAGTTCC CTCAGCCGTT ACCTGTGTGT GGTGATATCA AAGTAGAGTT CTTCCACAAA
            781 CAGAACAAGA TGCTAAAAAA GGACAAAATG TTTCACTTTT GGGTAAATAC ATTCTTCATA
            841 CCAGGACCAG AGGAAACCTC AGAAAAAGTA GAAAATGGAA GTCTATGTGA TCAAGAAATC
            901 GATAGCATTT GCAGTATAGA GCGTGCAGAT AATGACAAGG AATATCTAGT ACTTACTTTA
            961 ACAAAAAATG ATCTTGACAA AGCAAATAAA GACAAAGCCA ACCGATACTT TTCTCCAAAT
            1021 TTTAAGGTGA AGCTGTACTT CACAAAAACA GTAGAGGAGC CGTCAAATCC AGAGGCTAGC
            1081 AGTTCAACTT CTGTAACACC AGATGTTAGT GACAATGAAC CTGATCATTA TAGATATTCT
            1141 GACACCACTG ACTCTGATCC AGAGAATGAA CCTTTTGATG AAGATCAGCA TACACAAATT
            1201 ACAAAAGTC
PTEN        Protein Sequence
(SEQ ID NO: 1 MTAIIKEIVS RNKRRYQEDG FDLDLTYIYP NIIAMGFPAE RLEGVYRNNI DDVVRFLDSK
39)         61 HKNHYKIYNL CAERHYDTAK FNCRVAQYPF EDHNPPQLEL IKPFCEDLDQ WLSEDDNHVA
            121 AIHCKAGKGR TGVMICAYLL HRGKFLKAQE ALDFYGEVRT RDKKGVTIPS QRRYVYYYSY
            181 LLKNHLDYRP VALLFHKMMF ETIPMFSGGT CNPQFVVCQL KVKIYSSNSG PTRREDKFMY
            241 FEFPQPLPVC GDIKVEFFHK QNKMLKKDKM FHFWVNTFFI PGPEETSEKV ENGSLCDQEI
            301 DSICSIERAD NDKEYLVLTL TKNDLDKANK DKANRYFSPN FKVKLYFTKT VEEPSNPEAS
            361 SSTSVTPDVS DNEPDHYRYS DTTDSDPENE PFDEDQHTQI TKV
TP53        DNA Sequence
(SEQ ID NO: 1 ATGGAGGAGC CGCAGTCAGA TCCTAGCGTC GAGCCCCCTC TGAGTCAGGA AACATTTTCA
40)         61 GACCTATGGA AACTACTTCC TGAAAACAAC GTTCTGTCCC CCTTGCCGTC CCAAGCAATG
            121 GATGATTTGA TGCTGTCCCC GGACGATATT GAACAATGGT TCACTGAAGA CCCAGGTCCA
            181 GATGAAGCTC CCAGAATGCC AGAGGCTGCT CCCCCCGTGG CCCCTGCACC AGCAGCTCCT
            241 ACACCGGCGG CCCCTGCACC AGCCCCCTCC TGGCCCCTGT CATCTTCTGT CCCTTCCCAG
            301 AAAACCTACC AGGGCAGCTA CGGTTTCCGT CTGGGCTTCT TGCATTCTGG GACAGCCAAG
            361 TCTGTGACTT GCACGTACTC CCCTGCCCTC AACAAGATGT TTTGCCAACT GGCCAAGACC
            421 TGCCCTGTGC AGCTGTGGGT TGATTCCACA CCCCCGCCCG GCACCCGCGT CCGCGCCATG
            481 GCCATCTACA AGCAGTCACA GCACATGACG GAGGTTGTGA GGCGCTGCCC CCACCATGAG
            541 CGCTGCTCAG ATAGCGATGG TCTGGCCCCT CCTCAGCATC TTATCCGAGT GGAAGGAAAT
            601 TTGCGTGTGG AGTATTTGGA TGACAGAAAC ACTTTTCGAC ATAGTGTGGT GGTGCCCTAT
            661 GAGCCGCCTG AGGTTGGCTC TGACTGTACC ACCATCCACT ACAACTACAT GTGTAACAGT
            721 TCCTGCATGG GCGGCATGAA CCGGAGGCCC ATCCTCACCA TCATCACACT GGAAGACTCC
            781 AGTGGTAATC TACTGGGACG GAACAGCTTT GAGGTGCGTG TTTGTGCCTG TCCTGGGAGA
            841 GACCGGCGCA CAGAGGAAGA GAATCTCCGC AAGAAAGGGG AGCCTCACCA CGAGCTGCCC
            901 CCAGGGAGCA CTAAGCGAGC ACTGCCCAAC AACACCAGCT CCTCTCCCCA GCCAAAGAAG
            961 AAACCACTGG ATGGAGAATA TTTCACCCTT CAGATCCGTG GGCGTGAGCG CTTCGAGATG
            1021 TTCCGAGAGC TGAATGAGGC CTTGGAACTC AAGGATGCCC AGGCTGGGAA GGAGCCAGGG
            1081 GGGAGCAGGG CTCACTCCAG CCACCTGAAG TCCAAAAAGG GTCAGTCTAC CTCCCGCCAT
            1141 AAAAAACTCA TGTTCAAGAC AGAAGGGCCT GACTCAGAC
TP53        Protein Sequence
(SEQ ID NO: 1 MEEPQSDPSV EPPLSQETFS DLWKLLPENN VLSPLPSQAM DDLMLSPDDI EQWFTEDPGP
41)         61 DEAPRMPEAA PPVAPAPAAP TPAAPAPAPS WPLSSSVPSQ KTYQGSYGFR LGFLHSGTAK
            121 SVTCTYSPAL NKMFCQLAKT CPVQLWVDST PPPGTRVRAM AIYKQSQHMT EVVRRCPHHE
            181 RCSDSDGLAP PQHLIRVEGN LRVEYLDDRN TFRHSVVVPY EPPEVGSDCT TIHYNYMCNS
            241 SCMGGMNRRP ILTIITLEDS SGNLLGRNSF EVRVCACPGR DRRTEEENLR KKGEPHHELP
            301 PGSTKRALPN NTSSSPQPKK KPLDGEYFTL QIRGRERFEM FRELNEALEL KDAQAGKEPG
            361 GSRAHSSHLK SKKGQSTSRH KKLMFKTEGP DSD
EGFR        DNA Sequence
(SEQ ID NO: 1 ATGCGACCCT CCGGGACGGC CGGGGCAGCG CTCCTGGCGC TGCTGGCTGC GCTCTGCCCG
42)         61 GCGAGTCGGG CTCTGGAGGA AAAGAAAGTT TGCCAAGGCA CGAGTAACAA GCTCACGCAG
            121 TTGGGCACTT TTGAAGATCA TTTTCTCAGC CTCCAGAGGA TGTTCAATAA CTGTGAGGTG
            181 GTCCTTGGGA ATTTGGAAAT TACCTATGTG CAGAGGAATT ATGATCTTTC CTTCTTAAAG
            241 ACCATCCAGG AGGTGGCTGG TTATGTCCTC ATTGCCCTCA ACACAGTGGA GCGAATTCCT
            301 TTGGAAAACC TGCAGATCAT CAGAGGAAAT ATGTACTACG AAAATTCCTA TGCCTTAGCA
            361 GTCTTATCTA ACTATGATGC AAATAAAACC GGACTGAAGG AGCTGCCCAT GAGAAATTTA
            421 CAGGAAATCC TGCATGGCGC CGTGCGGTTC AGCAACAACC CTGCCCTGTG CAACGTGGAG
            481 AGCATCCAGT GGCGGGACAT AGTCAGCAGT GACTTTCTCA GCAACATGTC GATGGACTTC
            541 CAGAACCACC TGGGCAGCTG CCAAAAGTGT GATCCAAGCT GTCCCAATGG GAGCTGCTGG
            601 GGTGCAGGAG AGGAGAACTG CCAGAAACTG ACCAAAATCA TCTGTGCCCA GCAGTGCTCC
            661 GGGCGCTGCC GTGGCAAGTC CCCCAGTGAC TGCTGCCACA ACCAGTGTGC TGCAGGCTGC
            721 ACAGGCCCCC GGGAGAGCGA CTGCCTGGTC TGCCGCAAAT TCCGAGACGA AGCCACGTGC
            781 AAGGACACCT GCCCCCCACT CATGCTCTAC AACCCCACCA CGTACCAGAT GGATGTGAAC
            841 CCCGAGGGCA AATACAGCTT TGGTGCCACC TGCGTGAAGA AGTGTCCCCG TAATTATGTG
            901 GTGACAGATC ACGGCTCGTG CGTCCGAGCC TGTGGGGCCG ACAGCTATGA GATGGAGGAA
            961 GACGGCGTCC GCAAGTGTAA GAAGTGCGAA GGGCCTTGCC GCAAAGTGTG TAACGGAATA
            1021 GGTATTGGTG AATTTAAAGA CTCACTCTCC ATAAATGCTA CGAATATTAA ACACTTCAAA
            1081 AACTGCACCT CCATCAGTGG CGATCTCCAC ATCCTGCCGG TGGCATTTAG GGGTGACTCC
            1141 TTCACACATA CTCCTCCTCT GGATCCACAG GAACTGGATA TTCTGAAAAC CGTAAAGGAA
            1201 ATCACAGGGT TTTTGCTGAT TCAGGCTTGG CCTGAAAACA GGACGGACCT CCATGCCTTT
            1261 GAGAACCTAG AAATCATACG CGGCAGGACC AAGCAACATG GTCAGTTTTC TCTTGCAGTC
            1321 GTCAGCCTGA ACATAACATC CTTGGGATTA CGCTCCCTCA AGGAGATAAG TGATGGAGAT
            1381 GTGATAATTT CAGGAAACAA AAATTTGTGC TATGCAAATA CAATAAACTG GAAAAAACTG
            1441 TTTGGGACCT CCGGTCAGAA AACCAAAATT ATAAGCAACA GAGGTGAAAA CAGCTGCAAG
            1501 GCCACAGGCC AGGTCTGCCA TGCCTTGTGC TCCCCCGAGG GCTGCTGGGG CCCGGAGCCC
            1561 AGGGACTGCG TCTCTTGCCG GAATGTCAGC CGAGGCAGGG AATGCGTGGA CAAGTGCAAC
            1621 CTTCTGGAGG GTGAGCCAAG GGAGTTTGTG GAGAACTCTG AGTGCATACA GTGCCACCCA
            1681 GAGTGCCTGC CTCAGGCCAT GAACATCACC TGCACAGGAC GGGGACCAGA CAACTGTATC
            1741 CAGTGTGCCC ACTACATTGA CGGCCCCCAC TGCGTCAAGA CCTGCCCGGC AGGAGTCATG
            1801 GGAGAAAACA ACACCCTGGT CTGGAAGTAC GCAGACGCCG GCCATGTGTG CCACCTGTGC
            1861 CATCCAAACT GCACCTACGG ATGCACTGGG CCAGGTCTTG AAGGCTGTCC AACGAATGGG
            1921 CCTAAGATCC CGTCCATCGC CACTGGGATG GTGGGGGCCC TCCTCTTGCT GCTGGTGGTG
            1981 GCCCTGGGGA TCGGCCTCTT CATGCGAAGG CGCCACATCG TTCGGAAGCG CACGCTGCGG
            2041 AGGCTGCTGC AGGAGAGGGA GCTTGTGGAG CCTCTTACAC CCAGTGGAGA AGCTCCCAAC
            2101 CAAGCTCTCT TGAGGATCTT GAAGGAAACT GAATTCAAAA AGATCAAAGT GCTGGGCTCC
            2161 GGTGCGTTCG GCACGGTGTA TAAGGGACTC TGGATCCCAG AAGGTGAGAA AGTTAAAATT
            2221 CCCGTCGCTA TCAAGGAATT AAGAGAAGCA ACATCTCCGA AAGCCAACAA GGAAATCCTC
            2281 GATGAAGCCT ACGTGATGGC CAGCGTGGAC AACCCCCACG TGTGCCGCCT GCTGGGCATC
            2341 TGCCTCACCT CCACCGTGCA GCTCATCACG CAGCTCATGC CCTTCGGCTG CCTCCTGGAC
            2401 TATGTCCGGG AACACAAAGA CAATATTGGC TCCCAGTACC TGCTCAACTG GTGTGTGCAG
            2461 ATCGCAAAGG GCATGAACTA CTTGGAGGAC CGTCGCTTGG TGCACCGCGA CCTGGCAGCC
            2521 AGGAACGTAC TGGTGAAAAC ACCGCAGCAT GTCAAGATCA CAGATTTTGG GCTGGCCAAA
            2581 CTGCTGGGTG CGGAAGAGAA AGAATACCAT GCAGAAGGAG GCAAAGTGCC TATCAAGTGG
            2641 ATGGCATTGG AATCAATTTT ACACAGAATC TATACCCACC AGAGTGATGT CTGGAGCTAC
            2701 GGGGTGACTG TTTGGGAGTT GATGACCTTT GGATCCAAGC CATATGACGG AATCCCTGCC
            2761 AGCGAGATCT CCTCCATCCT GGAGAAAGGA GAACGCCTCC CTCAGCCACC CATATGTACC
            2821 ATCGATGTCT ACATGATCAT GGTCAAGTGC TGGATGATAG ACGCAGATAG TCGCCCAAAG
            2881 TTCCGTGAGT TGATCATCGA ATTCTCCAAA ATGGCCCGAG ACCCCCAGCG CTACCTTGTC
            2941 ATTCAGGGGG ATGAAAGAAT GCATTTGCCA AGTCCTACAG ACTCCAACTT CTACCGTGCC
            3001 CTGATGGATG AAGAAGACAT GGACGACGTG GTGGATGCCG ACGAGTACCT CATCCCACAG
            3061 CAGGGCTTCT TCAGCAGCCC CTCCACGTCA CGGACTCCCC TCCTGAGCTC TCTGAGTGCA
            3121 ACCAGCAACA ATTCCACCGT GGCTTGCATT GATAGAAATG GGCTGCAAAG CTGTCCCATC
            3181 AAGGAAGACA GCTTCTTGCA GCGATACAGC TCAGACCCCA CAGGCGCCTT GACTGAGGAC
            3241 AGCATAGACG ACACCTTCCT CCCAGTGCCT GAATACATAA ACCAGTCCGT TCCCAAAAGG
            3301 CCCGCTGGCT CTGTGCAGAA TCCTGTCTAT CACAATCAGC CTCTGAACCC CGCGCCCAGC
            3361 AGAGACCCAC ACTACCAGGA CCCCCACAGC ACTGCAGTGG GCAACCCCGA GTATCTCAAC
            3421 ACTGTCCAGC CCACCTGTGT CAACAGCACA TTCGACAGCC CTGCCCACTG GGCCCAGAAA
            3481 GGCAGCCACC AAATTAGCCT GGACAACCCT GACTACCAGC AGGACTTCTT TCCCAAGGAA
            3541 GCCAAGCCAA ATGGCATCTT TAAGGGCTCC ACAGCTGAAA ATGCAGAATA CCTAAGGGTC
            3601 GCGCCACAAA GCAGTGAATT TATTGGAGCA
EGFR        Protein Sequence
(SEQ ID NO: 1 MRPSGTAGAA LLALLAALCP ASRALEEKKV CQGTSNKLTQ LGTFEDHFLS LQRMFNNCEV
43)         61 VLGNLEITYV QRNYDLSFLK TIQEVAGYVL IALNTVERIP LENLQIIRGN MYYENSYALA
            121 VLSNYDANKT GLKELPMRNL QEILHGAVRF SNNPALCNVE SIQWRDIVSS DFLSNMSMDF
            181 QNHLGSCQKC DPSCPNGSCW GAGEENCQKL TKIICAQQCS GRCRGKSPSD CCHNQCAAGC
            241 TGPRESDCLV CRKFRDEATC KDTCPPLMLY NPTTYQMDVN PEGKYSFGAT CVKKCPRNYV
            301 VTDHGSCVRA CGADSYEMEE DGVRKCKKCE GPCRKVCNGI GIGEFKDSLS INATNIKHFK
            361 NCTSISGDLH ILPVAFRGDS FTHTPPLDPQ ELDILKTVKE ITGFLLIQAW PENRTDLHAF
            421 ENLEIIRGRT KQHGQFSLAV VSLNITSLGL RSLKEISDGD VIISGNKNLC YANTINWKKL
            481 FGTSGQKTKI ISNRGENSCK ATGQVCHALC SPEGCWGPEP RDCVSCRNVS RGRECVDKCN
            541 LLEGEPREFV ENSECIQCHP ECLPQAMNIT CTGRGPDNCI QCAHYIDGPH CVKTCPAGVM
            601 GENNTLVWKY ADAGHVCHLC HPNCTYGCTG PGLEGCPTNG PKIPSIATGM VGALLLLLVV
            661 ALGIGLFMRR RHIVRKRTLR RLLQERELVE PLTPSGEAPN QALLRILKET EFKKIKVLGS
            721 GAFGTVYKGL WIPEGEKVKI PVAIKELREA TSPKANKEIL DEAYVMASVD NPHVCRLLGI
            781 CLTSTVQLIT QLMPFGCLLD YVREHKDNIG SQYLLNWCVQ IAKGMNYLED RRLVHRDLAA
            841 RNVLVKTPQH VKITDFGLAK LLGAEEKEYH AEGGKVPIKW MALESILHRI YTHQSDVWSY
            901 GVTVWELMTF GSKPYDGIPA SEISSILEKG ERLPQPPICT IDVYMIMVKC WMIDADSRPK
            961 FRELIIEFSK MARDPQRYLV IQGDERMHLP SPTDSNFYRA LMDEEDMDDV VDADEYLIPQ
            1021 QGFFSSPSTS RTPLLSSLSA TSNNSTVACI DRNGLQSCPI KEDSFLQRYS SDPTGALTED
            1081 SIDDTFLPVP EYINQSVPKR PAGSVQNPVY HNQPLNPAPS RDPHYQDPHS TAVGNPEYLN
            1141 TVQPTCVNST FDSPAHWAQK GSHQISLDNP DYQQDFFPKE AKPNGIFKGS TAENAEYLRV
            1201 APQSSEFIGA
PI K3R1     DNA Sequence
(SEQ ID NO: 1 ATGAGTGCTG AGGGGTACCA GTACAGAGCG CTGTATGATT ATAAAAAGGA AAGAGAAGAA
44)         61 GATATTGACT TGCACTTGGG TGACATATTG ACTGTGAATA AAGGGTCCTT AGTAGCTCTT
            121 GGATTCAGTG ATGGACAGGA AGCCAGGCCT GAAGAAATTG GCTGGTTAAA TGGCTATAAT
            181 GAAACCACAG GGGAAAGGGG GGACTTTCCG GGAACTTACG TAGAATATAT TGGAAGGAAA
            241 AAAATCTCGC CTCCCACACC AAAGCCCCGG CCACCTCGGC CTCTTCCTGT TGCACCAGGT
            301 TCTTCGAAAA CTGAAGCAGA TGTTGAACAA CAAGCTTTGA CTCTCCCGGA TCTTGCAGAG
            361 CAGTTTGCCC CTCCTGACAT TGCCCCGCCT CTTCTTATCA AGCTCGTGGA AGCCATTGAA
            421 AAGAAAGGTC TGGAATGTTC AACTCTATAC AGAACACAGA GCTCCAGCAA CCTGGCAGAA
            481 TTACGACAGC TTCTTGATTG TGATACACCC TCCGTGGACT TGGAAATGAT CGATGTGCAC
            541 GTTTTGGCTG ACGCTTTCAA ACGCTATCTC CTGGACTTAC CAAATCCTGT CATTCCAGCA
            601 GCCGTTTACA GTGAAATGAT TTCTTTAGCT CCAGAAGTAC AAAGCTCCGA AGAATATATT
            661 CAGCTATTGA AGAAGCTTAT TAGGTCGCCT AGCATACCTC ATCAGTATTG GCTTACGCTT
            721 CAGTATTTGT TAAAACATTT CTTCAAGCTC TCTCAAACCT CCAGCAAAAA TCTGTTGAAT
            781 GCAAGAGTAC TCTCTGAAAT TTTCAGCCCT ATGCTTTTCA GATTCTCAGC AGCCAGCTCT
            841 GATAATACTG AAAACCTCAT AAAAGTTATA GAAATTTTAA TCTCAACTGA ATGGAATGAA
            901 CGACAGCCTG CACCAGCACT GCCTCCTAAA CCACCAAAAC CTACTACTGT AGCCAACAAC
            961 GGTATGAATA ACAATATGTC CTTACAAGAT GCTGAATGGT ACTGGGGAGA TATCTCGAGG
            1021 GAAGAAGTGA ATGAAAAACT TCGAGATACA GCAGACGGGA CCTTTTTGGT ACGAGATGCG
            1081 TCTACTAAAA TGCATGGTGA TTATACTCTT ACACTAAGGA AAGGGGGAAA TAACAAATTA
            1141 ATCAAAATAT TTCATCGAGA TGGGAAATAT GGCTTCTCTG ACCCATTAAC CTTCAGTTCT
            1201 GTGGTTGAAT TAATAAACCA CTACCGGAAT GAATCTCTAG CTCAGTATAA TCCCAAATTG
            1261 GATGTGAAAT TACTTTATCC AGTATCCAAA TACCAACAGG ATCAAGTTGT CAAAGAAGAT
            1321 AATATTGAAG CTGTAGGGAA AAAATTACAT GAATATAACA CTCAGTTTCA AGAAAAAAGT
            1381 CGAGAATATG ATAGATTATA TGAAGAATAT ACCCGCACAT CCCAGGAAAT CCAAATGAAA
            1441 AGGACAGCTA TTGAAGCATT TAATGAAACC ATAAAAATAT TTGAAGAACA GTGCCAGACC
            1501 CAAGAGCGGT ACAGCAAAGA ATACATAGAA AAGTTTAAAC GTGAAGGCAA TGAGAAAGAA
            1561 ATACAAAGGA TTATGCATAA TTATGATAAG TTGAAGTCTC GAATCAGTGA AATTATTGAC
            1621 AGTAGAAGAA GATTGGAAGA AGACTTGAAG AAGCAGGCAG CTGAGTATCG AGAAATTGAC
            1681 AAACGTATGA ACAGCATTAA ACCAGACCTT ATCCAGCTGA GAAAGACGAG AGACCAATAC
            1741 TTGATGTGGT TGACTCAAAA AGGTGTTCGG CAAAAGAAGT TGAACGAGTG GTTGGGCAAT
            1801 GAAAACACTG AAGACCAATA TTCACTGGTG GAAGATGATG AAGATTTGCC CCATCATGAT
            1861 GAGAAGACAT GGAATGTTGG AAGCAGCAAC CGAAACAAAG CTGAAAACCT GTTGCGAGGG
            1921 AAGCGAGATG GCACTTTTCT TGTCCGGGAG AGCAGTAAAC AGGGCTGCTA TGCCTGCTCT
            1981 GTAGTGGTGG ACGGCGAAGT AAAGCATTGT GTCATAAACA AAACAGCAAC TGGCTATGGC
            2041 TTTGCCGAGC CCTATAACTT GTACAGCTCT CTGAAAGAAC TGGTGCTACA TTACCAACAC
            2101 ACCTCCCTTG TGCAGCACAA CGACTCCCTC AATGTCACAC TAGCCTACCC AGTATATGCA
            2161 CAGCAGAGGC GA
PIK3R1      Protein Sequence
(SEQ ID NO: 1 MSAEGYQYRA LYDYKKEREE DIDLHLGDIL TVNKGSLVAL GFSDGQEARP EEIGWLNGYN
45)         61 ETTGERGDFP GTYVEYIGRK KISPPTPKPR PPRPLPVAPG SSKTEADVEQ QALTLPDLAE
            121 QFAPPDIAPP LLIKLVEAIE KKGLECSTLY RTQSSSNLAE LRQLLDCDTP SVDLEMIDVH
            181 VLADAFKRYL LDLPNPVIPA AVYSEMISLA PEVQSSEEYI QLLKKLIRSP SIPHQYWLTL
            241 QYLLKHFFKL SQTSSKNLLN ARVLSEIFSP MLFRFSAASS DNTENLIKVI EILISTEWNE
            301 RQPAPALPPK PPKPTTVANN GMNNNMSLQD AEWYWGDISR EEVNEKLRDT ADGTFLVRDA
            361 STKMHGDYTL TLRKGGNNKL IKIFHRDGKY GFSDPLTFSS VVELINHYRN ESLAQYNPKL
            421 DVKLLYPVSK YQQDQVVKED NIEAVGKKLH EYNTQFQEKS REYDRLYEEY TRTSQEIQMK
            481 RTAIEAFNET IKIFEEQCQT QERYSKEYIE KFKREGNEKE IQRIMHNYDK LKSRISEIID
            541 SRRRLEEDLK KQAAEYREID KRMNSIKPDL IQLRKTRDQY LMWLTQKGVR QKKLNEWLGN
            601 ENTEDQYSLV EDDEDLPHHD EKTWNVGSSN RNKAENLLRG KRDGTFLVRE SSKQGCYACS
            661 VVVDGEVKHC VINKTATGYG FAEPYNLYSS LKELVLHYQH TSLVQHNDSL NVTLAYPVYA
            721 QQRR
PI K3CA     DNA Sequence
(SEQ ID NO: 1 ATGCCTCCAC GACCATCATC AGGTGAACTG TGGGGCATCC ACTTGATGCC CCCAAGAATC
46)         61 CTAGTAGAAT GTTTACTACC AAATGGAATG ATAGTGACTT TAGAATGCCT CCGTGAGGCT
            121 ACATTAATAA CCATAAAGCA TGAACTATTT AAAGAAGCAA GAAAATACCC CCTCCATCAA
            181 CTTCTTCAAG ATGAATCTTC TTACATTTTC GTAAGTGTTA CTCAAGAAGC AGAAAGGGAA
            241 GAATTTTTTG ATGAAACAAG ACGACTTTGT GACCTTCGGC TTTTTCAACC CTTTTTAAAA
            301 GTAATTGAAC CAGTAGGCAA CCGTGAAGAA AAGATCCTCA ATCGAGAAAT TGGTTTTGCT
            361 ATCGGCATGC CAGTGTGTGA ATTTGATATG GTTAAAGATC CAGAAGTACA GGACTTCCGA
            421 AGAAATATTC TGAACGTTTG TAAAGAAGCT GTGGATCTTA GGGACCTCAA TTCACCTCAT
            481 AGTAGAGCAA TGTATGTCTA TCCTCCAAAT GTAGAATCTT CACCAGAATT GCCAAAGCAC
            541 ATATATAATA AATTAGATAA AGGGCAAATA ATAGTGGTGA TCTGGGTAAT AGTTTCTCCA
            601 AATAATGACA AGCAGAAGTA TACTCTGAAA ATCAACCATG ACTGTGTACC AGAACAAGTA
            661 ATTGCTGAAG CAATCAGGAA AAAAACTCGA AGTATGTTGC TATCCTCTGA ACAACTAAAA
            721 CTCTGTGTTT TAGAATATCA GGGCAAGTAT ATTTTAAAAG TGTGTGGATG TGATGAATAC
            781 TTCCTAGAAA AATATCCTCT GAGTCAGTAT AAGTATATAA GAAGCTGTAT AATGCTTGGG
            841 AGGATGCCCA ATTTGATGTT GATGGCTAAA GAAAGCCTTT ATTCTCAACT GCCAATGGAC
            901 TGTTTTACAA TGCCATCTTA TTCCAGACGC ATTTCCACAG CTACACCATA TATGAATGGA
            961 GAAACATCTA CAAAATCCCT TTGGGTTATA AATAGTGCAC TCAGAATAAA AATTCTTTGT
            1021 GCAACCTACG TGAATGTAAA TATTCGAGAC ATTGATAAGA TCTATGTTCG AACAGGTATC
            1081 TACCATGGAG GAGAACCCTT ATGTGACAAT GTGAACACTC AAAGAGTACC TTGTTCCAAT
            1141 CCCAGGTGGA ATGAATGGCT GAATTATGAT ATATACATTC CTGATCTTCC TCGTGCTGCT
            1201 CGACTTTGCC TTTCCATTTG CTCTGTTAAA GGCCGAAAGG GTGCTAAAGA GGAACACTGT
            1261 CCATTGGCAT GGGGAAATAT AAACTTGTTT GATTACACAG ACACTCTAGT ATCTGGAAAA
            1321 ATGGCTTTGA ATCTTTGGCC AGTACCTCAT GGATTAGAAG ATTTGCTGAA CCCTATTGGT
            1381 GTTACTGGAT CAAATCCAAA TAAAGAAACT CCATGCTTAG AGTTGGAGTT TGACTGGTTC
            1441 AGCAGTGTGG TAAAGTTCCC AGATATGTCA GTGATTGAAG AGCATGCCAA TTGGTCTGTA
            1501 TCCCGAGAAG CAGGATTTAG CTATTCCCAC GCAGGACTGA GTAACAGACT AGCTAGAGAC
            1561 AATGAATTAA GGGAAAATGA CAAAGAACAG CTCAAAGCAA TTTCTACACG AGATCCTCTC
            1621 TCTGAAATCA CTGAGCAGGA GAAAGATTTT CTATGGAGTC ACAGACACTA TTGTGTAACT
            1681 ATCCCCGAAA TTCTACCCAA ATTGCTTCTG TCTGTTAAAT GGAATTCTAG AGATGAAGTA
            1741 GCCCAGATGT ATTGCTTGGT AAAAGATTGG CCTCCAATCA AACCTGAACA GGCTATGGAA
            1801 CTTCTGGACT GTAATTACCC AGATCCTATG GTTCGAGGTT TTGCTGTTCG GTGCTTGGAA
            1861 AAATATTTAA CAGATGACAA ACTTTCTCAG TATTTAATTC AGCTAGTACA GGTCCTAAAA
            1921 TATGAACAAT ATTTGGATAA CTTGCTTGTG AGATTTTTAC TGAAGAAAGC ATTGACTAAT
            1981 CAAAGGATTG GGCACTTTTT CTTTTGGCAT TTAAAATCTG AGATGCACAA TAAAACAGTT
            2041 AGCCAGAGGT TTGGCCTGCT TTTGGAGTCC TATTGTCGTG CATGTGGGAT GTATTTGAAG
            2101 CACCTGAATA GGCAAGTCGA GGCAATGGAA AAGCTCATTA ACTTAACTGA CATTCTCAAA
            2161 CAGGAGAAGA AGGATGAAAC ACAAAAGGTA CAGATGAAGT TTTTAGTTGA GCAAATGAGG
            2221 CGACCAGATT TCATGGATGC TCTACAGGGC TTTCTGTCTC CTCTAAACCC TGCTCATCAA
            2281 CTAGGAAACC TCAGGCTTGA AGAGTGTCGA ATTATGTCCT CTGCAAAAAG GCCACTGTGG
            2341 TTGAATTGGG AGAACCCAGA CATCATGTCA GAGTTACTGT TTCAGAACAA TGAGATCATC
            2401 TTTAAAAATG GGGATGATTT ACGGCAAGAT ATGCTAACAC TTCAAATTAT TCGTATTATG
            2461 GAAAATATCT GGCAAAATCA AGGTCTTGAT CTTCGAATGT TACCTTATGG TTGTCTGTCA
            2521 ATCGGTGACT GTGTGGGACT TATTGAGGTG GTGCGAAATT CTCACACTAT TATGCAAATT
            2581 CAGTGCAAAG GCGGCTTGAA AGGTGCACTG CAGTTCAACA GCCACACACT ACATCAGTGG
            2641 CTCAAAGACA AGAACAAAGG AGAAATATAT GATGCAGCCA TTGACCTGTT TACACGTTCA
            2701 TGTGCTGGAT ACTGTGTAGC TACCTTCATT TTGGGAATTG GAGATCGTCA CAATAGTAAC
            2761 ATCATGGTGA AAGACGATGG ACAACTGTTT CATATAGATT TTGGACACTT TTTGGATCAC
            2821 AAGAAGAAAA AATTTGGTTA TAAACGAGAA CGTGTGCCAT TTGTTTTGAC ACAGGATTTC
            2881 TTAATAGTGA TTAGTAAAGG AGCCCAAGAA TGCACAAAGA CAAGAGAATT TGAGAGGTTT
            2941 CAGGAGATGT GTTACAAGGC TTATCTAGCT ATTCGACAGC ATGCCAATCT CTTCATAAAT
            3001 CTTTTCTCAA TGATGCTTGG CTCTGGAATG CCAGAACTAC AATCTTTTGA TGACATTGCA
            3061 TACATTCGAA AGACCCTAGC CTTAGATAAA ACTGAGCAAG AGGCTTTGGA GTATTTCATG
            3121 AAACAAATGA ATGATGCACA TCATGGTGGC TGGACAACAA AAATGGATTG GATCTTCCAC
            3181 ACAATTAAAC AGCATGCATT GAAC
PI K3CA     Protein Sequence
(SEQ ID NO: 1 MPPRPSSGEL WGIHLMPPRI LVECLLPNGM IVTLECLREA TLITIKHELF KEARKYPLHQ
47)         61 LLQDESSYIF VSVTQEAERE EFFDETRRLC DLRLFQPFLK VIEPVGNREE KILNREIGFA
            121 IGMPVCEFDM VKDPEVQDFR RNILNVCKEA VDLRDLNSPH SRAMYVYPPN VESSPELPKH
            181 IYNKLDKGQI IVVIWVIVSP NNDKQKYTLK INHDCVPEQV IAEAIRKKTR SMLLSSEQLK
            241 LCVLEYQGKY ILKVCGCDEY FLEKYPLSQY KYIRSCIMLG RMPNLMLMAK ESLYSQLPMD
            301 CFTMPSYSRR ISTATPYMNG ETSTKSLWVI NSALRIKILC ATYVNVNIRD IDKIYVRTGI
            361 YHGGEPLCDN VNTQRVPCSN PRWNEWLNYD IYIPDLPRAA RLCLSICSVK GRKGAKEEHC
            421 PLAWGNINLF DYTDTLVSGK MALNLWPVPH GLEDLLNPIG VTGSNPNKET PCLELEFDWF
            481 SSVVKFPDMS VIEEHANWSV SREAGFSYSH AGLSNRLARD NELRENDKEQ LKAISTRDPL
            541 SEITEQEKDF LWSHRHYCVT IPEILPKLLL SVKWNSRDEV AQMYCLVKDW PPIKPEQAME
            601 LLDCNYPDPM VRGFAVRCLE KYLTDDKLSQ YLIQLVQVLK YEQYLDNLLV RFLLKKALTN
            661 QRIGHFFFWH LKSEMHNKTV SQRFGLLLES YCRACGMYLK HLNRQVEAME KLINLTDILK
            721 QEKKDETQKV QMKFLVEQMR RPDFMDALQG FLSPLNPAHQ LGNLRLEECR IMSSAKRPLW
            781 LNWENPDIMS ELLFQNNEII FKNGDDLRQD MLTLQIIRIM ENIWQNQGLD LRMLPYGCLS
            841 IGDCVGLIEV VRNSHTIMQI QCKGGLKGAL QFNSHTLHQW LKDKNKGEIY DAAIDLFTRS
            901 CAGYCVATFI LGIGDRHNSN IMVKDDGQLF HIDFGHFLDH KKKKFGYKRE RVPFVLTQDF
            961 LIVISKGAQE CTKTREFERF QEMCYKAYLA IRQHANLFIN LFSMMLGSGM PELQSFDDIA
            1021 YIRKTLALDK TEQEALEYFM KQMNDAHHGG WTTKMDWIFH TIKQHALN
GBM DM      DNA Sequence
construct 1 1 ATGCTGAGAG TGGAATACCT GGACGACCGG AACACCTTCC GGCACTCTAT GGTGGTGCCT
insert      61 TACGAGCCTC CTGAAGTGGG CAGCGATTGC ACCACCAGAG GCAGAAAGAG AAGAAGCGCC
(SEQ ID NO: 121 CACTACATCG ACGGCCCTCA CTGCGTGAAA ACCTGTCCTG CCGTGGTCAT GGGCGAGAAC
48)         181 AATACCCTCG TGTGGAAGTA CGCCGACGCC AGAGGTCGCA AGAGAAGATC CATGGCCATC
            241 TACAAGCAGA GCCAGCACAT GACCGAGGTC GTGCGGCACT GTCCTCACAG AGAGAGATGC
            301 AGCGATAGCG ACGGACTGGC CCCTAGAGGC CGGAAAAGAA GATCTACCAC CATCCACTAC
            361 AACTACATGT GCAACAGCAG CTGCATGGGC AGCATGAACT GGCGGCCTAT CCTGACCATC
            421 ATCACCCTGG AAGATAGCCG GGGCAGAAAG CGGAGATCTG AGCAAGAGGC CCTGGAATAC
            481 TTTATGAAGC AAGTGAACGA CGCCCGGCAC GGCGGCTGGA CAACAAAGAT GGATTGGATC
            541 TTCCACACCA TCAGAGGACG GAAGCGGCGG AGCGACGATA ATCATGTGGC CGCCATCCAC
            601 TGCAAGGCCG GCAAAGGACA GACCGACGTG ATGATCTGTG CCTACCTGCT GCACCGGGGC
            661 AAGTTCAGAG GAAGAAAACG CAGAAGCGAG GACAGCAGCG GCAACCTGCT GGGCAGAAAT
            721 AGCTTCGAGG TGCACGTGTA CGCCTGTCCT GGCAGAGACA GAAGAACCGA GGAAGAGAAT
            781 CGCGGAAGAA AGAGGCGGAG CAGCACCAAG ATGCACGGCG ACTACACCCT GACACTGCGG
            841 AAGGGCAGAA ACAACAAGCT GATCAAGATC TTTCACCGCG ACGGGAAGTA CGGACGCGGA
            901 CGCAAGCGCA GATCTGTGCG GACCAGAGAC AAGAAAGGCG TGACAATCCC CAGCCAGCGG
            961 CACTACGTGT ACTACTACAG CTATCTGCTG AAGAACCACC TGGACTATCG CGGCCGTAAA
            1021 AGGCGCTCTG TGCAGCTGTG GGTCGACAGC ACACCTCCTC CAGGCACAAG AGTGCACGCC
            1081 ATGGCTATCT ATAAGCAATC CCAGCATATG ACGGAAGTGG TG
GBM DM      Protein Sequence*
construct 1 1 MLRVEYLDDR NTFRHSMVVP YEPPEVGSDC TTRGRKRRSA HYIDGPHCVK TCPAVVMGEN
insert      61 NTLVWKYADA RGRKRRSMAI YKQSQHMTEV VRHCPHRERC SDSDGLAPRG RKRRSTTIHY
(SEQ ID NO: 121 NYMCNSSCMG SMNWRPILTI ITLEDSRGRK RRSEQEALEY FMKQVNDARH GGWTTKMDWI
49)         181 FHTIRGRKRR SDDNHVAAIH CKAGKGQTDV MICAYLLHRG KFRGRKRRSE DSSGNLLGRN
            241 SFEVHVYACP GRDRRTEEEN RGRKRRSSTK MHGDYTLTLR KGRNNKLIKI FHRDGKYGRG
            301 RKRRSVRTRD KKGVTIPSQR HYVYYYSYLL KNHLDYRGRK RRSVQLWVDS TPPPGTRVHA
            361 MAIYKQSQHM TEVV
GBM DM      DNA Sequence
construct 2 1 ATGTTTCTGA GCCTGCAGCG GATGTTCAAC AACTGCGAGG TGGTGCTGCG GAACCTGGAA
insert      61 ATCACCTACG TGCAGCGGAA CTACGACCTG AGCTTCCGGG GCAGAAAGCG GAGAAGCACC
(SEQ ID NO: 121 TACCAGATGG ACGTGAACCC CGAGGGCAAG TACAGCTTCG GCGATACCTG CGTGAAGAAG
50)         181 TGCCCCAGAA ACTACGTGGT CACCGACCAC AGAGGCAGAA AGAGGCGGAG CATTCTGGAC
            241 GAGGCCTACG TGATGGCCAG CGTGGACAAT CCCCACATGT GTAGACTGCT GGGCATCTGC
            301 CTGACCAGCA CCGTGCAGCT GATCAGAGGC CGGAAGAGAA GAAGCCTGAA CACCGTCGAG
            361 AGAATCCCTC TGGAAAACCT GCAGATCATC AAGGGCAACA TGTACTACGA GAACAGCTAC
            421 GCCCTGGCCG TGCTGAGCAG AGGACGCAAA AGAAGATCTG GCCCTGGCCT GGAAGGCTGC
            481 CCTACAAATG GACCTAAGAT CCCCTGTATC GCTACCGGCA TGGTTGGAGC ACTGTTGCTG
            541 CTGCTGGTTG TGCGGGGAAG AAAGAGAAGA TCCGCCGCTG GCTGTACAGG CCCCAGAGAA
            601 TCTGATTGCC TCGTGTGCTG CAAGTTCCGC GACGAGGCCA CATGCAAGGA CACCTGTCCT
            661 CCACTGAGAG GACGGAAGCG GAGATCTGCC ACCTGTGTGA AAAAGTGTCC TCGCAACTAC
            721 GTCGTGACCG ATTACGGCAG CTGCGTCAGA GCTTGTGGCG CCGATAGCTA CGAGATGGAA
GBM DM      Protein Sequence*
construct 2 1 MFLSLQRMFN NCEVVLRNLE ITYVQRNYDL SFRGRKRRST YQMDVNPEGK YSFGDTCVKK
insert      61 CPRNYVVTDH RGRKRRSILD EAYVMASVDN PHMCRLLGIC LTSTVQLIRG RKRRSLNTVE
(SEQ ID NO: 121 RIPLENLQII KGNMYYENSY ALAVLSRGRK RRSGPGLEGC PTNGPKIPCI ATGMVGALLL
51)         181 LLVVRGRKRR SAAGCTGPRE SDCLVCCKFR DEATCKDTCP PLRGRKRRSA TCVKKCPRNY
            241 TDYGSCVR ACGADSYEME
*Driver mutation is highlighted in bold. The furin cleavage sequence is underlined.

Immune Responses to EGFR, TP53, PTEN, PIK3CA, and PIK3R1 GBM Driver Mutations (SEQ ID NO: 49) Encoded by GBM Construct 1 Expressed by the GB-1 Cell Line

GB-1 modified to (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression of TGFβ1 and CD276; was stably transduced with lentiviral particles to express ten peptide sequences encoding EGFR driver mutation G598V, TP53 driver mutations R175H, H179R, G245S, R248W, R273H, C275Y, V216M, and R158H, PTEN driver mutations R130Q, G132D, and R173H, PIK3CA driver mutations M1043V and H1047R, and PIK3R1 driver mutation G376R.

Immune responses to TP53, PTEN, PIK3R1, PIK3CA, and EGFR driver mutations were evaluated by IFNγ ELISpot. Specifically, 1.5×10⁶ of the parental, unmodified GB-1 or modified GB-1 described above were co-cultured with 1.5×10⁶ iDCs from seven HLA diverse donors (n=4/donor). HLA-A, HLA-B, and HLA-C alleles for each of the seven donors are in Table 2-11. CD14-PBMCs primed with DC loaded with unmodified GB-1 or modified GB-1 were isolated from co-culture on day 6. Primed CD14-PBMCs were stimulated with peptide pools, 15-mers overlapping by 9 amino acids, designed to span the length of the inserted driver mutations, excluding the furin cleavage sequences (Thermo Scientific Custom Peptide Service) for 24 hours in the ELISpot assay prior to detection of IFNγ production.

TABLE 2-11
Healthy Donor MHC-I characteristics
	Donor #	HLA-A	HLA-B	HLA-C
	1	*26:01 *68:02	*08:01 *15:03	*03:04 *12:03
	2	*03:01 *32:01	*07:02 *15:17	*07:01 *07:02
	3	*01:01 *32:01	*35:01 *40:06	*04:01 *15:02
	4	*32:01 *68:05	*27:05 *39:08	*01:02 *07:02
	5	*02:01 *33:01	*07:02 *14:02	*07:02 *08:02
	6	*03:01 *30:02	*07:02 *35:01	*04:01 *07:02
	7	*03:01 *03:01	*07:02 *18:01	*07:02 *12:03

FIG. 1 demonstrates priming Donor CD14-PBMCs with the GB-1 cell line modified as described above and herein generates more potent immune responses against GBM driver mutations compared to priming with unmodified, parental GB-1. Modified GB-1 significantly increased immune responses against TP53 driver mutations R175H and H179R (p=0.037), V216M (p=0.005), G245S and R248W (p=0.037), R273H and C275Y (p=0.005) (FIG. 1A), PTEN driver mutations R130W and R132D (p=0.001) and R173H (p=0.001) (FIG. 1B), PIK3R1 driver mutation G376R (p=0.001) (FIG. 1C), PIK3CA driver mutations M1043V and H1047R (p=0.005) (FIG. 1D), and EGFR driver mutation G598V (p=0.001) (FIG. 1E). IFNγ responses against TP53 driver mutation R158H induced by modified GB-1 were more robust relative to unmodified GB-1 (FIG. 1A) but did not reach statistical significance. Statistical analysis was completed using the Mann-Whitney U test. IFNγ responses to the 10 peptides encoding 15 GBM driver mutations expressed by unmodified and modified GB-1 are described for each Donor in Table 2-12.

TABLE 2-12
Immune responses to TP53, PTEN, PIK3R1, PIK3CA, and EGFR GBM driver mutations
	GBM	Unmodified GB-1 (SFU ± SEM)
	TP53		R175H		G245S	R273H
	mutation	R158H	H179R	V216M	R248W	C275Y
	Donor 1	190 ± 117	0 ± 0	380 ± 240	0 ± 0	0 ± 0
	Donor 2	210 ± 133	0 ± 0	50 ± 30	0 ± 0	0 ± 0
	Donor 3	0 ± 0	63 ± 28	170 ± 79	160 ± 67	0 ± 0
	Donor 4	0 ± 0	0 ± 0	160 ± 67	0 ± 0	0 ± 0
	Donor 5	0 ± 0	0 ± 0	140 ± 127	140 ± 127	0 ± 0
	Donor 6	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
	Donor 7	60 ± 48	0 ± 0	80 ± 67	0 ± 0	145 ± 106
	Average	66 ± 36	9 ± 9	110 ± 50	43 ± 28	21 ± 21
GBM	Modified GB-1 (SFU ± SEM)
TP53		R175H		G245S	R273H
mutation	R158H	H179R	V216M	R248W	C275Y
Donor 1	1,780 ± 1,365	810 ± 504	940 ± 669	660 ± 583	1,000 ± 621
Donor 2	5,120 ± 877	3,030 ± 1,116	5,110 ± 712	1,830 ± 586	3,350 ± 786
Donor 3	1,690 ± 825	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 4	2,580 ± 1,373	1,200 ± 645	3,800 ± 2,005	2,470 ± 167	2,870 ± 1,533
Donor 5	1,080 ± 331	955 ± 532	1,870 ± 829	1,080 ± 340	510 ± 383
Donor 6	1,548 ± 527	1,168 ± 492	1,110 ± 594	1,220 ± 395	1,700 ± 376
Donor 7	0 ± 0	810 ± 552	0 ± 0	0 ± 0	155 ± 127
Average	1,971 ± 603	1,139 ± 349	1,833 ± 734	1,037 ± 345	1,369 ± 500
	Unmodified GB-1 (SFU ± SEM)
	GBM	PTEN			PIK3CA
	Driver	R130Q	PTEN	PIK3R1	M1043V	EGFR
	mutation	R132D	R173H	G376R	H14047R	G598V
	Donor 1	270 ± 168	160 ± 71	55 ± 33	0 ± 0	263 ± 156
	Donor 2	0 ± 0	150 ± 57	0 ± 0	0 ± 0	0 ± 0
	Donor 3	70 ± 25	0 ± 0	80 ± 73	0 ± 0	0 ± 0
	Donor 4	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
	Donor 5	55 ± 33	0 ± 0	0 ± 0	0 ± 0	0 ± 0
	Donor 6	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
	Donor 7	0 ± 0	0 ± 0	80 ± 52	0 ± 0	340 ± 189
	Average	56 ± 37	44 ± 29	31 ± 15	0 ± 0	86 ± 56
	Modified GB-1 (SFU ± SEM)
GBM	PTEN			PIK3CA
Driver	R130Q	PTEN	PIK3R1	M1043V	EGFR
mutation	R132D	R173H	G376R	H14047R	G598V
Donor 1	750 ± 455	3,430 ± 1,892	3,118 ± 1,311	785 ± 594	2,583 ± 1,441
Donor 2	3,180 ± 905	4,520 ± 884	3,240 ± 451	3,680 ± 1,479	2,450 ± 1,450
Donor 3	0 ± 0	900 ± 521	830 ± 480	450 ± 303	0 ± 0
Donor 4	2,290 ± 1,055	3,020 ± 591	3,275 ± 1,717	2,210 ± 704	870 ± 463
Donor 5	535 ± 309	800 ± 495	820 ± 255	1,010 ± 402	588 ± 283
Donor 6	385 ± 168	1,910 ± 494	850 ± 270	2,270 ± 204	720 ± 305
Donor 7	340 ± 227	1,348 ± 457	1,165 ± 587	0 ± 0	430 ± 332
Average	1,069 ± 449	2,275 ± 534	1,900 ± 466	1,486 ± 487	1,091 ± 382

Immune Responses to GBM EGFR Driver Mutations (SEQ ID NO: 51) Encoded by GBM Construct 2 Expressed by the LN-229 Cell Line

LN-229 modified to (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ1 and CD276; and (iii) express modPSMA; was modified with lentiviral particles expressing seven peptide sequences encoding EGFR driver mutations A289D, V774M, R108K, S645C, R252C, H304Y and G63R.

Immune responses to EGFR driver mutations were evaluated by IFNγ ELISpot. Specifically, 1.5×106 of the parental, unmodified LN-229 cell line or the modified LN-229 cell described above and herein were co-cultured with 1.5×106 iDCs from six HLA diverse donors (n=4/donor). HLA-A, HLA-B, and HLA-C alleles for each of the seven donors are in Table 2-13. CD14-PBMCs primed with DCs loaded with unmodified LN-229 or modified LN-229 were isolated from co-culture on day 6. Primed CD14-PBMCs were stimulated with peptide pools, 15-mers overlapping by 9 amino acids, designed to span the length of the inserted driver mutations, excluding the furin cleavage sequences (Thermo Scientific Custom Peptide Service) for 24 hours in the ELISpot assay prior to detection of IFNγ production.

TABLE 2-13
Healthy Donor MHC-I characteristics
	Donor #	HLA-A	HLA-B	HLA-C
	1	*01:01 *01:01	*27:05 *37:01	*01:02 *06:02
	2	*01:01 *30:01	*08:01 *13:02	*06:02 *07:01
	3	*01:01 *32:01	*35:01 *40:06	*04:01 *15:02
	4	*01:01 *03:01	*07:02 *44:02	*05:01 *07:02
	5	*01:01 *32:01	*08:01 *14:01	*07:01 *08:02
	6	*29:01 *29:02	*44:03 *50:01	*06:02 *16:01

FIG. 2 describes immune responses to seven EGFR driver mutations encoding peptides inserted into GBM vaccine-A LN-229 cell line by six HLA-diverse donors determined by IFNγ ELISpot. Modified LN-229 induced IFNγ responses against EGFR driver mutations that were greater in magnitude compared to the unmodified LN-229 cell line (Table 2-14). The trend of increased magnitude of IFNγ responses induced by modified LN-229 against the seven EGFR driver mutations did not reach statistical significance compared to unmodified LN-229 cell line. Statistical significance was determined using the Mann-Whitney U test.

TABLE 2-14
Immune responses to EGFR driver mutations
GBM
EGFR
Mutation	G63R	A289D	V774M	R108K	S645C	R252C	H304Y
Unmodified LN-229 (SFU ± SEM)
Donor 1	65 ± 38	210 ± 134	0 ± 0	0 ± 0	90 ± 44	0 ± 0	90 ± 41
Donor 2	320 ± 114	260 ± 83	135 ± 56	90 ± 53	185 ± 70	270 ± 118	118 ± 85
Donor 3	240 ± 54	170 ± 93	210 ± 43	210 ± 10	100 ± 42	160 ± 59	160 ± 28
Donor 4	850 ± 255	445 ± 275	400 ± 349	360 ± 309	340 ± 219	390 ± 283	0 ± 0
Donor 5	180 ± 62	180 ± 107	0 ± 0	0 ± 0	440 ± 286	380 ± 222	130 ± 94
Donor 6	0 ± 0	50 ± 33	340 ± 240	170 ± 101	660 ± 502	600 ± 499	0 ± 0
Average	276 ± 124	219 ± 53	181 ± 69	138 ± 57	303 ± 91	300 ± 85	83 ± 28
Modified LN-229 (SFU ± SEM)
Donor 1	805 ± 795	1,990 ± 1,334	1,893 ± 688	205 ± 135	1,400 ± 652	930 ± 538	0 ± 0
Donor 2	1,780 ± 957	2,185 ± 833	615 ± 346	1,960 ± 932	1,445 ± 637	830 ± 483	0 ± 0
Donor 3	3,570 ± 1,721	1,160 ± 386	728 ± 305	1,600 ± 1,043	0 ± 0	570 ± 506	0 ± 0
Donor 4	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 5	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 6	820 ± 426	730 ± 423	0 ± 0	590 ± 397	250 ± 212	0 ± 0	0 ± 0
Average	1,163 ± 552	1,011 ± 387	539 ± 302	726 ± 348	516 ± 289	388 ± 180	0 ± 0

Genetic modifications completed for GBM vaccine-A and GBM vaccine-B cell lines are described in Table 2-15 below. Where indicated, expression of CD276 was decreased by gene knock out (KO) using electroporation of zinc-finger nucleases (i.e., zinc finger nuclease pair specific for CD276 targeting the genomic DNA sequence: GGCAGCCCTGGCATGggtgtgCATGTGGGTGCAGCC; SEQ ID NO: 52) or by lentiviral transduction of CD276 shRNA, ccggtgctggagaaagatcaaacagctcgagctgtttgatctttctccagcatttttt (SEQ ID NO: 53). All other genetic modifications were completed by lentiviral transduction, including TGFβ1 shRNA (shTGFβ1, mature antisense sequence: TTTCCACCATTAGCACGCGGG (SEQ ID NO: 54) and TGFβ2 shRNA (mature antisense sequence: AATCTGATATAGCTCAATCCG (SEQ ID NO: 55).

GBM Vaccine-A

LN-229 (ATCC, CRL-2611) was modified to reduce expression of CD276 (zinc-finger nuclease; SEQ ID NO: 52), knockdown (KD) secretion of transforming growth factor-beta 1 (TGFβ1) (shRNA; SEQ ID NO: 54), and to express granulocyte macrophage-colony stimulating factor (GM-CSF) (SEQ ID NO: 7, SEQ ID NO: 8), membrane-bound CD40L (mCD40L) (SEQ ID NO: 2, SEQ ID NO: 3), interleukin 12 p70 (IL-12) (SEQ ID NO: 9, SEQ ID NO: 10) and modPSMA (SEQ ID NO: 29, SEQ ID NO: 30), and peptide sequences encoding EGFR driver mutations A289D, V774M, R108K, S645C, R252C, H304Y and G63R (GBM DM construct 2; SEQ ID NO: 50, SEQ ID NO: 51).

GB-1 (JCRB, IF050489) was modified to reduce expression of CD276 (zinc-finger nuclease; SEQ ID NO: 52), reduce secretion of TGFβ1 (shRNA; SEQ ID NO: 54), and to express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), mCD40L (SEQ ID NO: 2, SEQ ID NO: 3), IL-12 (SEQ ID NO: 9, SEQ ID NO: 10), and peptide sequences encoding EGFR driver mutation G598V, TP53 driver mutations R175H, H179R, G245S, R248W, R273H, C275Y, V216M, and R158H, PTEN driver mutations R130Q, G132D, and R173H, PIK3CA driver mutations M1043V and H1047R, and PIK3R1 driver mutation G376R (GBM DM construct 1; SEQ ID NO: 48, SEQ ID NO: 49).

SF-126 (JCRB, IF050286) was modified to reduce expression of CD276 (zinc-finger nuclease; SEQ ID NO: 52), reduce secretion of TGFβ1 (shRNA; SEQ ID NO: 54) and transforming growth factor-beta 2 (TGFβ2) (shRNA; SEQ ID NO: 55), and to express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), mCD40L (SEQ ID NO: 2, SEQ ID NO: 3), IL-12 (SEQ ID NO: 9, SEQ ID NO: 10) and modTERT (SEQ ID NO: 28).

GBM Vaccine-B

DBTRG-05MG (ATCC, CRL-2020) was modified to reduce expression of CD276 (shRNA; SEQ ID NO: 53), reduce secretion of TGFβ1 (shRNA; SEQ ID NO: 54), and to express GM-CSF (SEQ ID NO: 7; SEQ ID NO: 8), mCD40L (SEQ ID NO: 2, SEQ ID NO: 3) and IL-12 (SEQ ID NO: 9, SEQ ID NO: 10).

KNS 60 (JCRB, IF050357) was modified to reduce expression of CD276 (zinc-finger nuclease; SEQ ID NO: 52), reduce secretion of TGFβ1 (shRNA; SEQ ID NO: 54) and TGFβ2 (shRNA; SEQ ID NO: 55), and to express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), mCD40L (SEQ ID NO: 2, SEQ ID NO: 3), IL-12 (SEQ ID NO: 9, SEQ ID NO: 10), modMAGEA1 (SEQ ID NO: 31, SEQ ID NO: 32), EGFRvIII (SEQ ID NO: 31, SEQ ID NO: 32), and HCMV pp65 (SEQ ID NO: 31, SEQ ID NO: 32).

DMS 53 (ATCC, CRL-2062) was cell line modified to reduce expression of CD276 (zinc-finger nuclease; SEQ ID NO: 52), reduce secretion of TGFβ2 (shRNA; SEQ ID NO: 55), and to express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8) and mCD40L (SEQ ID NO: 2, SEQ ID NO: 3).

TABLE 2-15
Glioblastoma Multiforme vaccine cell line nomenclature and genetic modifications
								Tumor-
								Associated
	Cell	CD276	TGFβ1	TGFβ2				Antigens	Driver
Cocktail	Line	KO/KD	KD	KD	GM-CSF	mCD40L	IL-12	(TAAs)	Mutations
A	LN-229	SEQ ID	SEQ ID	—	SEQ ID	SEQ ID	SEQ ID	modPSMA	EGFR
		NO: 52	NO: 54		NO: 8	NO: 3	NO: 10	(SEQ ID NO: 30)	(SEQ ID NO: 51)
A	GB-1*	SEQ ID	SEQ ID	—	SEQ ID	SEQ ID	SEQ ID	—	EGFR, TP53, PTEN,
		NO: 52	NO: 54		NO: 8	NO: 3	NO: 10		PIK3CA, PIK3R1
									(SEQ ID NO: 49)
A	SF-126	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	modTERT	—
		NO: 52	NO: 54	NO: 55	NO: 8	NO: 3	NO: 10	(SEQ ID NO: 28)
B	DBTRG-	SEQ ID{circumflex over ( )}	SEQ ID	—	SEQ ID	SEQ ID	SEQ ID		—
	05MG*	NO: 53	NO: 54		NO: 8	NO: 3	NO: 10
B	KNS 60	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	modMAGEA1	—
		NO: 52	NO: 54	NO: 55	NO: 8	NO: 3	NO: 10	EGFRvIII
								HCMV pp65
								(SEQ ID NO: 32)
B	DMS 53*	SEQ ID	—	SEQ ID	SEQ ID	SEQ ID	—	—	—
		NO: 52		NO: 55	NO: 8	NO: 3
—, not completed/not required.
*Cell line identified as CSC-like.
{circumflex over ( )}CD276 KD. mCD40L, membrane bound CD40L.

Example 3: Prostate Cancer (PCa) Driver Mutation Identification, Selection and Design

Example 3 describes the process for identification, selection, and design of driver mutations expressed by PCa patient tumors and that expression of these driver mutations by PCa vaccine component cell lines can generate a PCa anti-tumor response in an HLA diverse population.

Example 31 of WO/2021/113328 first described a PCa vaccine that included two cocktails, each including three modified cell lines as follows. Cocktail A: (a) PC3 is modified to (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ1, TGFβ2 and CD276; and (iii) express modTBXT and modMAGEC2; (b) NEC8 is modified to (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression of CD276; (c) NTERA-2cl-D1 is modified to (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression of CD276; and Cocktail B: (a) DMS 53 is modified to (i) increase expression of GM-CSF and membrane bound CD40L; and (ii) decrease expression of TGFβ2 and CD276; (b) DU145 modified to (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression of CD276; and (iii) express modPSMA; (c) LNCAP is modified to (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; and (ii) decrease expression of CD276.

As described herein, driver mutations have now been identified and included in certain cell lines of the PCa vaccine and potent immune responses have been detected.

Identification of Frequently Mutated Oncogenes in PCa

Table 3-1 below shows the selected oncogenes that exhibit greater than 5% mutation frequency (percentage of samples with one or more mutations) in 1499 PCa profiled patient samples.

TABLE 3-1
Oncogenes in PCa with greater than 5% mutation frequency
		Number of samples		Percentage of samples
	Total number	with one or more	Profiled	with one or more	Is Cancer Gene
Gene	of mutations	mutations	Samples	mutations	(source: OncoKB)
TP53	371	363	1500	24.20%	Yes
SPOP	138	136	1500	9.10%	Yes
KMT2D	123	107	1500	7.10%	Yes
KMT2C	103	92	1500	6.10%	Yes
FOXA1	98	95	1500	6.30%	Yes
AR	104	89	1500	5.90%	Yes

Identification of Driver Mutations in Selected GBM Oncogenes

The PCa driver mutations in TP53, SPOP and AR occurring in ≥0.5% of profiled patient samples (Frequency) are listed in Table 3-2. Among all PCa oncogenes listed in Table 3-1 above, missense mutations occurring at the same amino acid position in ≥0.5% of profiled patient samples were not found for KMT2D, KMT2C and FOXA1.

TABLE 3-2
Identified driver mutations in selected PCa oncogenes
	Driver	Number of samples	Total number of	Fre-
Gene	Mutations	with mutation	samples	quency
TP53	R282W	7	1500	0.50%
	R175H	8	1500	0.50%
	Y220C	12	1500	0.80%
	R273H	12	1500	0.80%
	R248Q	22	1500	1.50%
	R273C	24	1500	1.60%
SPOP	Y87C	7	1500	0.50%
	F102C	7	1500	0.50%
	F102V	7	1500	0.50%
	F133I	8	1500	0.50%
	W131G	16	1500	1.10%
	F133L	20	1500	1.30%
	F133V	20	1500	1.30%
AR	W742C	11	1500	0.70%
	H875Y	19	1500	1.30%
	T878A	19	1500	1.30%
	L702H	20	1500	1.30%

Prioritization and Selection of Identified PCa Driver Mutations

The results of the completed CD4 and CD8 epitope analysis, the total number of HLA-A and HLA-B supertype-restricted 9-mer CD8 epitopes, the total number of CD4 epitopes and frequency (%) for each mutation are shown in Table 3-3. Ten PCa driver mutations encoded by ten peptide sequences were initially selected and included as vaccine targets. Among these ten selected driver mutations, AR T878A was endogenously expressed by one of PCa vaccine component cell lines (LNCaP) and therefore was excluded from the final construct insert design. Driver mutation AR T878A would be selected for inclusion in the final construct design if it was not expressed by LNCaP.

TABLE 3-3
Prioritization and selection of identified PCa driver mutations
		Number of total	Frequency	Number of total	Included as a
	Driver	CD8 epitopes	(%)	CD4 epitopes	vaccine target?
Gene	mutations	(SB + WB)	(n = 1500)	(SB + WB)	Yes (Y) or No (N)
TP53	R175H	2	0.5	0	Y
	Y220C	2	0.8	0	Y
	R248Q	0	1.5	0	N
	R273C	1	1.6	0	Y
	R273H	1	0.8	6	N
	R282W	0	0.5	14	N
SPOP	Y87C	4	0.5	0	Y
	F102C	5	0.5	0	N
	F102V	5	0.5	7	Y
	W131G	1	1.1	0	N
	F133I	1	0.5	72	N
	F133L	3	1.3	23	Y
	F133V	1	1.3	50	N
	W131G			32
	F133L	0	2.4		N
AR	L702H	4	1.3	0	Y
	W742C	10	0.7	0	Y
	H875Y	13	1.3	49	Y
	T878A	9	1.3	0	Y (LNCaP)

The total number of CD8 epitopes for each HLA-A and HLA-B supertype introduced by 9 selected PCa driver mutations (encoded by 9 peptide sequences) is shown in Table 3-4.

TABLE 3-4
CD8 epitopes introduced by 9 selected PCa driver
mutations encoded by 9 peptide sequences
		HLA-A	HLA-B	Total number
		Supertypes	Supertypes	of CD8
Gene	Mutations	(n = 5)	(n = 7)	epitopes
TP53	R175H	1	1	2
	Y220C	0	2	2
	R273C	0	1	1
SPOP	Y87C	2	2	4
	F102V	0	5	5
	F133L	2	1	3
AR	L702H	2	2	4
	W742C	4	6	10
	H875Y	5	8	13

The total number of CD4 epitopes for Class II locus DRB1, DRB 3/4/5, DQA1/DQB1 and DPB1 introduced by 9 selected PCa driver mutations (encoded by 9 peptide sequences) are shown in Table 3-5.

TABLE 3-5
CD4 epitopes introduced by 9 selected PCa driver
mutations encoded by 9 peptide sequences
			DRB	DQA1		Total number
		DRB1	3/4/5	DQB1	DPB1	of CD4
Gene	Mutations	(n = 26)	(n = 6)	(n = 8)	(n = 6)	epitopes
TP53	R175H	0	0	0	0	0
	Y220C	0	0	0	0	0
	R273C	0	0	0	0	0
SPOP	Y87C	0	0	0	0	0
	F102V	0	0	0	7	7
	F133L	4	5	1	13	23
AR	L702H	0	0	0	0	0
	W742C	0	0	0	0	0
	H875Y	18	11	1	19	49

Generation of the Construct Encoding 9 Selected PCa Driver Mutations

The 9 selected PCa driver mutations shown in Table 3-6 were assembled into a single construct insert. Once the construct insert was assembled, the analysis of PCa patient sample coverage was performed as described in Example 1 and herein. Results indicated that the PCa patient sample coverage by the insert encoding nine driver mutations was 7.2% (Table 3-7). When the driver mutation T878A that was carried by one of PCa vaccine component cell lines was also included, the total PCa patient sample coverage by all ten identified PCa driver mutations was 8.2% (Table 3-8).

TABLE 3-6
Generation of the construct encoding
9 selected PCa driver mutations
					Total CD4
		Frequency	Total CD8	Total CD4	and CD8
Gene	Mutations	(%)	epitopes	epitopes	epitopes
TP53	R175H	0.5	2	0	2
	Y220C	0.8	2	0	2
	R273C	1.6	1	0	1
SPOP	Y87C	0.5	4	0	4
	F102V	0.5	5	7	12
	F133L	1.3	3	23	26
AR	L702H	1.3	4	0	4
	W742C	0.7	10	0	10
	H875Y	1.3	13	49	62

TABLE 3-7
PCa patient sample coverage by the construct encoding driver mutations
		Total number of
Coverage (Construct Insert Only)	Driver Mutation Target Gene	Samples with	Total Sample
Sample Description	TP53	SPOP	AR	Driver Mutations	(n = 1713)
# of samples with one DM	41	31	40	112	6.5%
Samples	0	0	5	5	0.3%
with ≥2 DMs from
same antigen
Samples with ≥2 DMs from				6	0.4%
different antigens
			Total	123	7.2%

TABLE 3-8
PCa patient sample coverage by the construct encoding driver
mutations and the cell line carrying driver mutation AR T878A
	Total number of
Coverage (Construct Insert & Cell Line)	Driver Mutation Target Gene	Samples with	Total Sample
Sample Description	TP53	SPOP	AR	Driver Mutations	(n = 1713)
# of samples	41	31	55	127	7.4%
with one DM
Samples	0	0	8	8	0.5%
with ≥2 DMs from
same antigen
Samples				6	0.4%
with ≥2 DMs from
different antigens
			Total	141	8.2%

Oncogene Sequences and Insert Sequences of the PCa Driver Mutation Construct

The DNA and protein sequences of oncogenes with selected driver mutations are included in Table 3-9. TP53 native DNA and protein sequences are described in Table 2-10. The construct (SEQ ID NO: 60 and SEQ ID NO: 61) insert gene encodes 336 amino acids containing the driver mutation sequences identified from TP53 (SEQ ID NO: 41), SPOP (SEQ ID NO: 57) and AR (SEQ ID NO: 59) that were separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37).

TABLE 3-9
Oncogene sequences and insert sequences for the PCa construct
SPOP             DNA Sequence
(SEQ ID NO:      1 ATGTCAAGGG TTCCAAGTCC TCCACCTCCG GCAGAAATGT CGAGTGGCCC CGTAGCTGAG
56)              61 AGTTGGTGCT ACACACAGAT CAAGGTAGTG AAATTCTCCT ACATGTGGAC CATCAATAAC
                 121 TTTAGCTTTT GCCGGGAGGA AATGGGTGAA GTCATTAAAA GTTCTACATT TTCATCAGGA
                 181 GCAAATGATA AACTGAAATG GTGTTTGCGA GTAAACCCCA AAGGGTTAGA TGAAGAAAGC
                 241 AAAGATTACC TGTCACTTTA CCTGTTACTG GTCAGCTGTC CAAAGAGTGA AGTTCGGGCA
                 301 AAATTCAAAT TCTCCATCCT GAATGCCAAG GGAGAAGAAA CCAAAGCTAT GGAGAGTCAA
                 361 CGGGCATATA GGTTTGTGCA AGGCAAAGAC TGGGGATTCA AGAAATTCAT CCGTAGAGAT
                 421 TTTCTTTTGG ATGAGGCCAA CGGGCTTCTC CCTGATGACA AGCTTACCCT CTTCTGCGAG
                 481 GTGAGTGTTG TGCAAGATTC TGTCAACATT TCTGGCCAGA ATACCATGAA CATGGTAAAG
                 541 GTTCCTGAGT GCCGGCTGGC AGATGAGTTA GGAGGACTGT GGGAGAATTC CCGGTTCACA
                 601 GACTGCTGCT TGTGTGTTGC CGGCCAGGAA TTCCAGGCTC ACAAGGCTAT CTTAGCAGCT
                 661 CGTTCTCCGG TTTTTAGTGC CATGTTTGAA CATGAAATGG AGGAGAGCAA AAAGAATCGA
                 721 GTTGAAATCA ATGATGTGGA GCCTGAAGTT TTTAAGGAAA TGATGTGCTT CATTTACACG
                 781 GGGAAGGCTC CAAACCTCGA CAAAATGGCT GATGATTTGC TGGCAGCTGC TGACAAGTAT
                 841 GCCCTGGAGC GCTTAAAGGT CATGTGTGAG GATGCCCTCT GCAGTAACCT GTCCGTGGAG
                 901 AACGCTGCAG AAATTCTCAT CCTGGCCGAC CTCCACAGTG CAGATCAGTT GAAAACTCAG
                 961 GCAGTGGATT TCATCAACTA TCATGCTTCG GATGTCTTGG AGACCTCTGG GTGGAAGTCA
                 1021 ATGGTGGTGT CACATCCCCA CTTGGTGGCT GAGGCATACC GCTCTCTGGC TTCAGCACAG
                 1081 TGCCCTTTTC TGGGACCCCC ACGCAAACGC CTGAAGCAAT CC
SPOP             Protein Sequence
(SEQ ID NO:      1 MSRVPSPPPP AEMSSGPVAE SWCYTQIKVV KFSYMWTINN FSFCREEMGE VIKSSTFSSG
57)              61 ANDKLKWCLR VNPKGLDEES KDYLSLYLLL VSCPKSEVRA KFKFSILNAK GEETKAMESQ
                 121 RAYRFVQGKD WGFKKFIRRD FLLDEANGLL PDDKLTLFCE VSVVQDSVNI SGQNTMNMVK
                 181 VPECRLADEL GGLWENSRFT DCCLCVAGQE FQAHKAILAA RSPVFSAMFE HEMEESKKNR
                 241 VEINDVEPEV FKEMMCFIYT GKAPNLDKMA DDLLAAADKY ALERLKVMCE DALCSNLSVE
                 301 NAAEILILAD LHSADQLKTQ AVDFINYHAS DVLETSGWKS MVVSHPHLVA EAYRSLASAQ
                 361 CPFLGPPRKR LKQS
AR               DNA Sequence
(SEQ ID          1 ATGGAAGTGC AGTTAGGGCT GGGAAGGGTC TACCCTCGGC CGCCGTCCAA GACCTACCGA
NO:58)           61 GGAGCTTTCC AGAATCTGTT CCAGAGCGTC CGCGAAGTGA TCCAGAACCC GGGCCCCAGG
                 121 CACCCAGAGG CCGCGAGCGC AGCACCTCCC GGCGCCAGTT TGCTGCTGCT GCAGCAGCAG
                 181 CAGCAGCAGC AGCAGCAGCA GCAGCAGCAG CAGCAGCAAG AGACTAGCCC CAGGCAGCAG
                 241 CAGCAGCAGC AGGGTGAGGA TGGTTCTCCC CAAGCCCATC GTAGAGGCCC CACAGGCTAC
                 301 CTGGTCCTGG ATGAGGAACA GCAACCTTCA CAGCCGCAGT CGGCCCTGGA GTGCCACCCC
                 361 GAGAGAGGTT GCGTCCCAGA GCCTGGAGCC GCCGTGGCCG CCAGCAAGGG GCTGCCGCAG
                 421 CAGCTGCCAG CACCTCCGGA CGAGGATGAC TCAGCTGCCC CATCCACGTT GTCCCTGCTG
                 481 GGCCCCACTT TCCCCGGCTT AAGCAGCTGC TCCGCTGACC TTAAAGACAT CCTGAGCGAG
                 541 GCCAGCACCA TGCAACTCCT TCAGCAACAG CAGCAGGAAG CAGTATCCGA AGGCAGCAGC
                 601 AGCGGGAGAG CGAGGGAGGC CTCGGGGGCT CCCACTTCCT CCAAGGACAA TTACTTAGGG
                 661 GGCACTTCGA CCATTTCTGA CAACGCCAAG GAGTTGTGTA AGGCAGTGTC GGTGTCCATG
                 721 GGCCTGGGTG TGGAGGCGTT GGAGCATCTG AGTCCAGGGG AACAGCTTCG GGGGGATTGC
                 781 ATGTACGCCC CACTTTTGGG AGTTCCACCC GCTGTGCGTC CCACTCCTTG TGCCCCATTG
                 841 GCCGAATGCA AAGGTTCTCT GCTAGACGAC AGCGCAGGCA AGAGCACTGA AGATACTGCT
                 901 GAGTATTCCC CTTTCAAGGG AGGTTACACC AAAGGGCTAG AAGGCGAGAG CCTAGGCTGC
                 961 TCTGGCAGCG CTGCAGCAGG GAGCTCCGGG ACACTTGAAC TGCCGTCTAC CCTGTCTCTC
                 1021 TACAAGTCCG GAGCACTGGA CGAGGCAGCT GCGTACCAGA GTCGCGACTA CTACAACTTT
                 1081 CCACTGGCTC TGGCCGGACC GCCGCCCCCT CCGCCGCCTC CCCATCCCCA CGCTCGCATC
                 1141 AAGCTGGAGA ACCCGCTGGA CTACGGCAGC GCCTGGGCGG CTGCGGCGGC GCAGTGCCGC
                 1201 TATGGGGACC TGGCGAGCCT GCATGGCGCG GGTGCAGCGG GACCCGGTTC TGGGTCACCC
                 1261 TCAGCCGCCG CTTCCTCATC CTGGCACACT CTCTTCACAG CCGAAGAAGG CCAGTTGTAT
                 1321 GGACCGTGTG GTGGTGGTGG GGGTGGTGGC GGCGGCGGCG GCGGCGGCGG CGGCGGCGAG
                 1381 GCGGGAGCTG TAGCCCCCTA CGGCTACACT CGGCCCCCTC AGGGGCTGGC GGGCCAGGAA
                 1441 AGCGACTTCA CCGCACCTGA TGTGTGGTAC CCTGGCGGCA TGGTGAGCAG AGTGCCCTAT
                 1501 CCCAGTCCCA CTTGTGTCAA AAGCGAAATG GGCCCCTGGA TGGATAGCTA CTCCGGACCT
                 1561 TACGGGGACA TGCGTTTGGA GACTGCCAGG GACCATGTTT TGCCCATTGA CTATTACTTT
                 1621 CCACCCCAGA AGACCTGCCT GATCTGTGGA GATGAAGCTT CTGGGTGTCA CTATGGAGCT
                 1681 CTCACATGTG GAAGCTGCAA GGTCTTCTTC AAAAGAGCCG CTGAAGGGAA ACAGAAGTAC
                 1741 CTGTGCGCCA GCAGAAATGA TTGCACTATT GATAAATTCC GAAGGAAAAA TTGTCCATCT
                 1801 TGTCGTCTTC GGAAATGTTA TGAAGCAGGG ATGACTCTGG GAGCCCGGAA GCTGAAGAAA
                 1861 CTTGGTAATC TGAAACTACA GGAGGAAGGA GAGGCTTCCA GCACCACCAG CCCCACTGAG
                 1921 GAGACAACCC AGAAGCTGAC AGTGTCACAC ATTGAAGGCT ATGAATGTCA GCCCATCTTT
                 1981 CTGAATGTCC TGGAAGCCAT TGAGCCAGGT GTAGTGTGTG CTGGACACGA CAACAACCAG
                 2041 CCCGACTCCT TTGCAGCCTT GCTCTCTAGC CTCAATGAAC TGGGAGAGAG ACAGCTTGTA
                 2101 CACGTGGTCA AGTGGGCCAA GGCCTTGCCT GGCCTCCGCA ACTTACACGT GGACGACCAG
                 2161 ATGGCTGTCA TTCAGTACTC CTGGATGGGG CTCATGGTGT TTGCCATGGG CTGGCGATCC
                 2221 TTCACCAATG TCAACTCCAG GATGCTCTAC TTCGCCCCTG ATCTGGTTTT CAATGAGTAC
                 2281 CGCATGCACA AGTCCCGGAT GTACAGCCAG TGTGTCCGAA TGAGGCACCT CTCTCAAGAG
                 2341 TTTGGATGGC TCCAAATCAC CCCCCAGGAA TTCCTGTGCA TGAAAGCCAT GCTACTCTTC
                 2401 AGCATTATTC CAGTGGATGG GCTGAAAAAT CAAAAATTCT TTGATGAACT TCGAATGAAC
                 2461 TACATCAAGG AACTCGATCG TATCATTGCA TGCAAAAGAA AAAATCCCAC ATCCTGCTCA
                 2521 AGACGCTTCT ACCAGCTCAC CAAGCTCCTG GACTCCGTGC ATCCTATTGC GAGAGAGCTG
                 2581 CATCAGTTCA CTTTTGACCT GCTAATCAAG TCACACATGG TGAGCGTGGA CTTTCCGGAA
                 2641 ATGATGGCAG AGATCATCTC TGTGCAAGTG CCCAAGATCC TTTCTGGGAA AGTCAAGCCC
                 2701 ATCTATTTCC ACACCCAG
AR               Protein Sequence
(SEQ ID          1 MEVQLGLGRV YPRPPSKTYR GAFQNLFQSV REVIQNPGPR HPEAASAAPP GASLLLLQQQ
NO: 59)          61 QQQQQQQQQQ QQQQQQQQQQ ETSPRQQQQQ QGEDGSPQAH RRGPTGYLVL DEEQQPSQPQ
                 121 SALECHPERG CVPEPGAAVA ASKGLPQQLP APPDEDDSAA PSTLSLLGPT FPGLSSCSAD
                 181 LKDILSEAST MQLLQQQQQE AVSEGSSSGR AREASGAPTS SKDNYLGGTS TISDNAKELC
                 241 KAVSVSMGLG VEALEHLSPG EQLRGDCMYA PLLGVPPAVR PTPCAPLAEC KGSLLDDSAG
                 301 KSTEDTAEYS PFKGGYTKGL EGESLGCSGS AAAGSSGTLE LPSTLSLYKS GALDEAAAYQ
                 361 SRDYYNFPLA LAGPPPPPPP PHPHARIKLE NPLDYGSAWA AAAAQCRYGD LASLHGAGAA
                 421 GPGSGSPSAA ASSSWHTLFT AEEGQLYGPC GGGGGGGGGG GGGGGGGGGG GGGEAGAVAP
                 481 YGYTRPPQGL AGQESDFTAP DVWYPGGMVS RVPYPSPTCV KSEMGPWMDS YSGPYGDMRL
                 541 ETARDHVLPI DYYFPPQKTC LICGDEASGC HYGALTCGSC KVFFKRAAEG KQKYLCASRN
                 601 DCTIDKFRRK NCPSCRLRKC YEAGMTLGAR KLKKLGNLKL QEEGEASSTT SPTEETTQKL
                 661 TVSHIEGYEC QPIFLNVLEA IEPGVVCAGH DNNQPDSFAA LLSSLNELGE RQLVHVVKWA
                 721 KALPGFRNLH VDDQMAVIQY SWMGLMVFAM GWRSFTNVNS RMLYFAPDLV FNEYRMHKSR
                 781 MYSQCVRMRH LSQEFGWLQI TPQEFLCMKA LLLFSIIPVD GLKNQKFFDE LRMNYIKELD
                 841 RIIACKRKNP TSCSRRFYQL TKLLDSVQPI ARELHQFTFD LLIKSHMVSV DFPEMMAEII
                 901 SVQVPKILSG KVKPIYFHTQ
PCa DM           DNA Sequence
construct insert 1 ATGTACCTCG ACGACCGGAA CACCTTCAGA CACAGCGTGG TGGTGCCTTG CGAGCCTCCT
(SEQ ID NO:      61 GAAGTGGGCA GCGATTGCAC CACCATCCAC TACAACAGAG GCCGGAAGCG GAGATCCATG
60)              121 GCCATCTACA AGCAGAGCCA GCACATGACC GAGGTCGTGC GGCACTGTCC TCACCACGAG
                 181 AGATGTAGCG ATAGCGACGG ACTGGCCCCT AGAGGCAGAA AGAGAAGATC CGAGGACAGC
                 241 AGCGGCAACC TGCTGGGCAG AAACAGCTTC GAAGTGTGCG TGTGTGCCTG TCCTGGCAGA
                 301 GACAGAAGGA CCGAGGAAGA GAACAGGGGC CGCAAGAGAA GAAGCAACCC TAAAGGCCTG
                 361 GACGAGGAAA GCAAGGACTA CCTGAGCCTG TGCCTGCTGC TGGTGTCCTG TCCTAAGTCT
                 421 GAAGTGCGGG CCAAGTTCCG GGGCAGAAAG CGGAGAAGTT ACCTGCTGCT CGTCAGCTGC
                 481 CCCAAGAGCG AAGTTCGCGC CAAAGTGAAG TTCAGCATCC TGAACGCCAA GGGCGAAGAG
                 541 ACAAAGGCCA TGAGAGGACG GAAACGGCGG AGCGCCATGG AATCTCAGAG GGCCTACAGA
                 601 TTCGTGCAGG GCAAAGACTG GGGCCTGAAG AAGTTTATCC GGCGGGACTT CCTGCTGGAT
                 661 GAGGCCAGAG GAAGAAAGCG CAGATCTTGT GCCGGCCACG ACAACAACCA GCCTGATAGC
                 721 TTTGCCGCTC TGCACAGCTC CCTGAACGAG CTGGGAGAAA GACAGCTGGT GCACGTTGTG
                 781 CGGGGAAGAA AGAGGCGGTC CAGAAACCTG CACGTGGACG ATCAGATGGC CGTGATCCAG
                 841 TACAGCTGCA TGGGCCTGAT GGTGTTCGCT ATGGGCTGGC GGAGCTTCAC CAACCGCGGA
                 901 CGGAAAAGAA GAAGCCTGAC AAAGCTGCTG GACAGCGTGC AGCCTATCGC CAGAGAGCTG
                 961 TACCAGTTCA CCTTCGACCT GCTGATCAAG AGCCACATGG TGTCCGTG
PCa DM           Protein Sequence*
construct insert 1 MYLDDRNTFR HSVVVPCEPP EVGSDCTTIH YNRGRKRRSM AIYKQSQHMT EVVRHCPHHE
(SEQ ID          61 RCSDSDGLAP RGRKRRSEDS SGNLLGRNSF EVCVCACPGR DRRTEEENRG RKRRSNPKGL
NO: 61)          121 DEESKDYLSL CLLLVSCPKS EVRAKFRGRK RRSYLLLVSC PKSEVRAKVK FSILNAKGEE
                 181 TKAMRGRKRR SAMESQRAYR FVQGKDWGLK KFIRRDFLLD EARGRKRRSC AGHDNNQPDS
                 241 FAALHSSLNE LGERQLVHVV RGRKRRSRNL HVDDQMAVIQ YSCMGLMVFA MGWRSFTNRG
                 301 RKRRSLTKLL DSVQPIAREL YQFTFDLLIK SHMVSV
*Driver mutation is highlighted in bold. The furin cleavage sequence is underlined.

Immune responses to TP53, SPOP and AR driver mutations (SEQ ID NO: 61) encoded by the PCa driver mutation Construct expressed by the PC3 cell line are described herein.

PC3 modified to (i) increase expression of GM-CSF, IL-12, and membrane bound CD40L; (ii) decrease expression of TGFβ1, TGFβ2 and CD276; and (iii) express modTBXT and modMAGEC2 was stably transduced with lentiviral particles to express nine peptide sequences encoding TP53 driver mutations Y220C, R175H and R273C, SPOP driver mutations Y87C, F102V and F133L, and AR driver mutations L702H, W742C and H875Y (SEQ ID NO: 61). Immune responses to TP53, SPOP and AR driver mutations were evaluated by IFNγ ELISpot. Specifically, 1.5×10⁶ of the parental, unmodified PC3 or modified PC3 described above were co-cultured with 1.5×10⁶ iDCs from six HLA diverse donors (n=4/donor). HLA-A, HLA-B, and HLA-C alleles for each of the six donors are described in Table 3-10. CD14-PBMCs primed with DCs loaded with unmodified PC3 or modified PC3 were isolated from co-culture on day 6. Primed CD14-PBMCs were stimulated with peptide pools, 15-mers overlapping by 9 amino acids, designed to span the length of the inserted driver mutations, excluding the furin cleavage sequences (Thermo Scientific Custom Peptide Service) for 24 hours in the ELISpot assay prior to detection of IFNγ production. For each driver mutation, the 15-mer peptides containing the driver mutation, and not flanking sequences, were pooled for stimulation of PBMCs in the IFNγ ELISpot assay.

TABLE 3-10
Healthy Donor MHC-I characteristics
	Donor #	HLA-A	HLA-B	HLA-C
	1	*02:01 *33:01	*07:02 *14:02	*07:02 *08:02
	2	*03:01 *25:01	*15:01 *44:02	*03:03 *05:01
	3	*02:01 *25:01	*18:01 *44:02	*12:02 *16:01
	4	*03:01 *11:01	*18:01 *57:01	*06:02 *07:02
	5	*01:01 *03:01	*07:02 *44:02	*05:01 *07:02
	6	*03:01 *31:01	*35:01 *40:01	*04:01 *07:02

FIG. 3 demonstrates priming donor CD14-PBMCs with the PC3 cell line modified as described above and herein induces stronger IFNγ responses to TP53 driver mutations Y220C, R175H and R273C (FIG. 3A), SPOP driver mutations Y87C, F102V and F133L (FIG. 3B), and AR driver mutations L702H, W742C and H875Y (FIG. 3C). IFNγ responses generated in individual Donors are described in Tables 3-11 (TP53 driver mutations), 3-12 (SPOP driver mutations) and 3-13 (AR driver mutations).

TABLE 3-11
Immune responses to TP53 driver mutations
PCa TP53
driver	Unmodified PC3 (SFU ± SEM)	Modified PC3 (SFU ± SEM)
mutation	Y220C	R175H	R273C	Y220C	R175H	R273C
Donor 1	180 ± 10	0 ± 0	0 ± 0	1,110 ± 865	630 ± 379	0 ± 0
Donor 2	115 ± 68	0 ± 0	0 ± 0	0 ± 0	1,303 ± 582	0 ± 0
Donor 3	0 ± 0	0 ± 0	0 ± 0	483 ± 247	205 ± 119	0 ± 0
Donor 4	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 5	150 ± 96	0 ± 0	90 ± 53	280 ± 254	210 ± 128	180 ± 155
Donor 6	0 ± 0	0 ± 0	100 ± 66	0 ± 0	0 ± 0	0 ± 0
Average	74 ± 34	0 ± 0	32 ± 20	312 ± 179	391 ± 205	30 ± 30

TABLE 3-12
Immune responses to SPOP driver mutations
PCa SPOP
driver	Unmodified PC3 (SFU ± SEM)	Modified PC3 (SFU ± SEM)
mutation	Y87C	F102V	F133L	Y87C	F102V	F133L
Donor 1	0 ± 0	150 ± 50	160 ± 123	2,200 ± 1,274	0 ± 0	660 ± 387
Donor 2	0 ± 0	0 ± 0	0 ± 0	248 ± 141	715 ± 276	0 ± 0
Donor 3	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	325 ± 188
Donor 4	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 5	0 ± 0	0 ± 0	100 ± 66	170 ± 160	0 ± 0	0 ± 0
Donor 6	0 ± 0	98 ± 62	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Average	0 ± 0	41 ± 27	43 ± 28	436 ± 355	119 ± 119	164 ± 112

TABLE 3-13
Immune responses to AR driver mutations
PCa AR
driver	Unmodified PC3 (SFU ± SEM)	Modified PC3 (SFU ± SEM)
mutation	L702H	W748C	H875Y	L702H	W748C	H875Y
Donor 1	140 ± 87	0 ± 0	120 ± 70	0 ± 0	700 ± 520	0 ± 0
Donor 2	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 3	0 ± 0	0 ± 0	0 ± 0	440 ± 254	580 ± 415	1,100 ± 639
Donor 4	0 ± 0	0 ± 0	0 ± 0	110 ± 64	0 ± 0	400 ± 236
Donor 5	110 ± 66	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 6	0 ± 0	0 ± 0	110 ± 85	0 ± 0	0 ± 0	1,350 ± 815
Average	42 ± 27	0 ± 0	38 ± 24	92 ± 72	213 ± 136	475 ± 248

Genetic modifications completed for PCa vaccine-A and PCa vaccine-B cell lines are described in Table 3-14 below. Where indicated, expression of CD276 was decreased by gene knock out (KO) using electroporation of zinc-finger nucleases (ZFNs) (SEQ ID NO: 52) as described herein. All other genetic modifications were completed by lentiviral transduction.

PCa Vaccine-A

PC3 (ATCC, CRL-1435) was modified to reduce expression of CD276 (zinc-finger nuclease; SEQ ID NO: 52), knockdown (KD) secretion of transforming growth factor-beta 1 (TGFβ1) (shRNA; SEQ ID NO: 54) and transforming growth factor-beta 2 (TGFβ2) (shRNA; SEQ ID NO: 55), and to express granulocyte macrophage-colony stimulating factor (GM-CSF) (SEQ ID NO: 7, SEQ ID NO: 8), membrane-bound CD40L (mCD40L) (SEQ ID NO: 2, SEQ ID NO: 3), interleukin 12 p70 (IL-12) (SEQ ID NO: 9, SEQ ID NO: 10), modTBXT (SEQ ID NO: 35, SEQ ID NO: 36), modMAGEC2 (SEQ ID NO: 35, SEQ ID NO: 36), and nine peptides encoding TP53 driver mutations Y220C, R175H and R273C, SPOP driver mutations Y87C, F102V and F133L, and AR driver mutations L702H, W742C and H875Y (as provided in PCa DM construct, SEQ ID NO: 60 and SEQ ID NO: 61).

NEC8 (JCRB, JCRB0250) was modified to reduce expression of CD276 (zinc-finger nuclease; SEQ ID NO: 52), and to express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), mCD40L (SEQ ID NO: 2, SEQ ID NO: 3), and IL-12 (SEQ ID NO: 9, SEQ ID NO: 10).

NTERA-2cl-D1 (ATCC, CRL-1973) was modified to reduce expression of CD276 (zinc-finger nuclease; SEQ ID NO: 52), and to express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), mCD40L (SEQ ID NO: 3, SEQ ID NO: 4), and IL-12 (SEQ ID NO: 9, SEQ ID NO: 10).

PCa Vaccine-B

DU145 (ATCC, HTB-81) was modified to reduce expression of CD276 (zinc-finger nuclease; SEQ ID NO: 52), and express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), mCD40L (SEQ ID NO: 2, SEQ ID NO: 3), IL-12 (SEQ ID NO: 9, SEQ ID NO: 10) and modPSMA (SEQ ID NO: 29, SEQ ID NO: 30).

LNCAP (ATCC, CRL-1740) was modified to reduce expression of CD276 (zinc-finger nuclease; SEQ ID NO: 52), and express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), mCD40L (SEQ ID NO: 2, SEQ ID NO: 3), IL-12 (SEQ ID NO: 9, SEQ ID NO: 10).

DMS 53 (ATCC, CRL-2062) was cell line modified to reduce expression of CD276 (zinc-finger nuclease; SEQ ID NO: 52), reduce secretion of TGFβ2 (shRNA; SEQ ID NO: 55), and express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8) and mCD40L (SEQ ID NO: 2, SEQ ID NO: 3).

TABLE 3-14
Prostate Cancer vaccine cell line nomenclature and genetic modifications
								Tumor-
								Associated
	Cell	CD276	TGFβ1	TGFβ2				Antigens	Driver
Cocktail	Line	KO	KD	KD	GM-CSF	mCD40L	IL-12	(TAAs)	Mutations
A	PC3	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	modTBXT	TP53, SPOP, AR
		NO: 52	NO: 54	NO: 55	NO: 8	NO: 3	NO: 10	modMAGEC2	(SEQ ID NO: 61)
								(SEQ ID NO: 36)
A	NEC8	SEQ ID	—	—	SEQ ID	SEQ ID	SEQ ID	—	—
		NO: 52			NO: 8	NO: 3	NO: 10
A	NTERA-	SEQ ID	—	—	SEQ ID	SEQ ID	SEQ ID		—
	2cl-D1	NO: 52			NO: 8	NO: 3	NO: 10
B	DU-145	SEQ ID	—	—	SEQ ID	SEQ ID	SEQ ID	modPSMA	—
		NO: 52			NO: 8	NO: 3	NO: 10	(SEQ ID NO: 30)
B	LNCaP	SEQ ID	—	—	SEQ ID	SEQ ID	SEQ ID	—	—
		NO: 52			NO: 8	NO: 3	NO: 10
B	DMS 53*	SEQ ID	—	SEQ ID	SEQ ID	SEQ ID		—	—
		NO: 52		NO: 55	NO: 8	NO: 3
—, not completed/not required.
*Cell line identified as CSC-like. mCD40L, membrane bound CD40L.

Example 4: Preparation of Non-Small Cell Lung Cancer Vaccines

Example 4 demonstrates reduction of CD276, TGFβ1 and TGFβ2 expression with concurrent expression of GM-CSF, membrane bound CD40L, and IL-12 in a NSCLC vaccine composition of two cocktails, each cocktail composed of three cell lines for a total of 6 cell lines, significantly increased the magnitude of cellular immune responses to at least eight full-length NSCLC tumor-associated antigens (TAAs) in an HLA-diverse population. This Example also describes the process for identification, selection, and design of driver mutations, EGFR activating mutations, EGFR and ALK acquired TKI resistance mutations expressed by NSCLC patient tumors. Expression of these mutations in certain cell lines of the NSCLC vaccine described above and herein can also generate a NSCLC anti-tumor response in an HLA diverse population.

As described herein, the first cocktail, NSCLC vaccine-A, is composed of cell line NCI-H460 also modified to express modBORIS and twenty NSCLC-specific driver mutations encoded by twelve peptides (Table 4-22), cell line NCI-H520, and cell line A549 also modified to express modTBXT, modWT1, KRAS driver mutations G12D and G12V (Table 26), and thirteen EGFR activating mutations encoded by twelve peptides (Table 4-30).

The second cocktail, NSCLC vaccine-B, is composed of cell line NCI-H23, also modified to express modMSLN, eight EGFR TKI acquired resistance mutations encoded by five peptides, twelve ALK TKI acquired resistance mutations encoded by seven peptides and modALK-IC (Table 4-44), cell line LK2, and cell line DMS 53.

The six NSCLC component cell lines collectively express at least twenty-four antigens, twenty-two NSCLC-specific driver mutations, thirteen EGFR activating mutations, eight EGFR acquired TKI resistance mutations, twelve ALK acquired TKI resistance mutations, and modALK intracellular domain that can provide an anti-NSCLC tumor response. Table 4-47, below, provides a summary of each cell line and the modifications associated with each cell line.

NSCLC Vaccine Components

Tumors and tumor cell lines are highly heterogeneous. The subpopulations within the tumor express different phenotypes with different biological potential and different antigenic profiles. For example, Cancer Stem Cells (CSCs) play a critical role in the metastasis, treatment resistance, and relapse of tumors. CSCs are relatively infrequent in solid tumors, and CSCs are identified by the expression and/or combinations of unique cell surface markers and stemness-related transcription factors that differ by tumor origin. Targeting the genes involved in cancer stem cell pathways is an important approach for cancer therapy. One advantage of a whole tumor cell vaccine is the ability to present a broad breadth of antitumor antigens to the immune system. By doing this, the immune response is generated against multiple antigens, bypassing issues related to antigen loss, which can lead to antigen escape (or immune relapse) and patient relapse (Keenan B P, et al., Semin Oncol. 2012; 39: 276-86).

The cell lines in the NSCLC vaccine described herein were selected to express a wide array of TAAs, including those known to be important specifically for NSCLC antitumor responses, such as MAGEA3 and PRAME, and TAAs known to be important for targets for NSCLC and other solid tumors, such as TERT. Prioritized TAAs for NSCLC were identified as described in Example 40 of WO/2021/113328 and herein. Expression of TAAs and NSCLC associated CSC-like markers by vaccine component cell lines were determined using RNA expression data sourced from the Broad Institute Cancer Cell Line Encyclopedia (CCLE). The HGNC gene symbol was included in the CCLE search and mRNA expression was downloaded for each TAA. Expression of a TAA or CSC marker by a cell line was considered positive if the RNA-seq value was >1.0. The six component cell lines expressed twelve to eighteen TAAs (FIG. 4A) and four to seven CSC markers (FIG. 4B).

As shown herein, to further enhance the breadth of TAAs, NCI-H460 was modified to express modBORIS and sixteen TP53 driver mutations, two PIK3CA driver mutations, and two KRAS driver mutations, A549 was modified to express modTBXT, modWT1, two KRAS driver mutations, and thirteen EGFR activating mutations, and NCI-H23 was modified to express modMSLN, eight EGFR acquired TKI resistance mutations, twelve ALK acquired TKI resistance mutations, and the modALK intracellular domain antigen. BORIS was not endogenously expressed in any of the six component cell lines at >1.0 FPKM. MSLN, TBXT and WT1 were expressed endogenously by one of six component cell lines at >1.0 FPKM. (FIG. 4A).

The present vaccine, after introduction of antigens as described above, expresses of all twenty-four prioritized TAAs with the potential to induce a NSCLC antitumor response. Some of these TAAs are known to be primarily enriched in NSCLC tumors and some can also induce an immune response to NSCLC and other solid tumors. RNA abundance of the twenty-four prioritized NSCLC TAAs was determined in 573 NSCLC patient samples with available mRNA data expression downloaded from the publicly available database, cBioPortal (cbioportal.org) (Cerami, E. et al. Cancer Discovery. 2012.; Gao, J. et al. Sci Signal. 2013.) (FIG. 4C). Five of the prioritized NSCLC TAAs were expressed by 100% of samples, 17 TAAs were expressed by 99.8% of samples, 18 TAAs were expressed by 99.1% of samples, 19 TAAs were expressed by 95.6% of samples, 20 TAAs were expressed by 83.2% of samples, 21 TAAs were expressed by 60.9% of samples, 22 TAAs were expressed by 40.1% of samples, 23 TAAs by 22.9% of samples, and 22 TAAs were expressed by 7.5% of samples (FIG. 4D).

Identification and design of antigens inserted into NSCLC vaccine cell lines was completed as described in Example 40 of WO/2021/113328. Identification, selection, and design of driver mutations targeting NSCLC tumors was completed as described in Example 1 and herein. Identification, selection, and design of vaccine inserts targeting NSCLC EGFR activating mutations, EGFR acquired TKI resistance mutations, and ALK acquired TKI resistance mutations was completed as described herein.

Expression of the transduced antigens modTBXT (SEQ ID NO: 18) (FIG. 5A) and modWT1 (SEQ ID NO: 18) (FIG. 5B) by A549, and modMSLN (SEQ ID NO: 22) by NCI-H23 (FIG. 5C) were detected by flow cytometry as described herein. Expression of the genes encoding modBORIS (SEQ ID NO: 20) and TP53, PIK3CA and KRAS driver mutations (SEQ ID NO: 79) by NCI-H460, KRAS G12D (SEQ ID NO: 24), G12V (SEQ ID NO: 26) and EGFR activating mutations (SEQ ID NO: 82) by A549, and EGFR TKI acquired resistance mutations (SEQ ID NO: 94), ALK TKI acquired resistance mutations (SEQ ID NO: 94) and modALK-IC (SEQ ID NO: 94) by NCI-H23 were detected by PCR. Genes encoding modTBXT, modWT1, KRAS G12D and KRAS G12V (SEQ ID NO: 18) were subcloned into the same lentiviral transfer vector separated by furin cleavage sites (SEQ ID NO: 37). Gene encoding EGFR activating mutations (SEQ ID NO: 82) was subcloned into the same lentiviral transfer vector separated by furin cleavage sites (SEQ ID NO: 37). Gene encoding NSCLC driver mutations (SEQ ID NO: 79) was subcloned into the same lentiviral transfer vector separated by furin cleavage sites (SEQ ID NO: 37). The gene encoding EGFR acquired TKI resistance mutations (SEQ ID NO: 94), ALK acquired TKI resistance mutations (SEQ ID NO: 94) and modALK-IC (SEQ ID NO: 94) was subcloned into the same lentiviral transfer vector separated by furin cleavage sites (SEQ ID NO: 37). Immune responses to the transduced antigens are described herein.

To maintain maximal heterogeneity of antigens and clonal subpopulations of each cell line, the modified cell lines utilized in the present vaccine have been established using antibiotic selection and flow cytometry and not through limiting dilution subcloning.

The cell lines in Table 4-1 are used in the present NSCLC vaccine.

TABLE 4-1
NSCLC vaccine cell lines and histology
	Cocktail	Cell Line Name	Lung Cancer Histology
	A	NCI-H520	Squamous
	A	A549	Adenocarcinoma
	A	NCI-H460	Large cell
	B	LK-2	Squamous
	B	NCI-H23	Adenocarcinoma
	B	DMS 53	Small cell carcinoma

CD276 Expression

Unmodified, parental NCI-H460, NCI-H520, A549, NCI-H23, LK-2, and DMS 53 cell lines expressed CD276. Expression of CD276 was decreased, or knocked out, by electroporation with a zinc finger nuclease (ZFN) pair specific for CD276 targeting the genomic DNA sequence: GGCAGCCCTGGCATGggtgtgCATGTGGGTGCAGCC (SEQ ID NO: 52). Following ZFN-mediated knockout of CD276, the cell lines were surface stained with PE α-human CD276 antibody (BioLegend, clone DCN.70) and full allelic knockout cells were enriched by cell sorting (BioRad S3e Cell Sorter). The sorted cells were plated in an appropriately sized vessel, based on the number of recovered cells, and expanded in culture. After cell enrichment for full allelic knockouts, cells were passaged 2-5 times and CD276 knockout percentage determined by flow cytometry. Specifically, expression of CD276 was determined by extracellular staining of CD276 modified and unmodified parental cell lines with PE α-human CD276 (BioLegend, clone DCN.70). Unstained cells and isotype control PE α-mouse IgG1 (BioLegend, clone MOPC-21) stained parental and CD276 KO cells served as controls. To determine the percent reduction of CD276 expression in the modified cell line, the MFI of the isotype control was subtracted from recorded MFI values of both the parental and modified cell lines. Percent reduction of CD276 expression is expressed as: (1-(MFI of the CD276KO cell line/MFI of the parental))×100). Reduction of CD276 expression by component cell lines is described in Table 4-2. These data demonstrate that gene editing of CD276 with ZFN resulted in greater than 96.9% knockout of CD276 in the six NSCLC vaccine component cell lines.

TABLE 4-2
Reduction of CD276 expression
	Unmodified Cell	Modified Cell	% Reduction
Cell line	Line MFI	Line MFI	CD276
NCI-H460	73,079	0	100
NCI-H520	171,117	21	≥99.9
A549	246,899	1358	99.5
NCI-H23	143,350	4438	96.9
LK-2	199,286	0	100
DMS 53	4,479	0	100
MFI is reported with isotype controls subtracted

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12

Cell lines were X-ray irradiated at 100 Gy prior to plating in 6-well plates at 2 cell densities (5.0e5 and 7.5e5) in duplicate. The following day, cells were washed with PBS and the media was changed to Secretion Assay Media (Base Media+5% CTS). After 48 hours, media was collected for ELISAs. The number of cells per well was counted using the Luna cell counter (Logos Biosystems). Total cell count and viable cell count were recorded. The secretion of cytokines in the media, as determined by ELISA, was normalized to the average number of cells plated in the assay for all replicates.

TGFβ1 secretion was determined by ELISA according to manufacturers instructions (Human TGFβ1 Quantikine ELISA, R&D Systems #SB100B). Four dilutions were plated in duplicate for each supernatant sample. If the results of the ELISA assay were below the LLD, the percentage decrease relative to parental cell lines was estimated by the number of cells recovered from the assay and the lower limit of detection, 15.4 pg/mL. If TGFβ1 was detected in >2 samples or dilutions the average of the positive values was reported with the n of samples run.

TGFβ2 secretion was determined by ELISA according to manufacturers instructions (Human TGFβ2 Quantikine ELISA, R&D Systems # SB250). Four dilutions were plated in duplicate for each supernatant sample. If the results of the ELISA assay were below the LLD, the percentage decrease relative to parental cell lines was estimated by the number of cells recovered from the assay and the lower limit of detection, 7.0 pg/mL. If TGFβ2 was detected in >2 samples or dilutions the average of the positive values was reported with the n of samples run.

GM-CSF secretion was determined by ELISA according to manufacturers instructions (GM-CSF Quantikine ELISA, R&D Systems #SGM00). Four dilutions were plated in duplicate for each supernatant sample. If the results of the ELISA assay were below the LLD, the percentage increase relative to parental cell lines was estimated by the number of cells recovered from the assay and the lower limit of detection, 3.0 pg/mL. If GM-CSF was detected in >2 samples or dilutions the average of the positive values was reported with the n of samples run.

IL-12 secretion was determined by ELISA according to manufacturer's instructions (LEGEND MAX Human IL-12 (p70) ELISA, Biolegend #431707). Four dilutions were plated in duplicate for each supernatant sample. If the results of the ELISA assay were below the LLD, the percentage increase was estimated by the number of cells recovered from the assay and the lower limit of detection, 1.2 pg/mL. If IL-12 was detected in >2 samples or dilutions the average of the positive values was reported with the n of samples run.

shRNA Downregulates TGF-β Secretion

Following CD276 knockout, TGFβ1 and TGFβ2 secretion levels were reduced using shRNA and resulting secretion levels determined as described above. Of the parental cell lines in NSCLC vaccine-A and NCI-H460, A549 and NCI-H520 secreted measurable levels of TGFβ1 and TGFβ2. Of the parental cell lines in NSCLC vaccine-B, NCI-H23 and DMS 53 secreted measurable levels of TGFβ1 and TGFβ2. LK-2 secreted detectable, but lower levels of TGFβ1 and TGFβ2.

NCI-H460 and A549 were transduced with the lentiviral particles encoding both TGFβ1 shRNA (shTGFβ1, mature antisense sequence: TTTCCACCATTAGCACGCGGG (SEQ ID NO: 54)) and the gene for expression of membrane bound CD40L (SEQ ID NO: 3) under the control of a different promoter. This allowed for simultaneous reduction of TGFβ1 and introduction of expression of membrane bound CD40L. NCI-H460 and A549 were subsequently transduced with the lentiviral particles encoding both TGFβ2 shRNA (mature antisense sequence: AATCTGATATAGCTCAATCCG (SEQ ID NO: 55) and GM-CSF (SEQ ID NO: 8) under the control of a different promoter. This allowed for simultaneous reduction of TGFβ2 and introduction of expression of GM-CSF.

DMS 53 and NCI-H23 were transduced with lentiviral particles encoding both TGFβ1 shRNA and the gene for expression of membrane bound CD40L concurrently with lentiviral particles encoding both TGFβ2 shRNA and GM-CSF. This allowed for simultaneous reduction of TGFβ1 and TGFβ2, and expression of CD40L and GM-CSF.

NCI-H520 and LK-2 cell lines were first transduced with lentiviral particles only expression shTGFβ1 and then subsequently transduced with lentiviral particles only expressing shTGFβ2. Cell lines modified with TGFβ1 shRNA and TGFβ2 shRNA are described by the clonal designation DK6.

TGFβ1 and TGFβ2 promote cell proliferation and survival. In some cell lines, as in some tumors, reduction of TGFβ signaling can induce growth arrest and lead to cell death. TGFβ1 secretion by LK-2 was not reduced by shRNA transduction. The LK-2 cell line secreted relatively lower levels of both TGFβ1 and TGFβ2 and potentially employed a compensatory mechanism to retain some TGFβ signaling likely necessary for proliferation and survival of this cell line.

Table 4-3 describes the percent reduction in TGFβ1 and/or TGFβ2 secretion in gene modified cell lines compared to unmodified, parental cell lines. Reduction of TGFβ1 ranged from 73% to 98%. Reduction of TGFβ2 ranged from 27% to 99%.

TABLE 4-3
TGF-β Secretion (pg/106 cells/24 hr) in Component Cell Lines
Cell Line	Cocktail	Clone	TGFβ1	TGFβ2
NCI-H520	A	Wild type	579	2294
NCI-H520	A	DK6	*<14	*<6
NCI-H520	A	Percent reduction	≥98%	≥99%
A549	A	Wild type	2237	1154
A549	A	DK6	596	841
A549	A	Percent reduction	73%	27%
NCI-H460	A	Wild type	673	2937
NCI-H460	A	DK6	*<14	1894
NCI-H460	A	Percent reduction	≥98%	36%
LK-2	B	Wild type	127	161
LK-2	B	DK6	136	69
LK-2	B	Percent reduction	NA	88%
NCI-H23	B	Wild type	877	130
NCI-H23	B	DK6	*<14	*<6
NCI-H23	B	Percent reduction	≥84%	≥95%
DMS 53	B	Wild type	205	806
DMS 53	B	DK6	*<14	*<6
DMS 53	B	Percent reduction	≥93%	≥99%
DK6: TGFβ1/TGFβ2 double knockdown;
ND = not detectable;
NA = not applicable;
*estimated using LLD, not detected

Based on a dose of 5×10⁵ of each component cell line, the total TGFβ1 and TGFβ2 secretion by the modified NSCLC vaccine-A and NSCLC vaccine-B and respective unmodified parental cell lines are shown in Table 4-4. The secretion of TGFβ1 by NSCLC vaccine-A was reduced by 82% and TGFβ2 by 57% pg/dose/24 hr. The secretion of TGFβ1 by NSCLC vaccine-B was reduced by 86% and TGFβ2 by 93% pg/dose/24 hr.

TABLE 4-4
TGF-β Secretion (pg/dose/24 hr)
by NSCLC vaccine-A and NSCLC vaccine-B
	Cocktail	Clones	TGFβ1	TGFβ2
	A	Unmodified	1,745	3,193
		DK6	312	1,371
		Percent reduction	82%	57%
	B	Wild type	605	549
		DK6	82	41
		Percent reduction	86%	93%

Membrane Bound CD40L (CD154) Expression

As described above, NCI-H23, A549, NCI-H460 and DMS 53 cell lines were transduced with lentiviral particles encoding the genes for TGFβ1 shRNA and membrane bound CD40L. NCI-H520 and LK-2 were transduced with lentiviral particles encoding the gene to express membrane bound CD40L (SEQ ID NO: 3). Cells were analyzed for cell surface expression of CD40L by flow cytometry. The unmodified and modified cells were stained with PE-conjugated human α-CD40L (BD Biosciences, clone TRAP1) or Isotype Control PE α-mouse IgG1 (BioLegend, clone MOPC-21). The MFI of the isotype control was subtracted from the CD40L MFI of both the unmodified and modified cell lines. If subtraction of the isotype control resulted in a negative value, an MFI of 1.0 was used to calculate the fold change in CD40L expression. Expression of membrane bound CD40L by all six vaccine component cell lines is described in Table 4-5. The data demonstrate CD40L expression on the cell membrane was substantially increased by all NSCLC vaccine cell lines.

TABLE 4-5
Membrane-bound CD40L (mCD40L) expression
	Unmodified Cell	Modified Cell	Fold Increase
Cell line	Line MFI	Line MFI	mCD40L
NCI-H460	0	1,756,541	1,756,541
NCI-H520	233	68,408	294
A549	0	1,786,775	1,786,775
NCI-H23	0	610,859	610,859
LK-2	0	65,788	65,788
DMS 53	0	4,317	4,317
MFI is reported with isotype controls subtracted

GM-CSF Expression

As described above, NCI-H23, A549, NCI-H460 and DMS 53 were transduced with lentiviral particles encoding genes to express TGFβ2 shRNA and GM-CSF. LK-2 and NCI-H520 cell lines were transduced with lentiviral particles only encoding the gene to express GM-CSF (SEQ ID NO: 8). GM-CSF expression was quantitated as described above. Table 4-6 shows all NSCLC vaccine cell lines express GM-CSF.

TABLE 4-6
GM-CSF expression by NSCLC vaccine-A and NSCLC vaccine-B
		GM-CSF	GM-CSF
	Cell Line	(ng/106 cells/24 hr)	(ng/dose/24 hr)
	NCI-H520	28	14
	A549	169	85
	NCI-H460	357	179
	Cocktail A Total	554	277
	LK-2	2	1
	NCI-H23	98	49
	DMS 53	30	15
	Cocktail B Total	130	65

Based on a dose of 5×10⁵ of each component cell line, total GM-CSF secretion by NSCLC vaccine-A was 277 ng per dose per 24 hours. GM-CSF secretion for NSCLC vaccine-B was 65 ng per dose per 24 hours. Total GM-CSF secretion per dose was therefore 342 ng per 24 hours.

IL-12 Expression

NCI-H23, A549, NCI-H460 and DMS 53 cell lines were transduced with lentivirus particles encoding the gene to express IL-12 p70. Expression of IL-12 by NSCLC vaccine cell lines was quantitated as described above and detailed in Table 4-7.

TABLE 4-7
IL-12 expression by NSCLC vaccine-A and NSCLC vaccine-B
		IL-12	IL-12
	Cell Line	(ng/106 cells/24 hr)	(ng/dose/24 hr)
	NCI-H520	NA	NA
	A549	65	33
	NCI-H460	91	46
	Cocktail A Total	156	79
	LK-2	NA	NA
	NCI-H23	145	73
	DMS 53	28	14
	Cocktail B Total	173	87

Based on a dose of 5×10⁵ of each component cell line, the total IL-12 secretion for NSCLC vaccine-A was 79 ng per dose per 24 hours. The total IL-12 secretion for NSCLC vaccine-B was 87 ng per dose per 24 hours. The total IL-12 secretion per unit dose was therefore 166 ng per 24 hours.

Immune Responses to Prioritized NSCLC TAAs Induced by DMS 53

WO/2021/113328 describes immune responses generated by vaccine compositions comprising cell line DMS 53 modified to reduce expression of CD276, reduce secretion of TGFβ2, and express GM-CSF and membrane bound CD40L. Further optimization of gene editing strategies allowed for inclusion of two additional adjuvant modifications to the DMS 53 cell line, reduction of TGFβ1 secretion and expression of IL-12. As described here in, immune responses to eight prioritized NSCLC TAAs significantly increased when DMS 53 was modified to reduce expression of CD276, reduce secretion of TGFβ1 and TGFβ2, express GM-CSF membrane bound CD40L and IL-12 compared to DMS 53 modified to reduce expression of CD276, reduce secretion of TGFβ2, and to express GM-CSF and membrane bound CD40L.

Immune responses to were evaluated by IFNγ ELISpot for six HLA diverse donors (n=4/donor). HLA-A, HLA-B, and HLA-C alleles for each of the six donors are in Table 4-8. Specifically, 1.5×10⁶ of DMS 53 modified cell line described above were co-cultured with 1.5×10⁶ autologous iDCs from six donors. CD14-PBMCs primed with DCs were isolated from co-culture on day 6 and stimulated with peptide pools designed to cover the full-length native antigens for 24 hours in the ELISpot assay prior to detection of IFNγ production. Custom peptide libraries of 15-mers overlapping by 9 amino acids were sourced from Thermo Scientific Custom Peptide Services for BORIS and 15-mer peptides overlapping by 11 amino acids were sourced for MSLN from GenScript. Commercially available peptide pools, 15-mers overlapping by 11 amino acids, were sourced as follows: TERT (JPT, PM-TERT), WT1 (JPT, PM-WT1), Brachyury (JPT, PM-BRAC), STEAP1 (JPT, PM-STEAP1), MAGE A3 (JPT, PM-MAGEA3), and Survivin (thinkpeptides, 7769_001-011).

TABLE 4-8
Healthy Donor MHC-I characteristics
	Donor#	HLA-A	HLA-B	HLA-C
	1	*01:01 *32:01	*35:01 *40:06	*04:01 *15:02
	2	*02:01 *11:01	*07:02 *37:01	*06:02 *07:02
	3	*03:01 *32:01	*07:02 *15:17	*07:01 *07:01
	4	*03:01 *03:01	*07:02 *15:01	*03:03 *07:02
	5	*03:01 *11:01	*44:03 *50:01	*06:02 *16:01
	6	*02:01 *02:05	*07:02 *41:02	*07:02 *17:01

DMS 53 modified to reduce expression of CD276, reduce secretion of TGFβ1 and TGFβ2, and express GM-CSF, membrane bound CD40L and IL-12 induced significantly more robust antigen specific IFNγ responses (10,662±5,289 SFU) than DMS 53 modified to reduce expression of CD276, reduce secretion of TGFβ2, and express GM-CSF and membrane bound CD40L (1,868±371 SFU) (p=0.015, Mann-Whitney U test) (FIG. 6A) (Table 4-9). FIG. 6B shows the total magnitude of IFNγ produced against eight NSCLC antigens by individual donors when CD14-PBMC were primed with autologous DCs loaded the different DMS 53 modified cell lines.

TABLE 4-9
IFNy responses generated by DMS 53 with
different genetic modifications
	DMS 53 cell line modifications (SFU ± SEM)
Donor #	CD276 KO, TGFβ2 KD,	CD276 KO, TGFβ1 KD, TGFβ2 KD,
(n = 4)	GM-CSF, mCD40L	GM-CSF, mCD40L, IL-12
1	2,383 ± 930	2,245 ± 791
2	250 ± 82	6,290 ± 1,412
3	2,630 ± 622	10,828 ± 1,584
4	1,510 ± 549	3,910 ± 1,619
5	1,830 ± 766	4,288 ± 1,800
6	2,603 ± 1,731	36,413 ± 5,602
Average	1,868 ± 371	10,662 ± 5,289

Expression of modTBXT and modWT1 (SEQ ID NO: 18) by the NSCLC Vaccine-A A549 Cell Line

As described above, NSCLC vaccine-A cell line A549 modified to reduce expression of CD276, reduce secretion of TGFβ1 and TGFβ2, and express GM-CSF, membrane bound CD40L and IL-12 was also transduced with lentiviral particles encoding the gene to express modTBXT and modWT1 antigens, and peptides encoding KRAS driver mutations G12V and G12D. Expression of TBXT and WT1 were confirmed by flow cytometry. Unmodified and antigen modified cells were stained intracellularly to detect the expression of each antigen as follows. For detection of modTBXT, cells were stained with rabbit anti-human TBXT antibody (Abcam ab209665, Clone EPR18113) (0.06 μg/test) or Rabbit Polyclonal Isotype Control (Biolegend 910801) followed by AF647-conjugated donkey anti-rabbit IgG antibody (Biolegend 406414) (0.125 μg/test). For detection of modWT1, cells were stained with rabbit anti-human WT1 antibody (AbCam ab89901, Clone CAN-R9) (0.06 μg/test) or Rabbit Polyclonal Isotype Control (Biolegend 910801) followed by AF647-conjugated donkey anti-rabbit IgG antibody (Biolegend 406414) (0.125 μg/test). The MFI of cells stained with the isotype control was subtracted from the MFI of the cells stained for TBXT or WT1. Fold increase in antigen expression was calculated as: (background subtracted modified MFI/background subtracted parental MFI). Subtraction of the MFI of the isotype control from the MFI of the TBXT and WT1 stained unmodified cell line resulted in negative value and fold increase of modTBXT and modWT1 expression by the antigen modified A549 cell line was calculated using 1 MFI. Expression of WT1 (FIG. 5A) by modified A549 (277,032 MFI) increased 277,032-fold over the unmodified cell line (0 MFI). Expression of TBXT by modified A549 (FIG. 5B) (173,733 MFI) increased 173,733-fold over the unmodified cell line (0 MFI).

Expression of modMSLN (SEQ ID NO: 22) by the NSCLC Vaccine-B NCI-H23 Cell Line

NSCLC vaccine-B cell line NCI-H23 modified to reduce the expression of CD276, reduce secretion of TGFβ1 and TGFβ2, and express GM-CSF, membrane bound CD40L and IL-12 was transduced with lentiviral particles encoding the gene for modMSLN. Expression of MSLN was confirmed by flow cytometry. Unmodified and antigen modified cells were surface stained with stained with PE conjugated rat anti-human MSLN antibody (R&D Systems, Clone 420411) (10 μL/test) or Isotype Control PE Rat IgG2a (Biolegend, Clone RTK2758). MFI of cells stained with isotype control was subtracted from the MFI of the cells stained for MSLN. Fold increase in antigen expression was calculated as: (background subtracted modified MFI/background subtracted parental MFI). Expression of MSLN increased by modified cell line NCI-H23 cell line (FIG. 5C) (13,453 MFI) 538-fold over that of the antigen unmodified cell line (25 MFI).

Immune Responses to Generated by Expression of modBORIS (SEQ ID NO: 20) by NSCLC Vaccine-A

IFNγ responses to BORIS were evaluated in the context of the NSCLC-vaccine A for six HLA diverse donors (Table 4-10). Specifically, 5×10⁵ of unmodified or NSCLC vaccine-A NCI-H520, A549 and NCI-H460 cell lines, a total of 1.5×10⁶ total modified cells, were co-cultured with 1.5×10⁶ iDCs from six HLA diverse donors (n=4/donor). CD14-PBMCs were isolated from co-culture with DCs on day 6 and stimulated with peptide pools, 15-mers overlapping by 9 amino acids, spanning the native BORIS protein sequence in the IFNγ ELISpot assay for 24 hours prior to detection of IFNγ producing cells. Peptides were purchased from Thermo Scientific Custom Peptide Service. NSCLC vaccine-A (2,299±223 SFU) induced significantly stronger BORIS specific IFNγ responses compared to unmodified control NSCLC vaccine-A (120±62 SFU) (p=0.002, Mann-Whitney U test) (FIG. 7A).

Immune Responses to Generated by Expression of modTBXT and modWT1 (SEQ ID NO: 18) by NSCLC Vaccine-A

IFNγ responses induced by modTBXT and modWT1 expressed by NSCLC vaccine-A cell line A549 were evaluated in the context of NSCLC-vaccine A as described above and herein for six HLA diverse donors (n=4/donor) (Table 4-10). IFNγ responses against TBXT and WT1 were evaluated in ELISpot by stimulating with 15-mer peptides, overlapping by 11 amino acids, spanning the native TBXT antigen (JPT, PM-BRAC) or native WT1 antigen (JPT, PM-WT1) proteins. NSCLC vaccine-A (1,791±252 SFU) significantly increased IFNγ responses to TBXT (1,791±252 SFU) compared unmodified controls (86±72 SFU) (p=0.002) (FIG. 7B). IFNγ responses to WT1 also significantly when CD14-PBMCs were primed with NSCLC vaccine-A (1,601±272 SFU) compared to the unmodified control cocktail (37±37 SFU) (p=0.002) (FIG. 7C). Statistical significance was determined using the Mann-Whitney U test.

Immune Responses to modMSLN in NSCLC Vaccine-B

IFNγ responses to the modMSLN antigen expressed NSCLC vaccine-A cell line NCI-H23 line were evaluated in the context of NSCLC vaccine-B as described above, and herein, for six HLA diverse donors (n=4/donor) (Table 4-10). IFNγ responses against native MSLN were evaluated in ELI Spot by stimulating with custom ordered 15-mer peptides, overlapping by 11 amino acids, designed to span the native MSLN protein (GeneScript). MSLN specific IFNγ responses were significantly stronger when CD14-PBMCs were primed with DCs loaded with NSCLC vaccine-B (3,193±698 SFU) compared to the unmodified control cocktail (208±101 SFU) (p=0.002, Mann-Whitney U test) (FIG. 7D).

TABLE 4-10
Healthy Donor MHC-I characteristics
	Donor #	HLA-A	HLA-B	HLA-C
	1	*01:01 *32:01	*35:01 *40:06	*04:01 *15:02
	2	*29:02 *31:01	*40:01 *55:01	*03:04 *16:01
	3	*29:01 *29:02	*44:03 *50:01	*06:02 *16:01
	4	*02:02 *30:02	*15:03 *57:03	*02:10 *07:18
	5	*02:01 *24:02	*08:01 *51:01	*03:04 *14:02
	6	*02:01 *30:02	*14:02 *57:02	*08:02 *18:02

Immune Responses to modBORIS, modWT1 and modTBXT to Neoepitopes in NSCLC Vaccine-A

Targeting neoepitopes to generate an antitumor response has the advantage that neoepitopes are tumor specific and not subject to central tolerance in the thymus. modBORIS, modWT1, modTBXT and modMSLN antigens expressed by the NSCLC vaccine encode neoepitopes with the potential to elicit immune responses greater in antigenic breadth and magnitude than native antigen proteins. Neoepitopes were introduced into the modBORIS, modWT1, modTBXT and modMSLN antigens expressed by the NSCLC vaccine by inclusion of non-synonymous mutations (NSMs) using the design strategy described in Example 40 of WO/2021/113328. Immune responses induced against a subset of neoepitopes are described herein.

MHC molecules are highly polymorphic and distinct epitopes or neoepitopes may be recognized by different individuals in the population. NetMHCpan 4.0 (services.healthtech.dtu.dk/service.php?NetMHCpan-4.0) (Jurtz V, et al. J Immunol. 2017) was used to predict neoepitopes that could potentially be recognized by six healthy donors (Table 4-10) encoded by modBORIS (SEQ ID NO: 20), modWT1 and modTBXT (SEQ ID NO: 18) antigens inserted into NSCLC vaccine-A. Epitope prediction was completed using donor specific HLA-A and HLA-B alleles. The number of modBORIS, modWT1 and modTBXT neoepitopes predicted to be recognized by each donor is described in Table 4-11.

TABLE 4-11
Donor specific HLA-A and HLA-B restricted neoepitopes
	Donor	Donor	Number of predicted HLA-A and HLA-B neoepitopes
	HLA-A	HLA-B	modBORIS	modWT1	modTBXT
Donor #	alleles	alleles	HLA-A	HLA-B	HLA-A	HLA-B	HLA-A	HLA-B
1	*01:01	*35:01	3	5	6	7	11	6
	*32:01	*40:06
2	*29:02	*40:01	2	5	3	8	3	6
	*31:01	*55:01
3	*29:01	*44:03	2	2	2	4	2	6
	*29:02	*50:01
4	*02:02	*15:03	3	5	4	6	10	8
	*30:02	*57:03
5	*02:01	*08:01	3	3	3	2	6	5
	*24:02	*51:01
6	*02:01	*14:02	4	4	5	7	8	9
	*30:02	*57:02

Immune responses to a subset of neoepitopes in Table 4-11 were evaluated in the context of NSCLC vaccine-A by IFNγ ELISpot as described above. Neoepitopes selected for further evaluation were predicted to be recognized by at least three of the six donors (Table 4-12). Donor CD14-PBMCs were co-cultured with autologous DCs loaded with unmodified or modified NSCLC vaccine-A. IFNγ responses were evaluated in the ELISpotPeptides, 15-mers overlapping by 9 amino acids, covering the full-length modBORIS, modWT, and modTBXT antigens were purchased from Thermo Scientific Custom Peptide Service. Individual peptides containing neoepitopes used for stimulation of CD14-PBMCs are identified in Table 4-12. Most MHC class-I epitopes are nine amino acids in length, but CD8+ T cell epitopes can range in length from eight to eleven amino acids. For this reason, peptides containing at least eight amino acids of the predicted nine amino acid neoepitope were used in the IFNγ ELI Spot assay.

TABLE 4-12
modBORIS, modWT1 and modTBXT neoepitopes and corresponding peptides evaluated in the IFNγ
ELISpot assay
		IFNγ ELISpot	Donors (Table 4-10) predicted to
Antigen	Neoepitope	15-mer peptide(s)	respond to neoepitope
modBORIS	RTVTLLWNY	RTVTLLWNYVNTHTG (SEQ	Donors 1, 2, 3, and 6
	(SEQ ID NO: 62)	ID NO: 63)
	LEENVMVAI (SEQ	LQFHALEENVMVAIE	Donors 1,2, and 3
	ID NO: 64)	EENVMVAIEDSKLAV (SEQ
		ID NO: 65)
	CSMCKYASM	THEKPFKCSMCKYAS	Donors 1, 2, 4, 5, and 6
	(SEQ ID NO: 66)	KCSMCKYASMEASKL (SEQ
		ID NO: 67)
modWT1	RYFKLSHLK (SEQ	CNKRYFKLSHLKMHS (SEQ	Donors 2, 4, 5, and 6
	ID NO: 68)	ID NO: 69)
modTBXT	LSLSSTHSY (SEQ	GGALSLSSTHSYDRY (SEQ	Donors 1, 2, 3, 5, and 6
	ID NO: 70)	ID NO: 71
	FPMYKGAAA	GFPMYKGAAAATDIV (SEQ	Donors 1, 2, 3, 4, and 6
	(SEQ ID NO: 72)	ID NO: 73)
	HLIASWTPV (SEQ	GHLIASWTPVSPPSM (SEQ	Donors 1, 2, 4, 5, and 6
	ID NO: 74)	ID NO: 75)

FIG. 8 demonstrates NSCLC vaccine-A can induce IFNγ responses against neoepitopes encoded by modBORIS, modWT1, and modTBXT. IFNγ responses against three modBORIS epitopes, one modWT1 neoepitope and three TBXT neoepitopes were evaluated in three to five donors (Table 4-12.1). Three of four donors responded to the modBORIS neoepitope RTVTLLWNY (SEQ ID NO: #) (FIG. 8A), one of three donors responded to the modBORIS neoepitope LEENVMVAI (SEQ ID NO: 64) (FIG. 8B), five of five donors responded to the modBORIS neoepitope CSMCKYASM (SEQ ID NO: 66) (FIG. 8C), three of four donors responded to the modWT1 neoepitope RYFKLSHLK (SEQ ID NO: 68) (FIG. 8D), four of five donors responded to the TBXT neoepitope LSLSSTHSY(SEQ ID NO: 70) (FIG. 8E), five of five donors responded to the TBXT neoepitope FPMYKGAAA (SEQ ID NO: 72) (FIG. 8F) and three of five donors responded to the TBXT neoepitope HLIASWTPV (SEQ ID NO: 74) (FIG. 8G).

Some IFNγ production was observed for some neoepitope peptides when donor CD14-PBMCs were primed with DCs loaded with the unmodified control cocktail in some donors. These responses could be attributed to cross-reactive T cell responses against epitopes derived from endogenous native antigens. NSCLC vaccine-A cell lines. IFNγ responses induced by the unmodified and modified NSCLC vaccine-A to modBORIS, modWT1 and modTBXT neoepitopes are summarized in Table 4-12.1.

TABLE 4-12.1
IFNγ responses to modBORIS, modWT1 and modTBXT neoepitopes
			Unmodified NSCLC	Modified
		Donor #	vaccine-A	NSCLC vaccine-A
Antigen	Neoepitope	(n = 4)	(SFU ± SEM)	(SFU ± SEM)
modBORIS	RTVTLLWNY	1	0 ± 0	0 ± 0
	(SEQ ID NO: 62	2	953 ± 354	4,073 ± 1,875
		3	0 ± 0	0 ± 0
		6	0 ± 0	910 ± 651
modBORIS	LEENVMVAI	1	0 ± 0	0 ± 0
	(SEQ ID NO: 64	2	455 ± 297	4,073 ± 1,875
		3	0 ± 0	0 ± 0
modBORIS	CSMCKYASM	1	0 ± 0	1,500 ± 397
	(SEQ ID NO: 66)	2	750 ± 307	3,310 ± 1,759
		4	290 ± 108	1,620 ± 890
		5	420 ± 189	4,320 ± 1,221
		6	349 ± 289	2,100 ± 1,095
modWT1	RYFKLSHLK	2	0 ± 0	1,600 ± 1,009
	(SEQ ID NO: 68)	4	275 ± 259	1,105 ± 986
		5	360 ± 157	2,940 ± 624
		6	0 ± 0	0 ± 0
modTBXT	LSLSSTHSY	1	0 ± 0	685 ± 285
	(SEQ ID NO: 70)	2	0 ± 0	4,840 ± 1,294
		3	0 ± 0	0 ± 0
		5	0 ± 0	3,910 ± 1,632
		6	0 ± 0	1,240 ± 1,032
modTBXT	FPMYKGAAA	1	0 ± 0	3,260 ± 724
	(SEQ ID NO: 72)	2	100 ± 63	1,480 ± 981
		3	0 ± 0	2,483 ± 956
		4	0 ± 0	1,955 ± 1,166
		6	0 ± 0	1,310 ± 1,212
modTBXT	HLIASWTPV	1	0 ± 0	4,950 ± 1,181
	(SEQ ID NO: 74)	2	415 ± 310	2,695 ± 1,884
		4	290 ± 169	0 ± 0
		5	0 ± 0	4,300 ± 1,162
		6	0 ± 0	0 ± 0

NSCLC Vaccine Induces Immune Responses Against Prioritized TAAs

IFNγ responses generated by NSCLC vaccine-A and NSCLC vaccine-B against eight NSCLC prioritized antigens was measured by ELISpot as described above and herein. CD14-PBMCs from six HLA-diverse healthy donors (Table 4-10) were co-cultured with autologous DCs loaded with unmodified or NSCLC vaccine-A and unmodified or NSCLC vaccine-B cocktails, for 6 days prior to stimulation with TAA-specific specific peptide pools designed to cover the full-length native antigen protein. IFNγ responses to BORIS, WT1, TBXT and MSLN were evaluated in ELISpot by stimulating primed CD14-PBMCs with peptides described above. Additional 15-mer peptide pools, overlapping by 11 amino acids, were sourced as follows: STEAP1 (PM-STEAP1), Survivin (thinkpeptides, 7769_001-011), MAGE A3 Mage A3 (JPT, PM-MAGEA3), and TERT (JPT, PM-TERT).

FIG. 9 demonstrates the NSCLC vaccine is capable of inducing antigen specific IFNγ responses by six HLA-diverse donors to eight NSCLC antigens 8.7-fold more robust (32,370±3,577 SFU) compared to the unmodified parental control (3,720±665 SFU) (FIG. 9A) (Table 4-13). The unit dose of NSCLC vaccine-A and NSCLC vaccine-B elicited IFNγ responses to seven antigens in one donor and eight antigens in five donors. NSCLC vaccine-A and NSCLC vaccine-B independently demonstrated 10.4-fold and 8.6-fold increases in antigen specific responses compared to unmodified controls, respectively. NSCLC vaccine-A significantly increased antigen specific responses (23,944±3,971 SFU) compared to the unmodified controls (1,343±233 SFU) (p=0.002) (FIG. 9B). NSCLC vaccine-B also significantly increased antigen specific responses (17,675±2,255 SFU) compared to the parental control cocktail (2,053±682 SFU) (p=0.005) (FIG. 9C). Statistical significance was determined using the Mann-Whitney U test. Antigen specific responses for individual donors induced by the NSCLC vaccine and unmodified control cell lines are shown in FIG. 10.

TABLE 4-13
IFNγ Responses to unmodified and modified NSCLC vaccine components
	Unmodified (SFU ± SEM)	Modified (SFU ± SEM)
Donor	NSCLC	NSCLC	NSCLC	NSCLC	NSCLC	NSCLC
# (n = 4)	vaccine-A	vaccine-B	Vaccine	vaccine-A	vaccine-B	Vaccine
1	780 ± 49	0 ± 0	1,440 ± 75	11,358 ± 719	12,700 ± 502	26,133 ± 1,109
2	1,690 ± 211	353 ± 44	2,233 ± 253	19,898 ± 931	25,245 ± 576	46,560 ± 1,370
3	1,088 ± 90	2,788 ± 260	4,130 ± 333	9,440 ± 418	12,440 ± 708	22,243 ± 1,015
4	2,223 ± 230	2,788 ± 286	5,020 ± 515	15,063 ± 547	23,330 ± 1,486	38,393 ± 2,011
5	1,485 ± 85	1,940 ± 122	3,775 ± 171	15,550 ± 338	14,350 ± 626	29,850 ± 899
6	785 ± 81	4,450 ± 96	5,723 ± 149	12,370 ± 409	17,985 ± 479	30,855 ± 829

Identification of Frequently Mutated Oncogenes in NSCLC to Identify NSCLC-Specific Driver Mutations

Driver mutations for NSCLC were identified, selected and constructs designed as described as described in Example 1 and herein. Expression of these driver mutations by the NSCLC vaccine-A NCI-H460 can generate a NSCLC anti-tumor response in an HLA diverse population.

Table 4-14 describes oncogenes that exhibit greater than 5% mutation frequency (percentage of samples with one or more mutations) in 2138 or 2179 NSCLC profiled patient samples.

TABLE 4-14
Oncogenes in NSCLC with greater than 5% mutation frequency
		Number of samples		Percentage of samples
	Total Number	with one or more	Profiled	with one or more	Is Cancer Gene
Gene	of mutations	mutations	Samples	mutations	(source: OncoKB)
TP53	1427	1334	2179	61.20%	Yes
LRP1B	1036	672	2138	31.40%	Yes
KRAS	429	420	2179	19.30%	Yes
PCLO	447	336	2179	15.40%	Yes
RELN	397	305	2179	14.00%	Yes
FAT4	340	270	2179	12.40%	Yes
KEAP1	242	238	2179	10.90%	Yes
FAT1	273	234	2179	10.70%	Yes
KMT2D	266	233	2179	10.70%	Yes
KMT2C	269	233	2179	10.70%	Yes
PTPRD	279	228	2138	10.70%	Yes
EGFR	265	225	2179	10.30%	Yes
RB1	231	219	2179	10.10%	Yes
NF1	227	205	2138	9.60%	Yes
CPS1	244	204	2179	9.40%	Yes
STK11	213	201	2179	9.20%	Yes
EPHA5	226	198	2138	9.30%	Yes
PTPRT	201	171	2179	7.80%	Yes
ZNF521	196	163	2179	7.50%	Yes
LRRK2	174	163	2138	7.60%	Yes
PIK3CA	166	161	2179	7.40%	Yes
ATM	181	159	2179	7.30%	Yes
CDKN2A	171	158	2179	7.30%	Yes
ERBB4	174	157	2179	7.20%	Yes
GRIN2A	164	152	2179	7.00%	Yes
HGF	172	152	2179	7.00%	Yes
EPHA3	168	149	2138	7.00%	Yes
KDR	162	148	2179	6.80%	Yes
PTPRB	164	148	2179	6.80%	Yes
MGA	170	147	2179	6.70%	Yes
NFE2L2	158	146	2179	6.70%	Yes
NOTCH1	154	140	2179	6.40%	Yes
PIK3CG	153	140	2138	6.50%	Yes
NTRK3	153	139	2138	6.50%	Yes
PREX2	149	138	2179	6.30%	Yes
PRKDC	143	135	2138	6.30%	Yes
MGAM	145	135	2179	6.20%	Yes
PDE4DIP	144	135	2179	6.20%	Yes
SETBP1	151	135	2179	6.20%	Yes
RUNX1T1	141	133	2179	6.10%	Yes
CREBBP	137	127	2179	5.80%	Yes
TRRAP	140	126	2179	5.80%	Yes
ROS1	126	123	2179	5.60%	Yes
SMARCA4	127	121	2179	5.60%	Yes
PTPRC	127	120	2179	5.50%	Yes
POLQ	136	120	2179	5.50%	Yes
EPHA7	123	116	2138	5.40%	Yes
ZFHX3	125	115	2179	5.30%	Yes
POLE	120	112	2179	5.10%	Yes
TPR	122	112	2179	5.10%	Yes
PDGFRA	119	110	2138	5.10%	Yes
ARID1A	120	109	2179	5.00%	Yes
EP400	114	108	2179	5.00%	Yes
RNF213	130	108	2179	5.00%	Yes

Identification of Driver Mutations in Selected NSCLC Oncogenes

The NSCLC driver mutations in TP53, KRAS, EGFR and PIK3CA occurring in ≥0.5% of profiled patient samples are shown in Table 4-15. There were no missense mutations occurring in ≥0.5% of profiled patient samples at the same amino acid position genes for the NSCLC oncogenes in Table 4-15 other than TP53, KRAS, EGFR and PIK3CA.

TABLE 4-15
Identified driver mutations in selected NSCLC oncogenes
	Driver	Number of samples	Total number of	Fre-
Gene	Mutation	with mutation	samples	quency
TP53	R110L	9	1959	0.50%
	H179R	9	1959	0.50%
	I251F	9	1959	0.50%
	C176F	10	1959	0.50%
	R249S	10	1959	0.50%
	R283P	10	1959	0.50%
	G245V	11	1959	0.60%
	R273C	11	1959	0.60%
	G154V	12	1959	0.60%
	Y163C	12	1959	0.60%
	R248Q	12	1959	0.60%
	R282W	12	1959	0.60%
	C141Y	13	1959	0.70%
	R175H	13	1959	0.70%
	H214R	13	1959	0.70%
	M237I	13	1959	0.70%
	R249M	13	1959	0.70%
	G245C	14	1959	0.70%
	R337L	16	1959	0.80%
	Y234C	18	1959	0.90%
	Y220C	21	1959	1.10%
	R273L	22	1959	1.10%
	V157F	26	1959	1.30%
	R158L	35	1959	1.80%
KRAS	G13D	11	1959	0.60%
	Q61L	11	1959	0.60%
	G12S	15	1959	0.80%
	G13C	19	1959	1.00%
	G12D	37	1959	1.90%
	G12A	38	1959	1.90%
	G12V	98	1959	5.00%
	G12C	166	1959	8.50%
EGFR	G719A	11	1959	0.60%
	L861Q	11	1959	0.60%
	L858R	58	1959	3.00%
PIK3CA	H1047R	11	1959	0.60%
	E542K	24	1959	1.20%
	E545K	33	1959	1.70%

Prioritization and Selection of Identified NSCLC Driver Mutations

Results of completed CD4 and CD8 epitope analysis, the total number of HLA-A and HLA-B supertype-restricted 9-mer CD8 epitopes, the total number of CD4 epitopes and frequency (%) for each mutation are shown in Table 4-16. Among all listed mutations, PIK3CA E545K, KRAS G12S and KRAS G12C were endogenous expressed by NSCLC vaccine component cell lines NCI-H460, A549 and NCI-H23 respectively, and were excluded from the final driver mutation insert design. KRAS G12D and KRAS G12V are two of the most frequently occurring KRAS mutations in NSCLC, and other solid tumor types, such as CRC, were excluded from the final driver mutation insert design below because these driver mutations were inserted into the NSCLC vaccine-A cell line NCI-H460 with modWT1 and modTBXT antigens as described herein. If KRAS G12D and KRAS G12V were not inserted into NCI-H460 they would be included in the current insert.

Two identified EGFR driver mutations identified, G719A and L858R, were also identified as initial EGFR activating mutations. These two mutations were included in the construct insert encoding EGFR activating mutations described in herein.

Taken together, as shown in Table 4-16, twenty NSCLC driver mutations encoded by twelve peptide sequences were selected and included as driver mutation vaccine targets.

TABLE 4-16
Prioritization and selection of identified NSCLC driver mutations
		Number of		Number of
		total CD8	Frequency	total CD4	Included as a
	Driver	epitopes	(%)	epitopes	vaccine target
Gene	mutations	(SB + WB)	(n = 1959)	(SB + WB)	Yes (Y) or No (N)
	R110L	12	0.5	6	Y
	C141Y	6	0.7	49	Y
	G154V	7	0.6	8	N
	V157F	8	1.3	46	N
	R158L	3	1.8	84	N
	G154V V157F R158L	13	3.7	98	Y
	Y163C	1	0.6	0	N
	G154V V157F R158L	11	4.3	11	N
	Y163C
	R175H	2	0.7	0	N
	C176F	4	0.5	46	N
	R175H C176F	4	1.2	79	Y
	H179R	1	0.5	8	N
TP53	R175H C176F H179R	3	1.7	70	N
	H214R	5	0.7	8	N
	Y220C	2	1.1	0	N
	H214R Y220C	5	1.8	1	Y
	Y234C	2	0.9	0	N
	M237I	1	0.7	136	N
	Y234C M237I	1	1.6	23	Y
	G245V	3	0.6	7	N
	G245C	1	0.7	0	N
	R248Q	0	0.6	0	N
	R249S	6	0.5	0	N
	R249M	8	0.7	3	N
	I251F	7	0.5	46	N
	G245V R249M I251F	15	1.8	56	Y
	R273C	1	0.6	0	N
	R273L	2	1.1	6	Y
	R282W	0	0.6	14	N
	R283P	0	0.5	1	N
	R337L	9	0.8	6	Y
	L858R	3	3	0	N
	L861Q	1	0.6	8	N
EGFR	L858R L861Q	2	3.6	28	Y
	G719A	4	0.6	0	Y
	E542K	1	1.2	0	Y
PIK3CA	E545K	0	1.7	0	NCI-H460
	H1047R	2	0.6	12	Y
	G12S	1	0.8	0	A549
	G12C	1	8.5	0	A549
	G12D	1	1.9	11	Y
	G12V	3	5	7	Y
KRAS	G12A	2	1.9	0	N
	G13D	0	0.6	11	N
	G13C	1	1	0	N
	G12AG13C	1	2.9	0	Y
	Q61L	0	0.6	6	No

The total number of CD8 epitopes for each HLA-A and HLA-B supertype introduced by 20 selected NSCLC driver mutations was determined as described in above encoded by 12 peptide sequences. Results of the epitope prediction analysis are shown in Table 4-17.

TABLE 4-17
CD8 epitopes introduced by 20 selected NSCLC driver
mutations encoded by 12 peptide sequences
		HLA-A	HLA-B	Total number
		Supertypes	Supertypes	of CD8
Gene	Mutations	(n = 5)	(n = 7)	epitopes
TP53	R110L	6	6	12
	C141Y	2	4	6
	G154V V157F R158L	5	8	13
	R175H C176F	2	2	4
	H214R Y220C	0	5	5
	Y234C M237I	1	0	1
	G245V R249M I251F	3	12	15
	R273L	0	2	2
	R337L	3	6	9
PIK3CA	E542K	1	0	1
	H1047R	0	2	2
KRAS	G12A, G13C	1	0	1

The total number of CD4 epitopes for Class II locus DRB1, DRB 3/4/5, DQA1/DQB1 and DPB1 introduced by 20 selected NSCLC driver mutations were determined as described in above encoded by 12 peptide sequences and the results shown in Table 4-18.

TABLE 4-18
CD4 epitopes introduced by 20 selected NSCLC driver
mutations encoded by 12 peptide sequences
						Total number
		DRB1	DRB3/4/5	DQA1/DQB1	DPB1	of CD4
Gene	Mutations	(n = 26)	(n = 6)	(n = 8)	(n = 6)	epitopes
TP53	R110L	0	0	0	6	6
	C141Y	18	11	1	19	49
	G154V V157F R158L	38	12	2	46	98
	R175H C176F	30	11	1	37	79
	H214R Y220C	0	0	0	1	1
	Y234C M237I	15	4	0	4	23
	G245V R249M I251F	24	8	1	23	56
	R273L	0	0	0	6	6
	R337L	0	0	0	6	6
PIK3CA	E542K	0	0	0	0	0
	H1047R	0	0	0	13	12
KRAS	G12A G13C	0	0	0	0	0

NSCLC Patient Sample Coverage by Selected Driver Mutations

Patient coverage analysis was completed as described in Example 1. As shown in Table 4-19, twenty selected NSCLC driver mutations were assembled into a single construct insert. Once the construct insert was assembled, the analysis of NSCLC patient sample coverage was performed as described above. The results indicated that the NSCLC patient sample coverage by the insert was 16.4% (Table 4-20). When the driver mutations endogenously expressed by the NSCLC vaccine component cell lines and the driver mutations previously inserted with other modifications were also included, the total NSCLC patient sample coverage was 32.1% (Table 4-21).

TABLE 4-19
Generation of the construct encoding
20 selected NSCLC driver mutations
	Driver	Frequency	Total	Total	CD4 &
Gene	mutations	(%)	CD8	CD4	CD8
TP53	R110L	0.5	12	6	18
	C141Y	0.7	6	49	55
	G154V V157F R158L	3.7	13	98	111
	R175H C176F	1.2	4	79	83
	H214R Y220C	1.8	5	1	6
	Y234C M237I	1.6	1	23	24
	G245V R249M I251F	1.8	15	56	71
	R273L	1.1	2	6	8
	R337L	0.8	9	6	15
PIK3CA	E542K	1.2	1	0	1
	H1047R	0.6	2	12	14
KRAS	G12A G13C	2.9	1	0	1

TABLE 4-20
NSCLC patient sample coverage by the construct encoding driver mutations
		Total number of
Coverage (Construct Insert Only)	Driver Mutation Target Gene	samples with	Total sample
Sample Description	TP53	KRAS	PIK3CA	driver mutations	(n = 1959)
# of samples	221	55	27	303	15.5%
with one DM
# of samples	9	0	0	9	0.5%
with ≥2 DMs
from same antigen
# of samples				10	0.5%
with ≥2 DMs
from different antigens
				322	16.4%

TABLE4-21
NSCLC patient sample coverage by all driver mutations
Coverage
(all driver mutations in		Total number of
constructs and cell lines)	Driver Mutation Target Gene	samples with	Total sample
Sample Description	TP53	KRAS	PIK3CA	driver mutations	(n = 1959)
# of samples	191	330	47	568	29.0%
with one DM
# of samples	9	7	0	16	0.8%
with≥2 DMs
from same antigen
# of samples				45	2.3%
with ≥2 DMs
from different antigens
				629	32.1%

Oncogene Sequences and Insert Sequences of the NSCLC Driver Mutation Construct

DNA and protein sequences of oncogenes with selected driver mutations were included in Table 4-22 below and Table 2-10 (TP53 and PIK3CA). The NSCLC driver mutation construct (SEQ ID NO: 78 and SEQ ID NO: 79) insert gene encodes 447 amino acids containing the selected driver mutation sequences separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37).

TABLE 4-22
Oncogene sequences and insert sequences for the NSCLC construct
KRAS (SEQ ID     DNA Sequence
NO: 76)          1 ATGACTGAAT ATAAACTTGT GGTAGTTGGA GCTGGTGGCG TAGGCAAGAG TGCCTTGACG
                 61 ATACAGCTAA TTCAGAATCA TTTTGTGGAC GAATATGATC CAACAATAGA GGATTCCTAC
                 121 AGGAAGCAAG TAGTAATTGA TGGAGAAACC TGTCTCTTGG ATATTCTCGA CACAGCAGGT
                 181 CAAGAGGAGT ACAGTGCAAT GAGGGACCAG TACATGAGGA CTGGGGAGGG CTTTCTTTGT
                 241 GTATTTGCCA TAAATAATAC TAAATCATTT GAAGATATTC ACCATTATAG AGAACAAATT
                 301 AAAAGAGTTA AGGACTCTGA AGATGTACCT ATGGTCCTAG TAGGAAATAA ATGTGATTTG
                 361 CCTTCTAGAA CAGTAGACAC AAAACAGGCT CAGGACTTAG CAAGAAGTTA TGGAATTCCT
                 421 TTTATTGAAA CATCAGCAAA GACAAGACAG AGAGTGGAGG ATGCTTTTTA TACATTGGTG
                 481 AGAGAGATCC GACAATACAG ATTGAAAAAA ATCAGCAAAG AAGAAAAGAC TCCTGGCTGT
                 541 GTGAAAATTA AAAAATGCAT TATAATG
KRAS SEQ ID      Protein Sequence
NO: 77)          1 MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET CLLDILDTAG
                 61 QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI KRVKDSEDVP MVLVGNKCDL
                 121 PSRTVDTKQA QDLARSYGIP FIETSAKTRQ RVEDAFYTLV REIRQYRLKK ISKEEKTPGC
                 181 VKIKKCIIM
NSCLC driver     DNA Sequence
mutation         1 ATGTCTAGCG TGCCAAGCCA GAAAACCTAC CAGGGCAGCT ACGGCTTCCT GCTGGGCTTT
construct insert 61 CTGCATAGCG GCACAGCCAA GAGCGTGACC TGTACCAGAG GCCGGAAGCG GAGAAGCTAC
(SEQ ID NO: 78)  121 AGCCCTGCTC TGAACAAGAT GTTCTGTCAG CTGGCCAAGA CATACCCCGT GCAGCTGTGG
                 181 GTCGACAGCA CACCTCCACC TGGCACAAGA AGAGGCCGCA AGAGAAGATC CAAGACCTGT
                 241 CCTGTCCAGC TCTGGGTTGA CTCTACCCCT CCTCCTGTGA CACGGTTCCT GGCCATGGCT
                 301 ATCTACAAGC AGAGCCAGCA CATGCGGGGC AGAAAGAGAA GAAGCGCCAT CTATAAGCAG
                 361 TCTCAGCACA TGACCGAGGT CGTGCGGCAC TTTCCTCACC ACGAGAGATG CAGCGATAGC
                 421 GACGGACTGG CTCCTCCTAG AGGCAGAAAA AGGCGGAGCG GCAACCTGAG AGTGGAATAC
                 481 CTGGACGACC GGAACACCTT TCGGAGAAGC GTGGTGGTGC CTTGCGAGCC TCCTGAAGTG
                 541 GGCTCTGATT GCAGAGGAAG AAAGCGGCGG AGCCCCTACG AACCACCAGA AGTTGGAAGC
                 601 GACTGCACCA CCATCCACTG CAACTACATC TGCAACAGCA GCTGCATGGG CGGCATGAAT
                 661 CGGAGAAGAG GACGGAAGAG GCGGTCCACA ACAATCCACT ACAATTACAT GTGTAACTCC
                 721 TCTTGTATGG GCGTGATGAA CAGGATGCCC TTCCTGACCA TCATCACCCT GGAAGATAGC
                 781 CGCGGCAGAA AGCGGAGATC CGAGGATAGC TCTGGCAATC TGCTGGGCAG AAACAGCTTC
                 841 GAGGTGCTCG TGTGTGCCTG TCCTGGCAGA GACAGAAGAA CCGAGGAAGA GAATCGCGGA
                 901 CGGAAACGCA GATCCCCTCT GGACGGCGAG TACTTCACAC TGCAGATCCG GGGCAGAGAA
                 961 CTGTTCGAGA TGTTCAGAGA GCTGAACGAG GCCCTGGAAC TGAAGGACCG CGGACGCAAA
                 1021 AGACGCAGCG ACAAAGAGCA GCTGAAGGCC ATCAGCACCA GAGATCCTCT GAGCAAGATC
                 1081 ACCGAGCAAG AAAAGGACTT CCTGTGGTCC CACCGGCACT ACCGCGGAAG AAAAAGAAGA
                 1141 TCCGAACAAG AGGCCCTCGA GTACTTTATG AAGCAGATGA ACGACGCCCG GCACGGCGGC
                 1201 TGGACAACAA AGATGGACTG GATCTTCCAC ACCATCCGGG GTCGCAAAAG AAGAAGCACC
                 1261 GAGTACAAGC TGGTGGTCGT GGGAGCTGCC TGTGTGGGAA AAAGCGCCCT GACAATCCAG
                 1321 CTGATCCAGA ACCACTTCGT G
NSCLC driver     Protein Sequence*
mutation         1 MSSVPSQKTY QGSYGFLLGF LHSGTAKSVT CTRGRKRRSY SPALNKMFCQ LAKTYPVQLW
construct insert 61 VDSTPPPGTR RGRKRRSKTC PVQLWVDSTP PPVTRFLAMA IYKQSQHMRG RKRRSAIYKQ
(SEQ ID NO: 79)  121 SQHMTEVVRH FPHHERCSDS DGLAPPRGRK RRSGNLRVEY LDDRNTFRRS VVVPCEPPEV
                 181 GSDCRGRKRR SPYEPPEVGS DCTTIHCNYI CNSSCMGGMN RRRGRKRRST TIHYNYMCNS
                 241 SCMGVMNRMP FLTIITLEDS RGRKRRSEDS SGNLLGRNSF EVLVCACPGR DRRTEEENRG
                 301 RKRRSPLDGE YFTLQIRGRE LFEMFRELNE ALELKDRGRK RRSDKEQLKA ISTRDPLSKI
                 361 TEQEKDFLWS HRHYRGRKRR SEQEALEYFM KQMNDARHGG WTTKMDWIFH TIRGRKRRST
                 421 EYKLVVVGAA CVGKSALTIQ LIQNHFV
*Driver mutation is highlighted in bold.
The furin cleavage sequence is underlined.

Immune Responses to Driver Mutations Induced by the NSCLC Vaccine-A NCI-H460 Cell Line

NSCLC vaccine-A cell line NCI-H460 modified to reduce expression of CD276, TGFβ1, TGFβ2 and express GM-CSF, membrane bound CD40L, IL-12, and modBORIS was transduced with lentiviral particles expressing twenty TP53, PIK3CA or KRAS driver mutations encoded by twelve peptide sequences separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37) as described above.

Immune responses to the inserted TP53, PIK3CA and KRAS driver mutations were determined by IFNγ ELISpot as described above and herein. Specifically, 1.5×10⁶ of unmodified NCI-H460 or the NSCLC vaccine-A NCI-H460 cell line modified to express TP53, PIK3CA, and KRAS driver mutations were co-cultured with 1.5×10⁶ iDCs from eight HLA diverse donors (n=4/donor). HLA-A, HLA-B, and HLA-C alleles for each donor are in Table 4-23. Peptides, 15-mers overlapping by 9 amino acids, were designed to cover the full amino acid sequences of the twelve individual driver mutations peptides. Only the 15-mer peptides containing the mutations were used to stimulate PBMCs in the IFNγ ELISpot assay.

TABLE 4-23
Healthy Donor MHC-I characteristics
	Donor #	HLA-A	HLA-B	HLA-C
	1	*02:01 *33:01	*07:02 *14:02	*07:02 *14:02
	2	*02:01 *03:01	*07:02 *14:02	*07:01 *07:02
	3	*02:01 *11:01	*07:02 *49:01	*03:04 *07:02
	4	*02:01 *03:01	*07:02 *41:02	*07:02 *17:01
	5	*02:01 *24:02	*08:01 *51:01	*03:04 *14:02
	6	*02:01 *30:02	*14:02 *57:02	*08:02 *18:02
	7	*02:01 *03:01	*13:02 *55:01	*03:04 *06:02
	8	*03:01 *24:02	*07:02 *15:09	*07:02 *07:04

FIGS. 11A-11C demonstrate immune responses against the twelve driver mutation encoding peptides expressed by NSCLC vaccine-A cell line NCI-H460 by at least two of eight HLA-diverse donors by IFNγ ELISpot. NSCLC vaccine-A NCI-H460 induced IFNγ responses against TP53, PIK3CA, and KRAS to all inserted driver mutation encoding peptides greater in magnitude relative to unmodified NCI-H460 cell line (Table 4-24). The magnitude of IFNγ responses induced by NSCLC vaccine-A NCI-H460 cell line significantly increased against the inserted driver mutation peptides encoding TP53 R110L (FIG. 11A) (p=0.004) TP53 C141Y (p=0.012) and TP53 G154V, V157F and R158L (p=0.039) (FIG. 11A), PIK3CA E542K (FIG. 11B) (p=0.026) and KRAS G12A and G13C (FIG. 11C) (p=0.026) compared to the unmodified NCI-H460 cell line. Statistical significance was determined using the Mann-Whitney U test. The NCI-H460 cell line endogenously expresses mRNA encoding TP53 (3.80 FPKM), PIK3CA (0.94 FPKM) and KRAS (1.72 FPKM) (CCLE, https://portals.broadinstitute.org/ccle). Immune responses induced by the unmodified NCI-H460 cell line could be attributed to cross-reactivity with epitopes presented from the endogenous TP53, PIK3CA and KRAS proteins.

TABLE 4-24
Immune responses to TP53, PIK3CA, and KRAS driver mutations
	Unmodified NCI-H460 (SFU ± SEM)	Modified NCI-H460 (SFU ± SEM)
			TP53				TP53
NSCLC			G154V	TP53			G154V	TP53
Driver	TP53	TP53	V157F	R175H	TP53	TP53	V157F	R175H
Mutation	R110L	C141Y	R158L	C176F	R110L	C141Y	R158L	C176F
Donor 1	220 ± 118	220 ± 94	0 ± 0	0 ± 0	880 ± 642	1,000 ± 836	2,690 ± 1,122	2,430 ± 1,184
Donor 2	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	3,440 ± 1,156	0 ± 0	2,542 ± 1,967
Donor 3	55 ± 52	0 ± 0	0 ± 0	70 ± 57	438 ± 210	310 ± 133	540 ± 278	660 ± 351
Donor 4	0 ± 0	0 ± 0	0 ± 0	0 ± 0	685 ± 310	0 ± 0	0 ± 0	0 ± 0
Donor 5	60 ± 38	0 ± 0	0 ± 0	0 ± 0	0 ± 0	1,470 ± 849	0 ± 0	0 ± 0
Donor 6	0 ± 0	0 ± 0	205 ± 115	0 ± 0	0 ± 0	0 ± 0	295 ± 111	670 ± 296
Donor 7	0 ± 0	75 ± 44	50 ± 38	0 ± 0	0 ± 0	870 ± 393	770 ± 656	910 ± 531
Donor 8	0 ± 0	70 ± 47	0 ± 0	0 ± 0	0 ± 0	120 ± 107	1,270 ± 1,116	0 ± 0
Average	58 ± 28	43 ± 25	32 ± 25	27 ± 19	165 ± 116	1,049 ± 389	774 ± 311	1,002 ± 346
	Unmodified NCI-H460 (SFU ± SEM)	Modified NCI-H460 (SFU ± SEM)
			TP53				TP53
NSCLC	TP53	TP53	G245V		TP53	TP53	G245V
Driver	H214R	Y234C	R249M	TP53	H214R	Y234C	R249M	TP53
Mutation	Y220C	M237I	I251F	R273L	Y220C	M237I	I251F	R273L
Donor 1	0 ± 0	0 ± 0	160 ± 135	0 ± 0	1,910 ± 609	2,900 ± 629	1,110 ± 468	1,630 ± 635
Donor 2	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	1,480 ± 904
Donor 3	0 ± 0	118 ± 52	0 ± 0	0 ± 0	600 ± 351	415 ± 246	0 ± 0	80 ± 62
Donor 4	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	1,715 ± 1,320	1,200 ± 812	0 ± 0
Donor 5	50 ± 30	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 6	0 ± 0	170 ± 81	0 ± 0	100 ± 66	0 ± 0	0 ± 0	1,160 ± 1,028	1,960 ± 1,854
Donor 7	0 ± 0	0 ± 0	0 ± 0	0 ± 0	1,550 ± 702	718 ± 335	0 ± 0	0 ± 0
Donor 8	120 ± 77	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Average	21 ± 15	36 ± 24	20 ± 20	16 ± 13	593 ± 269	504 ± 335	359 ± 185	644 ± 310
NSCLC	Unmodified NCI-H460 (SFU ± SEM)	Modified NCI-H460 (SFU ± SEM)
Driver	TP53	PIK3CA	PIK3CA	KRAS	TP53	PIK3CA	PIK3CA	KRAS
Mutation	R337L	E542K	H1047R	G12A G13C	R337L	E542K	H1047R	G12A G13C
Donor 1	0 ± 0	110 ± 85	0 ± 0	90 ± 77	3,325 ± 1,565	3,050 ± 1,636	2,310 ± 1,265	4,570 ± 1,881
Donor 2	200 ± 180	0 ± 0	0 ± 0	0 ± 0	2,027 ± 1,792	593 ± 337	0 ± 0	262 ± 236
Donor 3	55 ± 52	0 ± 0	0 ± 0	0 ± 0	238 ± 131	360 ± 157	0 ± 0	285 ± 205
Donor 4	0 ± 0	0 ± 0	0 ± 0	105 ± 61	760 ± 288	3,910 ± 1,028	2,460 ± 917	3,150 ± 2,088
Donor 5	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 6	85 ± 59	0 ± 0	0 ± 0	0 ± 0	2,728 ± 2,482	2,600 ± 1,253	2,183 ± 932	2,160 ± 944
Donor 7	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 8	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Average	43 ± 25	14 ± 14	0 ± 0	11 ± 11	1,115 ± 483	936 ± 429	562 ± 368	971 ± 573

Immune Responses to KRAS G12D and G12V Driver Mutations Induced by NSCLC Vaccine-A

The NCLC vaccine-A A549 cell line modified to reduce the expression of CD276, TGFβ1 and TGFβ2 and to express GM-CSF, membrane bound CD40L and IL-12 was transduced with lentiviral particles expressing modTBXT, modWT1, and two 28 amino acid peptides spanning the KRAS driver mutations G12D and G12V, respectively, separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37) as described above.

Immune responses against the KRAS driver mutations G12D and G12V induced by the modified NCI-H460 cell line were evaluated for in the context of NSCLC-vaccine A. Specifically, 5×10⁵ of the unmodified or modified NCI-H520, A549 and NCI-H460 cell lines, a total of 1.5×10⁶ total modified cells, were co-cultured with 1.5×10⁶ iDCs from six HLA diverse donors. HLA-A, HLA-B, and HLA-C alleles for each donor are in Table 4-10. Immune responses were evaluated by IFNγ ELISpot as described above and herein. Peptide pools, 15-mers overlapping by 9 amino acids, for 24 hours prior to detection of IFNγ producing cells. Peptides, 15-mers overlapping by 9 amino acids, were designed to cover the full amino acid sequences of KRAS G12D and G12V (Thermo Scientific Custom Peptide Service), excluding the furin cleavage sequences. Only the 15-mer peptides containing the G12D or G12V mutations were used to stimulate PBMCs in the IFNγ ELISpot assay.

FIG. 11D demonstrates NSCLC-vaccine A generates significantly more robust IFNγ responses against the inserted KRAS G12D (p=0.002) and G12V (p=0.002) driver mutation encoding peptides compared to unmodified NSCLC vaccine-A (Table 4-25). NSCLC vaccine-A induced IFNγ responses to KRAS G12D by four donors and KRAS G12V by six donors. Unmodified NSCLC vaccine-A induced IFNγ responses to KRAS G12D by two donors and KRAS G12V by one donor. Statistical significance was determined using the Mann-Whitney U test.

TABLE 4-25
Immune responses to KRAS driver mutations
	Unmodified NSCLC vaccine-A	Modified NSCLC vaccine-A
NSCLC	(SFU ± SEM)	(SFU ± SEM)
Driver	KRAS	KRAS	KRAS	KRAS
Mutation	G12D	G12V	G12D	G12V
Donor 1	0 ± 0	0 ± 0	0 ± 0	505 ± 319
Donor 2	780 ± 455	0 ± 0	2,920 ± 1,276	1,885 ± 1,117
Donor 3	0 ± 0	0 ± 0	855 ± 793	1,873 ± 1,023
Donor 4	0 ± 0	0 ± 0	1,555 ± 898	325 ± 322
Donor 5	230 ± 217	450 ± 268	1,780 ± 964	2,100 ± 1,224
Donor 6	0 ± 0	0 ± 0	0 ± 0	1,243 ± 435
Average	168 ± 128	75 ± 75	1,185 ± 463	1,322 ± 311

Selection of EGFR Activating Mutations for Expression by the NSCLC Vaccine

EGFR activating mutations are found in 20-30% of NSCLC patient tumors at diagnosis. NSCLC patients harboring the EGFR activating mutations such as exon 19 deletions, exon 21 L858R, exon 18 G719X, exon 21 L861Q, and potentially other less common mutations, are responsive to tyrosine kinase inhibitor (TKI) therapy. The most common initial activating mutations in EGFR are exon 19 deletions and exon 21 L858R. Together exon 19 deletions and the L858R point mutation account for approximately 70% of EGFR mutations in NSCLC at diagnosis. There are multiple variants of exon 19 deletions that are heterogenous in the length of the in frame deleted amino acid sequence. The most common exon 19 deletion subtype is Δ⁷⁴⁶ELREA⁷⁵⁰ (SEQ ID NO: 80). EGFR G719X accounts for approximately 3% of EGFR activating mutations and results from substitutions of the glycine at position 719 to other residues, primarily alanine (G719A), cysteine (G719C) or serine (G719S). Exon 21 L861Q accounts for approximately 2% of initial EGFR activating mutations.

Most NSCLC patients harboring activating mutations in exon 20 (exon 20 insertions) do not respond to FDA approved EGFR TKIs or irreversible inhibitors. Exon 20 insertions are heterogenous in frame inserts of one to seven amino acids. The frequency exon 20 insertions was reported to be between 4% and 11% of the subset of NSCLC patients with EGFR mutations in several studies. Specifically, Vyse and Huang et al reported that the frequency of EGFR exon 20 insertions was 4-10% of all observed EGFR mutations in NSCLC (Vyse, S. and Huang, PH. Signal Transduct. Target Ther. 4(5) (2019)). Arcila et al reported that exon 20 insertions account for at least 9% and potentially up to 11% of all EGFR-mutated cases, representing the third most common type of EGFR mutation after exon 19 deletions and L858R (Arcila, M E. et al. Mol. Ther. 12(2); 220-9 (2012)). Additionally, exon 20 insertions are largely mutually exclusive of other known oncogenic driver events that are characteristic of NSCLC, such as KRAS mutations. Ruan et al (Z. Ruan and N. Kannan. PNAS. August 2018, 115 (35) E8162-E8171) found 97 exon 20 insertions in 421 patient samples. The top 33 exon 20 insertions with the frequency 0.5% as reported by Ruan et al were identified for further evaluation (Table 4-26).

Identification, Selection and Prioritization NSCLC EGFR Activating Mutations

Once the EGFR activating mutations were identified, a similar process was completed for selecting and designing activating mutations as outlined in Example 1 and described herein.

The frequency of exon 19 deletions was determined in a non-redundant set of 2,268 NSCLC patient tumor samples as described herein. Eighty-five (3.7%) of the 2,268 samples harbored deletions in EGFR at the glutamic acid in amino acid position 746. Seventy-eight of the 2,268 samples (3.4%) contained the E746_A750del mutation, five samples (0.2%) contained the E746_S752delinsA mutation and two samples (0.1%) contained the E746_T751delinsA. The E746_A750del mutation was selected for further analysis because it occurred at the highest frequency of the three E746 deletion variants. Nineteen (0.8%) of the 2,268 NSCLC samples harbored an exon 19 deletion at the leucine at amino acid position 747 of EGFR. There were six different variants of exon 19 L747 deletions: L747_E749del (n=2), L747_A750del (n=1), L747_T751del (n=7), L747_S752del (n=4), L747_P753delinsS (n=3) and L747_A750delinsP (n=2). L747_T751del occurred most frequently of the L747 deletion variants and was selected for further analysis. L747_T751del occurred at a frequency of less than 0.5% (0.3%) in the 2,268 patient samples but was still included in the analysis as a representative of all exon 19 L747 deletion variants that cumulatively occurred in 0.8% of the 2,268 NSCLC samples.

The frequency of L858R and G719X was determined in the same non-redundant data set of 2,268 NSCLC samples. The L858R mutation was found in 121 samples (5.3%) and was included in further analysis. G719X occurred in 0.8% (n=17) of samples. The glycine at position 719 (G719X) was substituted with alanine in eleven samples, serine in four samples and cysteine in two samples. G719A was selected for further analysis because it occurred the most frequently of the G719X mutations and in 0.5% of the patient samples.

The frequency of each exon 20 insertion was determined using the occurrence of 97 distinct EGFR insertion mutations in 421 samples as reported by Ruan et al. The data was sourced from a publicly available supplementary data table downloaded Sep. 9, 2020 (https://www.pnas.org/content/115/35/E8162/tab-figures-data). For example, the insertion D770_N771insSVD was found in 53 of 421 NSCLC samples and the frequency of this insertion estimated as 12.6%. If more than one exon 20 insertion was counted in the data set the same number of times the frequency of each insertion was estimated by dividing by the number of insertions reported at that count. For example, the exon 20 insertions V769_D770insASV, S768_V769insVAS, and A767_S768insSVA were counted 83 times in the data set of 421 samples (19.7%) and the frequency the individual insertions estimated as 6.6%.

CD8 epitope analysis was first performed to select the most frequently occurring insertion mutation at each insertion point with CD8 epitopes. The insertion mutations that did not generate CD8 epitopes were excluded. The total number of HLA-A and HLA-B supertype-restricted 9-mer CD8 epitopes and estimated frequency (%) for each mutation were shown in Table 4-26. CD4 epitope analysis was also performed for the selected activating mutations that contained CD8 epitopes (Table 4-27).

TABLE 4-26
Prioritization and selection of identified NSCLC
EGFR activating mutations by CD8 epitope analysis
	Number of		Included as
	total CD8		a vaccine
EGFR activating	epitopes	Frequency	target
mutations	(SB + WB)	(%)	Yes (Y) or No (N)
D761 E762insEAFQ	7	2.6	Y
A763 Y764insFQEA	7	2.6	Y
A767 S768insSVA	8	6.6	Y
A767 S768insSVG	8	0.2	N
A767 S768insTLA	9	0.5	N
S768 V769insVAS	8	6.6	Y
V769 D770insASV	6	6.6	Y
V769 D770insGSV	6	0.2	N
V769 D770insGVV	6	0.7	N
V769 D770insMASVD	5	0.5	N
D770 N771insG	0	3.6	N
D770 N771insGD	0	0.5	N
D770 N771insGF	6	0.7	N
D770 N771insGL	4	0.5	N
D770 N771insGT	0	0.5	N
D770 N771insSVD	3	12.6	Y
D770repGY	1	3.1	N
N771 P772insH	3	0.7	N
N771 P772insN	0	1.4	N
N771 P772insV	3	0.5	N
N771repGF	7	0.5	Y
N771repGY	5	0.7	N
P772 H773insDNP	0	1.0	N
P772 H773insPR	4	2.6	Y
N772 P772insYNP	4	0.5	N
P772repSVDNR	2	1.2	N
H773 V774insAH	4	1.2	N
H773 V774insGNPH	0	0.7	N
H773 V774insH	3	3.8	Y
H773 V774insNPH	0	7.8	N
H773 V774insPH	4	2.9	N
H773repNPY	8	0.7	N
V774 C775insHV	3	3.1	Y
E746_A750del	0	3.4	N
L747_T751del	0	0.3	N
G719A	4	0.5	Y
L858R	3	5.3	N
L861Q	1	0.7	N
L858R L861Q	2	6.0	Y

TABLE 4-27
CD4 epitope analysis of selected EGFR activating mutations
	Number of		Included as
	total CD4		a vaccine
EGFR activating	epitopes	Frequency	target
mutations	(SB + WB)	(%)	Yes (Y) or No (N)
D761 E762insEAFQ	159	2.6	Y
A763 Y764insFQEA	158	2.6	Y
A767 S768insSVA	180	6.6	Y
S768 V769insVAS	188	6.6	Y
V769 D770insASV	152	6.6	Y
D770 N771insSVD	138	12.6	Y
N771repGF	124	0.5	Y
P772 H773insPR	86	2.6	Y
H773 V774insH	48	3.8	Y
V774 C775insHV	84	3.1	Y
G719A	0	0.5	Y
L858R L861Q	28	6.0	Y

Thirteen NSCLC activating mutations were selected and included as driver mutation vaccine targets. The total number of CD8 epitopes for each HLA-A and HLA-B supertype introduced by 13 selected NSCLC EGFR activating mutations encoded by 12 peptides was shown in Table 4-28.

TABLE 4-28
CD8 epitopes introduced by 13 selected NSCLC EGFR
activating mutations encoded by 12 peptides
	HLA-A	HLA-B
EGFR activating	Supertypes	Supertypes	Total CD8
mutations	(n = 5)	(n = 7)	epitopes
D761 E762insEAFQ	4	3	7
A763 Y764insFQEA	4	3	7
A767 S768insSVA	3	5	8
S768 V769insVAS	3	5	8
V769 D770insASV	2	4	6
D770 N771insSVD	2	1	3
N771repGF	4	3	7
P772 H773insPR	0	4	4
H773 V774insH	0	3	3
V774 C775insHV	0	3	3
G719A	1	3	4
L858R, L861Q	1	1	2

The total number of CD4 epitopes for Class II locus DRB1, DRB 3/4/5, DQA1/DQB1 and DPB1 introduced by 13 selected NSCLC EGFR activating mutations is shown in Table 4-29.

TABLE 4-29
CD4 epitopes introduced by 13 selected NSCLC EGFR
activating mutations encoded by 12 peptides
EGFR activating	DRB1	DRB3/4/5	DQA1/DQB1	DPB1	Total CD4
mutations	(n = 26)	(n = 6)	(n = 8)	(n = 6)	epitopes
D761 E762insEAFQ	70	20	40	29	159
A763 Y764insFQEA	82	19	37	20	158
A767 S768insSVA	91	31	28	30	180
S768 V769insVAS	101	32	29	26	188
V769 D770insASV	84	22	28	18	152
D770 N771insSVD	76	21	25	16	138
N771repGF	69	19	21	15	124
P772 H773insPR	47	11	22	6	86
H773 V774insH	25	8	12	3	48
V774 C775insHV	48	12	16	8	84
G719A	0	0	0	0	0
L858R, L861Q	9	0	8	11	28

NSCLC EGFR Activating Mutation Construct

The EGFR activating mutation construct (SEQ ID NO: 81 and SEQ ID NO: 82) insert gene encodes 448 amino acids encoding EGFR activating mutation sequences described in Table 4-30 separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37). Native EGFR DNA and protein sequences are described in Table 2-10.

TABLE 4-30
NSCLC EGFR activating mutation construct sequences
NSCLC EGFR       DNA Sequence
activating       1 ATGGCCACAT CTCCCAAGGC CAACAAAGAG ATCCTGGACG AGGCCTTCCA AGAGGCCTAC
mutation         61 GTGATGGCCA GCGTGGACAA TCCTCACGTG TGCAGAAGAG GCCGGAAGCG GAGAAGCAAA
construct insert 121 GCTAACAAAG AAATTCTCGA CGAAGCCTAT GTCATGGCCT CCGTGGCCTC TGTGGATAAC
(SEQ ID NO: 81)  181 CCACATGTGT GCAGACTGCT GGGCATCTGC AGAGGCCGCA AGAGAAGATC CAGAGAGGCT
                 241 ACAAGCCCTA AGGCAAACAA AGAAATACTG GATGAAGCTT TTCAAGAGGC TTATGTTATG
                 301 GCTTCCGTCG ACAACCCACA CGTGCGGGGC AGAAAGCGGC GGAGCAAAGA AATCCTTGAT
                 361 GAGGCATATG TGATGGCATC TGTGGACAGT GTGGATAATC CCCACGTCTG TCGGCTGCTG
                 421 GGAATTTGCC TGACCAGCAG AGGCAGAAAA AGACGGTCCC TGCGCATCCT GAAAGAGACA
                 481 GAGTTCAAGA AGATCAAGGT CCTGGCCAGC GGCGCCTTTG GCACAGTGTA CAAAGGCCTG
                 541 TGGATTCCCG AGCGCGGCAG AAAGAGAAGA AGCCTGGACG AAGCTTACGT TATGGCCAGT
                 601 GTCGATAACC CTCACCACGT GTGCCGCCTG CTCGGAATCT GTCTGACAAG CACCGTGCAG
                 661 CGGGGACGCA AGCGGAGATC TGTGCTGGTT AAGACCCCTC AGCACGTGAA GATCACCGAC
                 721 TTCGGCAGAG CTAAGCAGCT GGGCGCCGAG GAAAAAGAGT ATCACGCCGA AGGCAGAGGA
                 781 CGGAAGAGGC GCAGCAACAA AGAGATACTT GACGAAGCCT ACGTGATGGC TTCTGTGGAC
                 841 GGCTTCCCTC ACGTCTGTAG ACTCCTCGGC ATCTGCCTGA CCTCCACCAG AGGACGAAAA
                 901 CGCAGAAGCG AGATTCTTGA CGAGGCTTAC GTCATGGCAT CCGTGGATAA CCCTCCACGG
                 961 CATGTCTGTA GGCTGTTGGG GATCTGTCTC ACCTCTACCG TCCGGGGAAG AAAAAGGCGG
                 1021 AGCGCCAACA AAGAAATTTT GGATGAGGCC TACGTTATGG CCTCTGTGGC TAGCGTGGAC
                 1081 AACCCGCATG TTTGTCGCCT GCTTGGGATC TGCCTCAGAG GAAGAAAGCG GAGGTCTAAC
                 1141 AAAGAAATAT TGGACGAGGC TTATGTGATG GCTAGCGTGG CCTCCGTGGA CAATCCCCAT
                 1201 GTCTGTAGAT TGCTCGGGAT ATGTCTGACC AGGGGTCGCA AGCGCCGATC TCTCGATGAG
                 1261 GCTTATGTCA TGGCCAGTGT GGACAACCCA CACGTCCACG TGTGCAGGCT GCTTGGTATT
                 1321 TGCCTCACCT CCACCGTGCA GCTG
NSCLC EGFR       Protein Sequence*
activating       1 MATSPKANKE ILDEAFQEAY VMASVDNPHV CRRGRKRRSK ANKEILDEAY VMASVASVDN
mutation         61 PHVCRLLGIC RGRKRRSREA TSPKANKEIL DEAFQEAYVM ASVDNPHVRG RKRRSKEILD
construct insert 121 EAYVMASVDS VDNPHVCRLL GICLTSRGRK RRSLRILKET EFKKIKVLAS GAFGTVYKGL
(SEQ ID NO: 82)  181 WIPERGRKRR SLDEAYVMAS VDNPHHVCRL LGICLTSTVQ RGRKRRSVLV KTPQHVKITD
                 241 FGRAKQLGAE EKEYHAEGRG RKRRSNKEIL DEAYVMASVD GFPHVCRLLG ICLTSTRGRK
                 301 RRSEILDEAY VMASVDNPPR HVCRLLGICL TSTVRGRKRR SANKEILDEA YVMASVASVD
                 361 NPHVCRLLGI CLRGRKRRSN KEILDEAYVM ASVASVDNPH VCRLLGICLT RGRKRRSLDE
                 421 AYVMASVDNP HVHVCRLLGI CLTSTVQL
*Activating mutation is highlighted in bold.
The furin cleavage sequence is underlined.

Immune responses to EGFR activating mutations

The NSCLC vaccine-A A549 cell line modified to expression of CD276, reduce secretion of TGFβ1 and TGFβ2, and express GM-CSF, membrane bound CD40L, IL-12, modWT1 and modTBXT, and peptides encoding KRAS driver mutations G12D and G12V was transduced with lentiviral particles encoding the gene to express thirteen EGFR activating mutations encoded by twelve peptides separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37).

Immune responses to EGFR activating mutations were evaluated by IFNγ ELISpot. Specifically, 1.5×10⁶ of unmodified A549 or NSCLC vaccine-A A549 modified to express EGFR activating mutations were co-cultured with 1.5×10⁶ iDCs from eight HLA diverse donors (n=4/donor). The HLA-A, HLA-B, and HLA-C alleles for each of the eight donors are in Table 4-10. CD14-PBMCs were isolated from co-culture with DCs on day 6 and stimulated with peptide pools, 15-mers overlapping by 9 amino acids, for each EGFR activating mutation (Thermo Scientific Custom Peptide Service) for 24 hours prior to detection of IFNγ producing cells. Peptides, 15-mers overlapping by 9 amino acids, were designed to cover the full amino acid sequence of the twelve peptides encoding EGFR activating mutations, excluding the furin cleavage sequences, but only 15-mer peptides containing the EGFR mutations were used to stimulate PBMCs in the IFNγ ELISpot assay.

FIG. 12 demonstrates IFNγ production against all twelve EGFR activating mutations are more robust for NSCLC vaccine-A A549 compared to unmodified A549 (Table 4-30.1). The magnitude of IFNγ responses induced by the modified NSCLC vaccine-A A549 cell line against the A767 S768insSVA (p=0.016), H773 V774insH (p=0.039, N771 repGF (p=0.047), S768 V769insVAS (p=0.008) and V769 D770insASV (p=0.016) EGFR activating mutations was significantly greater compared to unmodified A549. Statistical significance was determined using the Mann-Whitney U test.

TABLE 4-30.1
Immune responses to EGFR activating mutations
	Unmodified A549 (SFU ± SEM)	Modified A549 (SFU ± SEM)
NSCLC	A763	A767	D761	D770	A763	A767	D761	D770
EGFR	Y764ins	S768ins	E762ins	N771ins	Y764ins	S768ins	E762ins	N771ins
mutation	FQEA	SVA	EAFQ	SVD	FQEA	SVA	EAFQ	SVD
Donor 1	80 ± 46	0 ± 0	0 ± 0	0 ± 0.	0 ± 0	0 ± 0	0 ± 0	1,585 ± 677
Donor 2	140 ± 81	0 ± 0	78 ± 43	0 ± 0	0 ± 0	415 ± 240	0 ± 0	0 ± 0
Donor 3	60 ± 48	0 ± 0	0 ± 0	100 ± 60	0 ± 0	120 ± 71	400 ± 309	220 ± 160
Donor 4	0 ± 0	0 ± 0	0 ± 0	0 ± 0	285 ± 262	275 ± 265	0 ± 0	0 ± 0
Donor 5	0 ± 0	0 ± 0	0 ± 0	160 ± 73	600 ± 376	570 ± 334	0 ± 0	0 ± 0
Donor 6	220 ± 116	275 ± 161	210 ± 137	140 ± 115	2,010 ± 826	1,075 ± 826	1,020 ± 742	0 ± 0
Donor 7	130 ± 94	0 ± 0	0 ± 0	145 ± 106	0 ± 0	1,185 ± 714	625 ± 509	805 ± 802
Donor 8	93 ± 74	0 ± 0	0 ± 0	0 ± 0	0 ± 0	960 ± 554	0 ± 0	0 ± 0
Average	74 ± 28	34 ± 34	36 ± 27	70 ± 27	437 ± 243	498 ± 134	178 ± 130	226 ± 196
NSCLC	Unmodified A549 (SFU ± SEM)	Modified A549 (SFU ± SEM)
EGFR		H773	L858R			H773	L858R
mutation	G719A	V774insH	L861Q	N771 rep GF	G719A	V774insH	L861Q	N771rep GF
Donor 1	0 ± 0	0 ± 0	0 ± 0	0 ± 0	2,540 ± 995	1,350 ± 632	0 ± 0	210 ± 197
Donor 2	0 ± 0	0 ± 0	230 ± 87	250 ± 237	345 ± 161	1,110 ± 822	0 ± 0	0 ± 0
Donor 3	115 ± 102	0 ± 0	0 ± 0	0 ± 0	625 ± 223	315 ± 196	100 ± 87	0 ± 0
Donor 4	0 ± 0	50 ± 19	0 ± 0	0 ± 0	0 ± 0	460 ± 396	0 ± 0	280 ± 165
Donor 5	0 ± 0	0 ± 0	0 ± 0	0 ± 0	190 ± 177	490 ± 331	820 ± 278	340 ± 228
Donor 6	0 ± 0	60 ± 42	460 ± 202	215 ± 131	0 ± 0	0 ± 0	2,390 ± 1,405	4,088 ± 1,380
Donor 7	0 ± 0	0 ± 0	0 ± 0	0 ± 0	683 ± 392	0 ± 0	600 ± 482	0 ± 0
Donor 8	0 ± 0	53 ± 13	93 ± 80	0 ± 0	0 ± 0	0 ± 0	596 ± 312	1,107 ± 194
Average	18 ± 14	20 ± 10	98 ± 59	58 ± 38	486 ± 303	527 ± 170	591 ± 288	796 ± 486
	Unmodified A549 (SFU ± SEM)	Modified A549 (SFU ± SEM)
NSCLC		S768	V769		P772	S768		V774
EGFR	P772	V769ins	D770ins	V774	H773ins	V769ins	V769	C775ins
mutation	H773insPR	VAS	ASV	C775insHV	PR	VAS	D770insASV	HV
Donor 1	310 ± 184	0 ± 0	0 ± 0	175 ± 141	160 ± 147	530 ± 319	1,100 ± 328	823 ± 368
Donor 2	230 ± 181	190 ± 126	170 ± 98	190 ± 153	0 ± 0	1,050 ± 373	540 ± 243	2,750 ± 1,504
Donor 3	0 ± 0	100 ± 58	0 ± 0	0 ± 0	0 ± 0	440 ± 222	238 ± 122	0 ± 0
Donor 4	0 ± 0	110 ± 75	0 ± 0	0 ± 0	340 ± 236	330 ± 240	0 ± 0	0 ± 0
Donor 5	320 ± 209	0 ± 0	0 ± 0	290 ± 169	0 ± 0	295 ± 279	980 ± 475	1,390 ± 618
Donor 6	145 ± 97	150 ± 137	340 ± 197	285 ± 200	3,620 ± 1,380	410 ± 253	2,680 ± 2,203	0 ± 0
Donor 7	0 ± 0	0 ± 0	0 ± 0	0 ± 0	605 ± 355	885 ± 304	0 ± 0	490 ± 297
Donor 8	0 ± 0	0 ± 0	0 ± 0	0 ± 0	267 ± 210	524 ± 335	0 ± 0	0 ± 0
Average	163 ± 53	69 ± 28	64 ± 45	154 ± 48	548 ± 441	484 ± 87	818 ± 308	794 ± 355

Identification and Prioritization of EGFR Acquired Tyrosine Kinase Inhibitor (TKI) Resistance Mutations for Expression by the NSCLC Vaccine

Table 4-31 describes EGFR TKI acquired resistance mutations identified through literature search.

TABLE 4-31
NSCLC EGFR TKI acquired mutations
EGFR acquired mutation Brief description
L692V                  Acquired resistance mutation to 3rd-generation EGFR TKIs.
E709K                  Acquired resistance mutation to 3rd-generation EGFR TKIs.
L718Q                  Acquired resistance mutation to 3rd-generation EGFR TKIs.
G724S                  Acquired resistance mutation to 3rd-generation EGFR TKIs.
T790M                  Acquired resistance mutation to 1st- or 2nd-generation EGFR TKIs.
C797S                  Acquired resistance mutation to 3rd-generation EGFR TKIs.
L798I                  Acquired resistance mutation to 3rd-generation EGFR TKIs.
L844V                  Acquired resistance mutation to 3rd-generation EGFR TKIs.

Once the EGFR acquired mutations were identified, the process for selection of EGFR TKI acquired mutations was completed as described in Example 1 and described herein.

Results of completed CD4 and CD8 epitope analysis, the total number of HLA-A and HLA-B supertype-restricted 9-mer CD8 epitopes and the total number of CD4 epitopes for each EGFR acquired mutation are shown in Table 4-32. Eight EGFR acquired mutations encoded by five peptide sequences were selected and included as vaccine targets based on the CD4 and CD8 epitope analysis results.

Information on frequencies of EGFR acquired mutations in patient samples was not available for resistance acquired mutations other than T790M. Tumor biopsies, from which the patient data are generated, are usually acquired prior to first line therapy to guide patient treatment and, therefore, would not include samples with acquired resistance mutations. The frequency of T790M in the available patient data (n=7 of 2,268) underestimates the frequency of T790M in the general patient population following 1st line treatment. Patients may not undergo a second tumor biopsy to evaluate T790M status because this mutation can also be detected using liquid biopsy approaches. For this reason, the presence of T790M would be underestimated the available patient data set. Several studies reported approximately 50% of patients acquired the T790M mutation following 1st-generation TKI treatment.

TABLE 4-32
Prioritization and selection of identified NSCLC
EGFR TKI acquired resistance mutations
	Number of	Number of	Included as
	total CD8	total CD4	a vaccine
EGFR acquired	epitopes	epitopes	target
mutations	(SB + WB)	(SB + WB)	Yes (Y) or No (N)
L692V	2	7	Y
E709K	4	0	Y
L718Q	1	n/a	N
G724S	5	0	N
G719A	0	4	N
L718Q G724S	6	0	Y
T790M	13	3	N
C797S	7	n/a	N
L798I	6	n/a	N
C797S L798I	8	n/a	N
T790M C797S L798I	19	72	Y
L844V	2	7	Y

The total number of CD8 epitopes for each HLA-A and HLA-B supertype introduced by 8 EGFR acquired mutations encoded by 5 peptide sequences was shown in Table 4-33.

TABLE 4-33
CD8 epitopes introduced by 8 selected NSCLC EGFR TKI acquired
resistance mutations encoded by 5 peptide sequences
	HLA-A	HLA-B	Total number
EGFR acquired	Supertypes	Supertypes	of CD8
mutations	(n = 5)	(n = 7)	epitopes
L692V	1	1	2
E709K	3	1	4
L718Q G724S	2	4	6
T790M C797S L798I	8	11	19
L844V	1	1	2

The total number of CD4 epitopes for Class II locus DRB1, DRB 3/4/5, DQA1/DQB1 and DPB1 introduced by 8 EGFR acquired mutations encoded by 5 peptide sequences was shown in Table 4-34.

TABLE 4-34
CD4 epitopes introduced by 8 selected NSCLC EGFR TKI acquired
resistance mutations encoded by 5 peptide sequences
					Total number
EGFR acquired	DRB1	DRB3/4/5	DQA1/DQB1	DPB1	of CD4
mutations	(n = 26)	(n = 6)	(n = 8)	(n = 6)	epitopes
L692V	0	0	0	7	7
E709K	0	0	0	0	0
L718Q G724S	0	0	0	0	0
T790M C797S L798I	41	8	1	22	72
L844V	0	0	0	7	7

EGFR Insert Sequences of the NSCLC EGFR Acquired Mutation Construct

The construct insert gene encodes 185 amino acids containing the EGFR acquired mutation sequences that were separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37). The native DNA and protein EGFR sequences are described in Table 2-10.

TABLE 4-35
NSCLC EGFR TKI acquired resistance mutations construct
NSCLC EGFR       DNA Sequence
acquired         1 ATGCTGACAT CTACCGTGCA GCTGATCATG CAGCTCATGC CCTTCGGCAG CATCCTGGAC
mutation         61 TATGTGCGCG AGCACAAGGA CAACATCGGC AGCCAGTACC GGGGCAGAAA GCGGAGATCT
construct insert 121 AGAACCCTGC GGAGACTGCT GCAAGAGCGC GAACTGGTGG AACCCGTTAC ACCTTCTGGC
(SEQ ID NO: 83)  181 GAGGCCCCTA ATCAGGCCCT GCTGAGAATC CTGAGAGGCC GGAAGAGAAG AAGCCCTAGC
                 241 GGAGAGGCTC CTAACCAGGC TTTGCTGCGG ATTCTGAAGA AAACCGAGTT CAAGAAGATC
                 301 AAGGTCCTCG GCAGCGGCGC CTTTGGCAGA GGCAGAAAAA GAAGATCCGA GGACAGACGG
                 361 CTGGTGCACA GAGATCTGGC CGCTAGAAAC GTGGTGGTCA AGACCCCTCA GCACGTGAAG
                 421 ATCACCGACT TCGGACTGGC CAGAGGACGG AAACGAAGAT CTCTGCTGCG CATCCTGAAA
                 481 GAGACAGAGT TTAAAAAGAT TAAGGTGCAA GGCTCCGGCG CCTTCAGCAC CGTGTACAAA
                 541 GGACTGTGGA TTCCC
NSCLC EGFR       Protein Sequence*
acquired         1 MLTSTVQLIM QLMPFGSILD YVREHKDNIG SQYRGRKRRS RTLRRLLQER ELVEPVTPSG
mutation         61 EAPNQALLRI LRGRKRRSPS GEAPNQALLR ILKKTEFKKI KVLGSGAFGR GRKRRSEDRR
construct insert 121 LVHRDLAARN VVVKTPQHVK ITDFGLARGR KRRSLLRILK ETEFKKIKVQ GSGAFSTVYK
(SEQ ID NO: 84)  181 GLWIP
*Acquired resistance mutation is highlighted in bold.
The furin cleavage sequence is underlined.

Identification of ALK TKI Acquired Resistance Mutations in NSCLC

Chromosomal rearrangements are the most common genetic alterations in ALK gene, which result in the creation of multiple fusion genes implicated in tumorigenesis, including ALK/EML4, ALK/RANBP2, ALK/ATIC, ALK/TFG, ALK/NPM1, ALK/SQSTM1, ALK/KIF5B, ALK/CLTC, ALK/TPM4 and ALK/MSN. Of the patients with NSCLC tested for ALK rearrangements, EML4 is a common fusion partner in NSCLC patients. ALK/EML4 was expressed in 2-9% of lung adenocarcinomas and expression of ALK fusion genes was mutually exclusive of expression of EGFR mutations. The fusion oncoprotein EML4-ALK contains an N-terminus derived from EML4 and a C-terminus containing the entire intracellular tyrosine kinase domain of ALK, which mediates the ligand-independent dimerization and/or oligomerization of ALK, resulting in constitutive kinase activity. The partner protein, which is the N-terminus of the fusion protein, controls the fusion protein's behavior by upregulating expression of ALK intracellular domain and activating its kinase activity. This activation continues through a series of proteins involved in multiple signaling pathways that are important for tumor cell proliferation or differentiation.

EML4-ALK-positive patients show approximately a 60-74% response rate to ALK inhibitors, such as crizotinib. While this treatment does have a positive outcome for many patients, the response is heterogeneous in some patients and other patients show little or no response to treatment. In addition, it is common that initially responsive patients regress within 1 to 2 years post-treatment due to the acquisition of secondary mutations and the activation of alternative pathways. ALK acquired mutations and/or amplification account for ˜30% of crizotinib (first generation ALK TKI) resistance in ALK-positive NSCLC. However, most crizotinib-resistant tumors remain ALK dependent with sensitivity to next-generation ALK TKIs. In contrast, 40% to 50% of cases resistant to second-generation ALK TKIs do not harbor on-target resistance mechanisms, and these are no longer ALK dependent. One important category of ALK-independent, or off-target, resistance mechanisms is the activation of bypass signaling track(s) through genetic alterations, autocrine signaling, or dysregulation of feedback signaling, resulting in the reactivation of downstream effectors required for tumor cell growth and survival.

ALK rearrangements can be found in various cancers, including, but not limited to colorectal cancer, breast cancer and ovarian cancer. Additionally, the ALK receptor tyrosine kinase can be activated in a wide range of cancers by both chromosomal translocations leading to ALK-fusion proteins or by mutations in the context of full-length ALK protein. For example, ALK mutation is found in 7% of sporadic neuroblastomas and 50% of familial neuroblastomas. The majority of the reported mutations in neuroblastomas are located within the ALK kinase domain and are present in 7-8% of all neuroblastoma cases. Frequently found mutations include ALK-F1174 (V, L, S, I, C), ALK-F1245 (C, I, L, V) and ALK-R1275 (L or Q) in the kinase domain, which account for around 85% of all ALK mutant cases. These mutations also occur in NSCLC. A vaccine targeting selected ALK acquired mutations in NSCLC may thus be effective against other tumor types.

Table 4-36 describes a list of ALK TKI acquired resistance mutations obtained through literature search as described above and herein.

TABLE 4-36
List of NSCLC ALK TKI acquired resistance mutations
ALK acquired
mutation Brief description
1151Tins Affects residues adjacent to the N-terminus of the αC helix,
         promotes ATP binding, and stabilizes active ALK.
L1152P   Affects residues adjacent to the N-terminus of the αC helix,
         promotes ATP binding, and stabilizes active ALK.
L1152R   Affects residues adjacent to the N-terminus of the αC helix,
         promotes ATP binding, and stabilizes active ALK.
C1156Y   Affects residues adjacent to the N-terminus of the αC helix,
         promotes ATP binding, and stabilizes active ALK.
I1171T   Promotes ATP binding and stabilizes active ALK. Frequently
         identified in alectinib-resistant cases, but not in
         ceritinib-resistant cases.
I1171N   Promotes ATP binding and stabilizes active ALK. Frequently
         identified in alectinib-resistant cases, but not in
         ceritinib-resistant cases.
I1171S   Promotes ATP binding and stabilizes active ALK. Frequently
         identified in alectinib-resistant cases, but not in
         ceritinib-resistant cases.
F1174L   Affects residues adjacent to the C-terminus of the αC helix,
         promotes ATP binding, stabilizes active ALK. Confers
         resistance to ceritinib but is sensitive to alectinib.
F1174S   Affects residues adjacent to the C-terminus of the αC helix,
         promotes ATP binding, stabilizes active ALK. Confers
         resistance to ceritinib but is sensitive to alectinib.
F1174C   Affects residues adjacent to the C-terminus of the αC helix,
         promotes ATP binding, stabilizes active ALK. Confers
         resistance to ceritinib but is sensitive to alectinib.
V1180L   Impairs affinity of crizotinib for the ATP binding site.
L1196M   First ALK resistance mutation reported. Considered the gatekeeper
         mutation. Mutation in the catalytic site that
         prevents crizotinib from binding. One of the most common
         resistance mutations detected in post-crizotinib treated
         samples.
L1198P   Promotes ATP binding and stabilizes active ALK.
G1202R   Solvent-front mutation. Impairs affinity of crizotinib for
         ATP binding site. Confers high-level resistance to first and
         second-generation ALK TKIs.
D1203N   Solvent-front mutation. Mechanism of resistance unknown.
S1206Y   Solvent-front mutation. Impairs affinity of crizotinib
         for ATP binding site.
S1206C   Solvent-front mutation. Impairs affinity of crizotinib
         for ATP binding site.
E1210K   E1210K/D1203N is a compound resistance mutation.
G1269A   Lies in the ATP-binding pocket and impairs affinity of crizotinib
         for ATP binding site. One of the most resistance
         mutations detected in post-crizotinib treated samples.
G1269S   Lies in the ATP-binding pocket and impairs affinity of
         crizotinib for ATP binding site.

Prioritization and Selection of Identified NSCLC ALK TKI Acquired Resistance Mutations

Once the ALK acquired mutations were identified as described above, a similar process for selecting and designing ALK acquired mutations for inclusion in the NSCLC vaccine as described in Example 1 and herein.

The total number of HLA-A and HLA-B supertype-restricted 9-mer CD8 epitopes was first determined to down select the ALK acquired mutations considered for inclusion in the final insert. The insertion mutations that did not generate CD8 epitopes were excluded from further analysis. Then the total number of CD4 epitopes for the down selected ALK acquired mutations was determined as described herein. The results of completed CD4 and CD8 epitope analysis are shown in Table 4-37. Twelve ALK acquired mutations encoded by seven peptide sequences were selected and included as vaccine targets based on the CD4 and CD8 epitope analysis results. The information on frequencies of ALK acquired mutations was not available for patient samples. Tumor biopsies, from which the patient data are generated, are most likely acquired prior to first line therapy to guide treatment and, therefore, would not include samples with acquired resistance mutations.

TABLE 4-37
Prioritization and selection of identified
NSCLC ALK TKI acquired resistance mutations
	Number of	Number of	Included as
	total CD8	total CD4	a vaccine
ALK acquired	epitopes	epitopes	target
mutations	(SB + WB)	(SB + WB)	Yes (Y) or No (N)
1151Tins	3	n/a	N
L1152P	0	n/a	N
L1152R	0	n/a	N
C1156Y	2	n/a	N
1151Tins C1156Y	4	58	Y
I1171T	4	n/a	N
I1171N	3	n/a	N
I1171S	4	n/a	N
F1174L	3	n/a	N
F1174S	0	n/a	N
F1174C	1	n/a	N
I1171T F1174L	5	31	N
I1171N F1174L	7	39	Y
I1171S F1174L	6	26	N
V1180L	5	75	Y
L1196M	7	n/a	N
L1198P	2	n/a	N
G1202R	4	n/a	N
D1203N	4	n/a	N
L1196M G1202R	8	22	N
L1196M D1203N	9	3	N
L1196M G1202R D1203N	11	40	Y
L1196M L1198P G1202R D1203N	8	62	N
S1206Y	4	93	Y
S1206C	0	n/a	N
E1210K	0	n/a	N
F1245C	2	0	Y
G1269A	2	0	N
G1269S	2	0	N
R1275Q	4	n/a	N
G1269A R1275Q	5	0	Y

The total number of CD8 epitopes for each HLA-A and HLA-B supertype introduced by 12 selected ALK acquired mutations encoded by 7 peptide sequences was shown in Table 4-38.

TABLE 4-38
CD8 epitopes introduced by 12 selected NSCLC ALK TKI acquired
resistance mutations encoded by 7 peptide sequences
	HLA-A	HLA-B	Total number
ALK acquired	Supertypes	Supertypes	of CD8
Mutations	(n = 5)	(n = 7)	epitopes
1151 Tins C1156Y	2	2	4
I1171N F1174L	4	3	7
V1180L	0	5	5
L1196M G1202R D1203N	2	9	11
S1206Y	2	2	4
F1245C	2	0	2
G1269A R1275Q	2	3	5

The total number of CD4 epitopes for Class II locus DRB1, DRB 3/4/5, DQA1/DQB1 and DPB1 introduced by 12 selected NSCLC ALK acquired mutations encoded by 7 peptide sequences was shown in Table 4-39.

TABLE 4-39
CD4 epitopes introduced by 12 selected NSCLC ALK acquired
resistance mutations encoded by 7 peptide sequences
					Total number
ALK acquired	DRB1	DRB3/4/5	DQA1/DQB1	DPB1	of CD4
Mutations	(n = 26)	(n = 6)	(n = 8)	(n = 6)	epitopes
1151Tins C1156Y	28	10	2	18	58
I1171N F1174L	21	3	0	15	39
V1180L	30	11	1	33	75
L1196M G1202R D1203N	15	6	0	19	40
S1206Y	48	17	3	25	93
F1245C	0	0	0	0	0
G1269A R1275Q	0	0	0	0	0

ALK Sequences and Insert Sequences of the NSCLC ALK TKI Acquired Resistance Mutation Construct

The construct insert gene encodes 261 amino acids containing the ALK acquired mutation sequences that were separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37). Native ALK DNA and protein sequence and the ALK acquired mutation insert sequence are escribed in Table 4-40.

TABLE 4-40
Native ALK sequences and insert sequences for the NSCLC ALK acquired mutation construct
ALK              DNA Sequence
(SEQ ID NO: 85)  1 ATGGGAGCCA TCGGGCTCCT GTGGCTCCTG CCGCTGCTGC TTTCCACGGC AGCTGTGGGC
                 61 TCCGGGATGG GGACCGGCCA GCGCGCGGGC TCCCCAGCTG CGGGGCCGCC GCTGCAGCCC
                 121 CGGGAGCCAC TCAGCTACTC GCGCCTGCAG AGGAAGAGTC TGGCAGTTGA CTTCGTGGTG
                 181 CCCTCGCTCT TCCGTGTCTA CGCCCGGGAC CTACTGCTGC CACCATCCTC CTCGGAGCTG
                 241 AAGGCTGGCA GGCCCGAGGC CCGCGGCTCG CTAGCTCTGG ACTGCGCCCC GCTGCTCAGG
                 301 TTGCTGGGGC CGGCGCCGGG GGTCTCCTGG ACCGCCGGTT CACCAGCCCC GGCAGAGGCC
                 361 CGGACGCTGT CCAGGGTGCT GAAGGGCGGC TCCGTGCGCA AGCTCCGGCG TGCCAAGCAG
                 421 TTGGTGCTGG AGCTGGGCGA GGAGGCGATC TTGGAGGGTT GCGTCGGGCC CCCCGGGGAG
                 481 GCGGCTGTGG GGCTGCTCCA GTTCAATCTC AGCGAGCTGT TCAGTTGGTG GATTCGCCAA
                 541 GGCGAAGGGC GACTGAGGAT CCGCCTGATG CCCGAGAAGA AGGCGTCGGA AGTGGGCAGA
                 601 GAGGGAAGGC TGTCCGCGGC AATTCGCGCC TCCCAGCCCC GCCTTCTCTT CCAGATCTTC
                 661 GGGACTGGTC ATAGCTCCTT GGAATCACCA ACAAACATGC CTTCTCCTTC TCCTGATTAT
                 721 TTTACATGGA ATCTCACCTG GATAATGAAA GACTCCTTCC CTTTCCTGTC TCATCGCAGC
                 781 CGATATGGTC TGGAGTGCAG CTTTGACTTC CCCTGTGAGC TGGAGTATTC CCCTCCACTG
                 841 CATGACCTCA GGAACCAGAG CTGGTCCTGG CGCCGCATCC CCTCCGAGGA GGCCTCCCAG
                 901 ATGGACTTGC TGGATGGGCC TGGGGCAGAG CGTTCTAAGG AGATGCCCAG AGGCTCCTTT
                 961 CTCCTTCTCA ACACCTCAGC TGACTCCAAG CACACCATCC TGAGTCCGTG GATGAGGAGC
                 1021 AGCAGTGAGC ACTGCACACT GGCCGTCTCG GTGCACAGGC ACCTGCAGCC CTCTGGAAGG
                 1081 TACATTGCCC AGCTGCTGCC CCACAACGAG GCTGCAAGAG AGATCCTCCT GATGCCCACT
                 1141 CCAGGGAAGC ATGGTTGGAC AGTGCTCCAG GGAAGAATCG GGCGTCCAGA CAACCCATTT
                 1201 CGAGTGGCCC TGGAATACAT CTCCAGTGGA AACCGCAGCT TGTCTGCAGT GGACTTCTTT
                 1261 GCCCTGAAGA ACTGCAGTGA AGGAACATCC CCAGGCTCCA AGATGGCCCT GCAGAGCTCC
                 1321 TTCACTTGTT GGAATGGGAC AGTCCTCCAG CTTGGGCAGG CCTGTGACTT CCACCAGGAC
                 1381 TGTGCCCAGG GAGAAGATGA GAGCCAGATG TGCCGGAAAC TGCCTGTGGG TTTTTACTGC
                 1441 AACTTTGAAG ATGGCTTCTG TGGCTGGACC CAAGGCACAC TGTCACCCCA CACTCCTCAA
                 1501 TGGCAGGTCA GGACCCTAAA GGATGCCCGG TTCCAGGACC ACCAAGACCA TGCTCTATTG
                 1561 CTCAGTACCA CTGATGTCCC CGCTTCTGAA AGTGCTACAG TGACCAGTGC TACGTTTCCT
                 1621 GCACCGATCA AGAGCTCTCC ATGTGAGCTC CGAATGTCCT GGCTCATTCG TGGAGTCTTG
                 1681 AGGGGAAACG TGTCCTTGGT GCTAGTGGAG AACAAAACCG GGAAGGAGCA AGGCAGGATG
                 1741 GTCTGGCATG TCGCCGCCTA TGAAGGCTTG AGCCTGTGGC AGTGGATGGT GTTGCCTCTC
                 1801 CTCGATGTGT CTGACAGGTT CTGGCTGCAG ATGGTCGCAT GGTGGGGACA AGGATCCAGA
                 1861 GCCATCGTGG CTTTTGACAA TATCTCCATC AGCCTGGACT GCTACCTCAC CATTAGCGGA
                 1921 GAGGACAAGA TCCTGCAGAA TACAGCACCC AAATCAAGAA ACCTGTTTGA GAGAAACCCA
                 1981 AACAAGGAGC TGAAACCCGG GGAAAATTCA CCAAGACAGA CCCCCATCTT TGACCCTACA
                 2041 GTTCATTGGC TGTTCACCAC ATGTGGGGCC AGCGGGCCCC ATGGCCCCAC CCAGGCACAG
                 2101 TGCAACAACG CCTACCAGAA CTCCAACCTG AGCGTGGAGG TGGGGAGCGA GGGCCCCCTG
                 2161 AAAGGCATCC AGATCTGGAA GGTGCCAGCC ACCGACACCT ACAGCATCTC GGGCTACGGA
                 2221 GCTGCTGGCG GGAAAGGCGG GAAGAACACC ATGATGCGGT CCCACGGCGT GTCTGTGCTG
                 2281 GGCATCTTCA ACCTGGAGAA GGATGACATG CTGTACATCC TGGTTGGGCA GCAGGGAGAG
                 2341 GACGCCTGCC CCAGTACAAA CCAGTTAATC CAGAAAGTCT GCATTGGAGA GAACAATGTG
                 2401 ATAGAAGAAG AAATCCGTGT GAACAGAAGC GTGCATGAGT GGGCAGGAGG CGGAGGAGGA
                 2461 GGGGGTGGAG CCACCTACGT ATTTAAGATG AAGGATGGAG TGCCGGTGCC CCTGATCATT
                 2521 GCAGCCGGAG GTGGTGGCAG GGCCTACGGG GCCAAGACAG ACACGTTCCA CCCAGAGAGA
                 2581 CTGGAGAATA ACTCCTCGGT TCTAGGGCTA AACGGCAATT CCGGAGCCGC AGGTGGTGGA
                 2641 GGTGGCTGGA ATGATAACAC TTCCTTGCTC TGGGCCGGAA AATCTTTGCA GGAGGGTGCC
                 2701 ACCGGAGGAC ATTCCTGCCC CCAGGCCATG AAGAAGTGGG GGTGGGAGAC AAGAGGGGGT
                 2761 TTCGGAGGGG GTGGAGGGGG GTGCTCCTCA GGTGGAGGAG GCGGAGGATA TATAGGCGGC
                 2821 AATGCAGCCT CAAACAATGA CCCCGAAATG GATGGGGAAG ATGGGGTTTC CTTCATCAGT
                 2881 CCACTGGGCA TCCTGTACAC CCCAGCTTTA AAAGTGATGG AAGGCCACGG GGAAGTGAAT
                 2941 ATTAAGCATT ATCTAAACTG CAGTCACTGT GAGGTAGACG AATGTCACAT GGACCCTGAA
                 3001 AGCCACAAGG TCATCTGCTT CTGTGACCAC GGGACGGTGC TGGCTGAGGA TGGCGTCTCC
                 3061 TGCATTGTGT CACCCACCCC GGAGCCACAC CTGCCACTCT CGCTGATCCT CTCTGTGGTG
                 3121 ACCTCTGCCC TCGTGGCCGC CCTGGTCCTG GCTTTCTCCG GCATCATGAT TGTGTACCGC
                 3181 CGGAAGCACC AGGAGCTGCA AGCCATGCAG ATGGAGCTGC AGAGCCCTGA GTACAAGCTG
                 3241 AGCAAGCTCC GCACCTCGAC CATCATGACC GACTACAACC CCAACTACTG CTTTGCTGGC
                 3301 AAGACCTCCT CCATCAGTGA CCTGAAGGAG GTGCCGCGGA AAAACATCAC CCTCATTCGG
                 3361 GGTCTGGGCC ATGGCGCCTT TGGGGAGGTG TATGAAGGCC AGGTGTCCGG AATGCCCAAC
                 3421 GACCCAAGCC CCCTGCAAGT GGCTGTGAAG ACGCTGCCTG AAGTGTGCTC TGAACAGGAC
                 3481 GAACTGGATT TCCTCATGGA AGCCCTGATC ATCAGCAAAT TCAACCACCA GAACATTGTT
                 3541 CGCTGCATTG GGGTGAGCCT GCAATCCCTG CCCCGGTTCA TCCTGCTGGA GCTCATGGCG
                 3601 GGGGGAGACC TCAAGTCCTT CCTCCGAGAG ACCCGCCCTC GCCCGAGCCA GCCCTCCTCC
                 3661 CTGGCCATGC TGGACCTTCT GCACGTGGCT CGGGACATTG CCTGTGGCTG TCAGTATTTG
                 3721 GAGGAAAACC ACTTCATCCA CCGAGACATT GCTGCCAGAA ACTGCCTCTT GACCTGTCCA
                 3781 GGCCCTGGAA GAGTGGCCAA GATTGGAGAC TTCGGGATGG CCCGAGACAT CTACAGGGCG
                 3841 AGCTACTATA GAAAGGGAGG CTGTGCCATG CTGCCAGTTA AGTGGATGCC CCCAGAGGCC
                 3901 TTCATGGAAG GAATATTCAC TTCTAAAACA GACACATGGT CCTTTGGAGT GCTGCTATGG
                 3961 GAAATCTTTT CTCTTGGATA TATGCCATAC CCCAGCAAAA GCAACCAGGA AGTTCTGGAG
                 4021 TTTGTCACCA GTGGAGGCCG GATGGACCCA CCCAAGAACT GCCCTGGGCC TGTATACCGG
                 4081 ATAATGACTC AGTGCTGGCA ACATCAGCCT GAAGACAGGC CCAACTTTGC CATCATTTTG
                 4141 GAGAGGATTG AATACTGCAC CCAGGACCCG GATGTAATCA ACACCGCTTT GCCGATAGAA
                 4201 TATGGTCCAC TTGTGGAAGA GGAAGAGAAA GTGCCTGTGA GGCCCAAGGA CCCTGAGGGG
                 4261 GTTCCTCCTC TCCTGGTCTC TCAACAGGCA AAACGGGAGG AGGAGCGCAG CCCAGCTGCC
                 4321 CCACCACCTC TGCCTACCAC CTCCTCTGGC AAGGCTGCAA AGAAACCCAC AGCTGCAGAG
                 4381 ATCTCTGTTC GAGTCCCTAG AGGGCCGGCC GTGGAAGGGG GACACGTGAA TATGGCATTC
                 4441 TCTCAGTCCA ACCCTCCTTC GGAGTTGCAC AAGGTCCACG GATCCAGAAA CAAGCCCACC
                 4501 AGCTTGTGGA ACCCAACGTA CGGCTCCTGG TTTACAGAGA AACCCACCAA AAAGAATAAT
                 4561 CCTATAGCAA AGAAGGAGCC ACACGACAGG GGTAACCTGG GGCTGGAGGG AAGCTGTACT
                 4621 GTCCCACCTA ACGTTGCAAC TGGGAGACTT CCGGGGGCCT CACTGCTCCT AGAGCCCTCT
                 4681 TCGCTGACTG CCAATATGAA GGAGGTACCT CTGTTCAGGC TACGTCACTT CCCTTGTGGG
                 4741 AATGTCAATT ACGGCTACCA GCAACAGGGC TTGCCCTTAG AAGCCGCTAC TGCCCCTGGA
                 4801 GCTGGTCATT ACGAGGATAC CATTCTGAAA AGCAAGAATA GCATGAACCA GCCTGGGCCC
ALK              Protein Sequence
(SEQ ID NO: 86)  1 MGAIGLLWLL PLLLSTAAVG SGMGTGQRAG SPAAGPPLQP REPLSYSRLQ RKSLAVDFVV
                 61 PSLFRVYARD LLLPPSSSEL KAGRPEARGS LALDCAPLLR LLGPAPGVSW TAGSPAPAEA
                 121 RTLSRVLKGG SVRKLRRAKQ LVLELGEEAI LEGCVGPPGE AAVGLLQFNL SELFSWWIRQ
                 181 GEGRLRIRLM PEKKASEVGR EGRLSAAIRA SQPRLLFQIF GTGHSSLESP TNMPSPSPDY
                 241 FTWNLTWIMK DSFPFLSHRS RYGLECSFDF PCELEYSPPL HDLRNQSWSW RRIPSEEASQ
                 301 MDLLDGPGAE RSKEMPRGSF LLLNTSADSK HTILSPWMRS SSEHCTLAVS VHRHLQPSGR
                 361 YIAQLLPHNE AAREILLMPT PGKHGWTVLQ GRIGRPDNPF RVALEYISSG NRSLSAVDFF
                 421 ALKNCSEGTS PGSKMALQSS FTCWNGTVLQ LGQACDFHQD CAQGEDESQM CRKLPVGFYC
                 481 NFEDGFCGWT QGTLSPHTPQ WQVRTLKDAR FQDHQDHALL LSTTDVPASE SATVTSATFP
                 541 APIKSSPCEL RMSWLIRGVL RGNVSLVLVE NKTGKEQGRM VWHVAAYEGL SLWQWMVLPL
                 601 LDVSDRFWLQ MVAWWGQGSR AIVAFDNISI SLDCYLTISG EDKILQNTAP KSRNLFERNP
                 661 NKELKPGENS PRQTPIFDPT VHWLFTTCGA SGPHGPTQAQ CNNAYQNSNL SVEVGSEGPL
                 721 KGIQIWKVPA TDTYSISGYG AAGGKGGKNT MMRSHGVSVL GIFNLEKDDM LYILVGQQGE
                 781 DACPSTNQLI QKVCIGENNV IEEEIRVNRS VHEWAGGGGG GGGATYVFKM KDGVPVPLII
                 841 AAGGGGRAYG AKTDTFHPER LENNSSVLGL NGNSGAAGGG GGWNDNTSLL WAGKSLQEGA
                 901 TGGHSCPQAM KKWGWETRGG FGGGGGGCSS GGGGGGYIGG NAASNNDPEM DGEDGVSFIS
                 961 PLGILYTPAL KVMEGHGEVN IKHYLNCSHC EVDECHMDPE SHKVICFCDH GTVLAEDGVS
                 1021 CIVSPTPEPH LPLSLILSVV TSALVAALVL AFSGIMIVYR RKHQELQAMQ MELQSPEYKL
                 1081 SKLRTSTIMT DYNPNYCFAG KTSSISDLKE VPRKNITLIR GLGHGAFGEV YEGQVSGMPN
                 1141 DPSPLQVAVK TLPEVCSEQD ELDFLMEALI ISKFNHQNIV RCIGVSLQSL PRFILLELMA
                 1201 GGDLKSFLRE TRPRPSQPSS LAMLDLLHVA RDIACGCQYL EENHFIHRDI AARNCLLTCP
                 1261 GPGRVAKIGD FGMARDIYRA SYYRKGGCAM LPVKWMPPEA FMEGIFTSKT DTWSFGVLLW
                 1321 EIFSLGYMPY PSKSNQEVLE FVTSGGRMDP PKNCPGPVYR IMTQCWQHQP EDRPNFAIIL
                 1381 ERIEYCTQDP DVINTALPIE YGPLVEEEEK VPVRPKDPEG VPPLLVSQQA KREEERSPAA
                 1441 PPPLPTTSSG KAAKKPTAAE ISVRVPRGPA VEGGHVNMAF SQSNPPSELH KVHGSRNKPT
                 1501 SLWNPTYGSW FTEKPTKKNN PIAKKEPHDR GNLGLEGSCT VPPNVATGRL PGASLLLEPS
                 1561 SLTANMKEVP LFRLRHFPCG NVNYGYQQQG LPLEAATAPG AGHYEDTILK SKNSMNQPGP
NSCLC ALK        DNA Sequence
acquired         1 ATGGACCCAT CTCCACTGCA AGTGGCCGTG AAAACCACAC TGCCCGAGGT GTACAGCGAG
mutation         61 CAGGACGAGC TGGACTTCCT GATGGAAGCC CTGATCATCC GGGGCAGAAA GCGGAGAAGC
construct insert 121 TGCTCCGAGC AGGATGAACT CGATTTTCTC ATGGAAGCTC TCATCAACAG CAAGCTGAAC
(SEQ ID NO: 87)  181 CACCAGAACA TCGTGCGGTG CATCGGCGTG TCCAGAGGCC GGAAGAGAAG ATCCAGATGT
                 241 ATCGGAGTGT CCCTGCAGAG CCTGCCTAGA TTCATTCTGA TGGAACTGAT GGCCGGACGG
                 301 AACCTGAAGT CCTTCCTGAG AGAGACACGC GGCAGAAAGA GGCGGAGCGC CAGAGATATT
                 361 GCCTGCGGCT GTCAGTACCT GGAAGAGAAC CACTGCATCC ACCGGGATAT CGCCGCCAGA
                 421 AACTGCCTGC TGACATGCCC CAGAGGAAGA AAACGGCGGA GCCTTATGGA AGCACTTATC
                 481 ATTAGCAAGT TCAATCACCA GAATATCCTC CGCTGCATTG GCGTCAGCCT GCAGTCTCTG
                 541 CCTCGCTTCA TCCTGAGAGG ACGGAAGCGG AGATCCCCAC GGTTTATCCT GCTGGAACTT
                 601 ATGGCAGGCG GCGACCTGAA ATACTTCCTG CGGGAAACCC GGCCTAGACC TAGCCAGCCA
                 661 TCTAGCCTGA GAGGCAGAAA AAGACGGTCC AATTGTCTGC TGACCTGTCC TGGACCTGGC
                 721 AGAGTGGCCA AGATCGCCGA TTTTGGCATG GCCCAGGACA TCTACCGGGC CAGCTACTAC
                 781 AGA
NSCLC ALK        Protein Sequence*
acquired         1 MDPSPLQVAV KTTLPEVYSE QDELDFLMEA LIIRGRKRRS CSEQDELDFL MEALINSKLN
mutation         61 HQNIVRCIGV SRGRKRRSRC IGVSLQSLPR FILMELMAGR NLKSFLRETR GRKRRSARDI
construct insert 121 ACGCQYLEEN HCIHRDIAAR NCLLTCPRGR KRRSLMEALI ISKFNHQNIL RCIGVSLQSL
(SEQ ID NO: 88)  181 PRFILRGRKR RSPRFILLEL MAGGDLKYFL RETRPRPSQP SSLRGRKRRS NCLLTCPGPG
                 241 RVAKIADFGM AQDIYRASYY R
*Acquired resistance mutation is highlighted in bold.
The furin cleavage sequence is underlined

Design of ALK Intracellular Domain as a Vaccine Target

All ALK fusion proteins, such as ALK/EML4, ALK/RANBP2, ALK/ATIC, ALK/TFG, ALK/NPM1, ALK/SQSTM1, ALK/KIF5B, ALK/CLTC, ALK/TPM4, and ALK/MSN, contain the entire intracellular tyrosine kinase domain of ALK (ALK-IC). The expression level of ALK-IC is upregulated by the N-terminus of the fusion protein. ALK is minimally expressed in normal tissues. Expression of the ALK protein or its intracellular domain is a characteristic of abnormal cells. As a result, ALK-IC is an ideal target in ALK-rearranged NSCLC and other tumor types.

To improve breadth and magnitude of vaccine-induced cellular immune responses, non-synonymous mutations (NSM) were introduced into ALK-IC as described previously in Example 40 of WO/2021/113328. The sequence identity between huALK-IC and modALK-IC is 95.6%. The HLA-A and HLA-B supertype-restricted epitopes for huALK-IC and ModALK-IC are summarized in Table 4-41. Seventy-two NSMs occurring 2 times were identified for ALK-IC and 25 NSMs were included in the ModALK-IC antigen sequence. Compared to native ALK-IC, ModALK-IC contains an additional 31 neoepitopes due to the introduction of NSMs.

TABLE 4-41
Epitopes in Native and Designed ALK-IC
HLA	Native	Designed
Supertype	SB	WB	Total	SB	WB	Total
A01	5	6	11	5	7	12
A02	3	4	7	5	6	11
A03	1	7	8	3	8	11
A24	4	9	13	4	10	14
A26	2	10	12	2	10	12
B07	11	14	25	12	17	29
B08	4	12	16	7	12	19
B27	1	7	8	2	10	12
B39	5	16	21	6	20	26
B44	4	9	13	5	9	14
B58	3	5	8	3	7	10
B62	0	8	8	1	10	11
Total Epitopes	43	107	150	55	126	181

Insert Sequences Encoding EGFR Acquired Mutations, ALK Acquired Mutations and Modified ALK Intracellular Domain

Table 4-42 describes the sequence of a construct insert gene encodes 830 amino acids containing the modified ALK intracellular domain and acquired mutation sequences that were separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37).

TABLE 4-42
Insert Sequences for the NSCLC ALK construct encoding acquired mutations and
modified intracellular domain (IC)
NSCLC ALK    DNA Sequence
construct    1 ATGGACCCAT CTCCACTGCA AGTGGCCGTG AAAACCACAC TGCCCGAGGT GTACAGCGAG
insert       61 CAGGACGAGC TGGACTTCCT GATGGAAGCC CTGATCATCC GGGGCAGAAA GCGGAGAAGC
ding enco    121 TGCTCCGAGC AGGATGAACT CGATTTTCTC ATGGAAGCTC TCATCAACAG CAAGCTGAAC
TKI acquired 181 CACCAGAACA TCGTGCGGTG CATCGGCGTG TCCAGAGGCC GGAAGAGAAG ATCCAGATGT
resistance   241 ATCGGAGTGT CCCTGCAGAG CCTGCCTAGA TTCATTCTGA TGGAACTGAT GGCCGGACGG
mutations    301 AACCTGAAGT CCTTCCTGAG AGAGACACGC GGCAGAAAGA GGCGGAGCGC CAGAGATATT
and IC       361 GCCTGCGGCT GTCAGTACCT GGAAGAGAAC CACTGCATCC ACCGGGATAT CGCCGCCAGA
(SEQ ID      421 AACTGCCTGC TGACATGCCC CAGAGGAAGA AAACGGCGGA GCCTTATGGA AGCACTTATC
NO: 89)      481 ATTAGCAAGT TCAATCACCA GAATATCCTC CGCTGCATTG GCGTCAGCCT GCAGTCTCTG
             541 CCTCGCTTCA TCCTGAGAGG ACGGAAGCGG AGATCCCCAC GGTTTATCCT GCTGGAACTT
             601 ATGGCAGGCG GCGACCTGAA ATACTTCCTG CGGGAAACCC GGCCTAGACC TAGCCAGCCA
             661 TCTAGCCTGA GAGGCAGAAA AAGACGGTCC AATTGTCTGC TGACCTGTCC TGGACCTGGC
             721 AGAGTGGCCA AGATCGCCGA TTTTGGCATG GCCCAGGACA TCTACCGGGC CAGCTACTAC
             781 AGACGCGGAC GCAAGAGAAG AAGCTACCGG CGGAAGCACC AAGAGCTGCA GGCAATGCAA
             841 ATGGAACTGC AGTCCCCTGA GTACAAGCTG AGCAAGCTGC GGACCAGCAC CATCATGACC
             901 GACTACAACC CCAACTACTG CTTCGCCGGC AAGACCAGCA GCATCTCCGA TCTGAAAGAG
             961 GTGCCCCGGA AGAACATCAC CCTGATCTGG GATCTTGGAC ACGGCGCCTT CGGAGAGGTG
             1021 TACGAGGGAC AAGTGTCCCG GATGCCTAAC GATCCATCTC CTATGAAGGT GGCCGTCAAG
             1081 ACCCTGCCTG AAGTGTGCTC TGAACAAGAT GAGCTTGACT TTTTGATGGA AGCACTCATT
             1141 ATCTCCAAGT TCAACCATCA AAACATCGTC AGATGCATTG GGGTGTCCCT CCAGTCCATG
             1201 CCACGGTTCA TTCTGCTTGA GTTGATGGTC GGAGGCGACC TCAAGAGCTT TCTGCGCGAG
             1261 ACAAGACCCA GGCCAAGCCA GCCTAGTTCT CTGGCCATGC TGGATCTGCT GCACGTGGCC
             1321 CTGGATATCG CTTGTGGCTG CCAGTATCTC GAGAAGAATC ACTTCATCCA CAGAGACATT
             1381 GCCGCTCGGA ATTGCCTGCT CACTTGCCCA GGACCTGGAC GCGTGGCCAA AATTGGAGAC
             1441 TTCGGAATGG CCCGCGATAT CTACAGAGTG TCCTACTACC GGAAGCGGGG CTGTGCCATG
             1501 CTGCCCATTA AGTGGATGCC ACCTGAGGCC TTCATGGAAG GCATCTTCAC CAGCAAGACC
             1561 GACACACTGA GCTTCGGCGT GCTGCTGTGG GAGATCTTTA GCGTGGGCTA CATGCCCTAT
             1621 CCTAGCAAGA GCAATCAAGA GGTGCTGGAA TTCGTGACCA GCGGCGGCAG AATGGACCCT
             1681 CCTAAGAATT GTCTGGGCCC CGTGTACCGG ATCATGACCC AGTGTTGGCA GCACCAGCCT
             1741 GAGGACAGAC CCAACTTCGC CATCATCCTC GAGCGGATCG AGTACTGCAC ACAGGACCCC
             1801 GACGTGATCA ACACAGCCCT GCCTATCGAG TACGGCCCTC TGGTGGAAGA GGAAGAGAAA
             1861 GTCCCCGTCA GACCCAAGAA TCCCGAAGGC GTTCCACCTC TGCTGGTGTC TCAGCAGGCC
             1921 AAGAGAGAAG AGGAACGGTC ACCAGCTGTG CCTCCACCAC TGCCTACAAC AAGCTCTGGA
             1981 AAGGCCGCCA AGAAGCCTAC AGCCGCCGAA ATTAGCGTGC GGGTGCCAAG AGGACCTGCT
             2041 GTGGAAGGCG GCCATGTGAA TATGGCCTTC AGCCAGAGCA ACCCTCCACT CGAGCTGCAC
             2101 AGAGTGCACC GGTTCAGAAA CAAGCCTACC AGCCTGTGGA ACCCTATGTA CGGCAGCTGG
             2161 TTCACCGAGA AGCCCACCAA GAAGAACAAC CCTATCGCCA AGAAAGAGCC CCACGACAGA
             2221 GGCAATCTGG GCCTCGAGGG AAGCTGTACC GTGCCTCCTA ATGTGGCCAC TGGTAGACTG
             2281 CCTGGCGCCT CTCTGCTGCT CGAACCTTCT CTGCTGACAG CCAACATGAA GAAGGTGCCC
             2341 CTGTTCCGGC TGAGGCACTT CCCTTGTGGC AACGTGAACT ACAGCTATCA GCAGCAGGGC
             2401 CTGCCTCTGG AAGCTGCTAC AGCTCCTGGC GCCGGACACT ACGAGGACAC CATCCTGAAG
             2461 TCTAAGAACA GCATGAACCA GCCTGGGCCT
NSCLC ALK    Protein Sequence*
construct    1 MDPSPLQVAV KTTLPEVYSE QDELDFLMEA LIIRGRKRRS CSEQDELDFL MEALINSKLN
insert       61 HQNIVRCIGV SRGRKRRSRC IGVSLQSLPR FILMELMAGR NLKSFLRETR GRKRRSARDI
encoding     121 ACGCQYLEEN HCIHRDIAAR NCLLTCPRGR KRRSLMEALI ISKFNHQNIL RCIGVSLQSL
acquired     181 PRFILRGRKR RSPRFILLEL MAGGDLKYFL RETRPRPSQP SSLRGRKRRS NCLLTCPGPG
mutations    241 RVAKIADFGM AQDIYRASYY RRGRKRRSYR RKHQELQAMQ MELQSPEYKL SKLRTSTIMT
and IC       301 DYNPNYCFAG KTSSISDLKE VPRKNITLIW DLGHGAFGEV YEGQVSRMPN DPSPMKVAVK
(SEQ ID      361 TLPEVCSEQD ELDFLMEALI ISKFNHQNIV RCIGVSLQSM PRFILLELMV GGDLKSFLRE
NO: 90)      421 TRPRPSQPSS LAMLDLLHVA LDIACGCQYL EKNHFIHRDI AARNCLLTCP GPGRVAKIGD
             481 FGMARDIYRV SYYRKRGCAM LPIKWMPPEA FMEGIFTSKT DTLSFGVLLW EIFSVGYMPY
             541 PSKSNQEVLE FVTSGGRMDP PKNCLGPVYR IMTQCWQHQP EDRPNFAIIL ERIEYCTQDP
             601 DVINTALPIE YGPLVEEEEK VPVRPKNPEG VPPLLVSQQA KREEERSPAV PPPLPTTSSG
             661 KAAKKPTAAE ISVRVPRGPA VEGGHVNMAF SQSNPPLELH RVHRFRNKPT SLWNPMYGSW
             721 FTEKPTKKNN PIAKKEPHDR GNLGLEGSCT VPPNVATGRL PGASLLLEPS LLTANMKKVP
             781 LFRLRHFPCG NVNYSYQQQG LPLEAATAPG AGHYEDTILK SKNSMNQPGP
*Acquired resistance mutation is highlighted in bold.
The furin cleavage sequence is underlined

Insert Sequences Encoding EGFR Acquired Mutations and ALK Acquired Mutations

The construct insert described in Table 4-43 gene encodes 452 amino acids containing the EGFR and ALK acquired mutation sequences that were separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37).

TABLE 4-43
Insert sequences for the NSCLC construct encoding EGFR and
ALK TKI acquired resistance mutations
NSCLC            DNA Sequence
construct insert 1 ATGCTGACAT CTACCGTGCA GCTGATCATG CAGCTCATGC CCTTCGGCAG CATCCTGGAC
encoding         61 TATGTGCGCG AGCACAAGGA CAACATCGGC AGCCAGTACC GGGGCAGAAA GCGGAGATCT
EGFR and ALK     121 AGAACCCTGC GGAGACTGCT GCAAGAGCGC GAACTGGTGG AACCCGTTAC ACCTTCTGGC
TKI acquired     181 GAGGCCCCTA ATCAGGCCCT GCTGAGAATC CTGAGAGGCC GGAAGAGAAG AAGCCCTAGC
resistance       241 GGAGAGGCTC CTAACCAGGC TTTGCTGCGG ATTCTGAAGA AAACCGAGTT CAAGAAGATC
mutations        301 AAGGTCCTCG GCAGCGGCGC CTTTGGCAGA GGCAGAAAAA GAAGATCCGA GGACAGACGG
(SEQ ID NO: 91)  361 CTGGTGCACA GAGATCTGGC CGCTAGAAAC GTGGTGGTCA AGACCCCTCA GCACGTGAAG
                 421 ATCACCGACT TCGGACTGGC CAGAGGACGG AAACGAAGAT CTCTGCTGCG CATCCTGAAA
                 481 GAGACAGAGT TTAAAAAGAT TAAGGTGCAA GGCTCCGGCG CCTTCAGCAC CGTGTACAAA
                 541 GGACTGTGGA TTCCCAGAGG AAGAAAGCGG CGGAGCGATC CATCTCCTCT GCAAGTGGCC
                 601 GTGAAAACCA CACTGCCCGA GGTGTACAGC GAGCAGGACG AGCTGGACTT CCTGATGGAA
                 661 GCCCTGATCA TCCGCGGCAG AAAGAGGCGG TCTTGCTCCG AGCAGGATGA ACTCGATTTT
                 721 TTGATGGAAG CTCTCATCAA CAGCAAGCTG AACCACCAGA ACATCGTGCG GTGCATCGGC
                 781 GTGTCCCGGG GACGCAAGAG AAGATCCAGA TGTATCGGAG TGTCCCTGCA GAGCCTGCCT
                 841 AGATTCATTC TGATGGAACT GATGGCCGGA CGGAACCTGA AGTCCTTCCT GAGAGAAACC
                 901 CGGGGACGCA AACGCAGAAG CGCCAGAGAT ATTGCCTGCG GCTGTCAGTA CCTGGAAGAG
                 961 AACCACTGCA TCCACCGGGA TATCGCCGCC AGAAACTGCC TGCTGACATG CCCTCGGGGA
                 1021 AGAAAAAGAC GGTCCCTCAT GGAAGCACTT ATCATTAGCA AGTTCAATCA CCAGAATATC
                 1081 CTCCGCTGCA TTGGCGTCAG CCTGCAGTCT CTGCCTCGCT TTATCCTGCG CGGTAGAAAA
                 1141 CGGCGCAGCC CCAGATTCAT CCTCCTCGAA CTTATGGCAG GCGGCGACCT GAAGTACTTT
                 1201 CTGCGCGAGA CTCGGCCCAG ACCTAGCCAG CCAAGTTCTC TGCGTGGACG GAAGCGGAGA
                 1261 AGCAATTGTC TGCTGACCTG TCCTGGACCT GGCAGAGTGG CCAAGATCGC CGATTTTGGC
                 1321 ATGGCCCAGG ACATCTACAG AGCCAGCTAC TACAGA
NSCLC            Protein Sequence*
construct insert 1 MLTSTVQLIM QLMPFGSILD YVREHKDNIG SQYRGRKRRS RTLRRLLQER ELVEPVTPSG
encoding         61 EAPNQALLRI LRGRKRRSPS GEAPNQALLR ILKKTEFKKI KVLGSGAFGR GRKRRSEDRR
EGFR and ALK     121 LVHRDLAARN VVVKTPQHVK ITDFGLARGR KRRSLLRILK ETEFKKIKVQ GSGAFSTVYK
TKI acquired     181 GLWIPRGRKR RSDPSPLQVA VKTTLPEVYS EQDELDFLME ALIIRGRKRR SCSEQDELDF
resistance       241 LMEALINSKL NHQNIVRCIG VSRGRKRRSR CIGVSLQSLP RFILMELMAG RNLKSFLRET
mutations        301 RGRKRRSARD IACGCQYLEE NHCIHRDIAA RNCLLTCPRG RKRRSLMEAL IISKFNHQNI
(SEQ ID NO: 92)  361 LRCIGVSLQS LPRFILRGRK RRSPRFILLE LMAGGDLKYF LRETRPRPSQ PSSLRGRKRR
                 421 SNCLLTCPGP GRVAKIADFG MAQDIYRASY YR
*Acquired resistance mutation is highlighted in bold.
The furin cleavage sequence is underlined

Insert Sequences Encoding EGFR Acquired Mutations, ALK Acquired Mutations and Modified ALK Intracellular Domain

The construct insert gene (SEQ ID NO: 93 and SEQ ID NO: 94) described in Table 4-44 encodes 1021 amino acids containing the EGFR and ALK acquired mutation sequences and modified ALK intracellular domain that were separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37).

TABLE 4-44
Insert Sequences for the NSCLC construct encoding EGFR and ALK acquired mutations and
modified ALK intracellular domain (IC)
NSCLC        DNA sequence
construct    1 ATGCTGACAT CTACCGTGCA GCTGATCATG CAGCTCATGC CCTTCGGCAG CATCCTGGAC
insert       61 TATGTGCGCG AGCACAAGGA CAACATCGGC AGCCAGTACC GGGGCAGAAA GCGGAGATCT
encoding     121 AGAACCCTGC GGAGACTGCT GCAAGAGCGC GAACTGGTGG AACCCGTTAC ACCTTCTGGC
EGFR and     181 GAGGCCCCTA ATCAGGCCCT GCTGAGAATC CTGAGAGGCC GGAAGAGAAG AAGCCCTAGC
ALK          241 GGAGAGGCTC CTAACCAGGC TTTGCTGCGG ATTCTGAAGA AAACCGAGTT CAAGAAGATC
acquired     301 AAGGTCCTCG GCAGCGGCGC CTTTGGCAGA GGCAGAAAAA GAAGATCCGA GGACAGACGG
mutations    361 CTGGTGCACA GAGATCTGGC CGCTAGAAAC GTGGTGGTCA AGACCCCTCA GCACGTGAAG
and modified 421 ATCACCGACT TCGGACTGGC CAGAGGACGG AAACGAAGAT CTCTGCTGCG CATCCTGAAA
ALKIC        481 GAGACAGAGT TTAAAAAGAT TAAGGTGCAA GGCTCCGGCG CCTTCAGCAC CGTGTACAAA
(SEQ ID      541 GGACTGTGGA TTCCCAGAGG AAGAAAGCGG CGGAGCGATC CATCTCCTCT GCAAGTGGCC
NO: 93)      601 GTGAAAACCA CACTGCCCGA GGTGTACAGC GAGCAGGACG AGCTGGACTT CCTGATGGAA
             661 GCCCTGATCA TCCGCGGCAG AAAGAGGCGG TCTTGCTCCG AGCAGGATGA ACTCGATTTT
             721 TTGATGGAAG CTCTCATCAA CAGCAAGCTG AACCACCAGA ACATCGTGCG GTGCATCGGC
             781 GTGTCCCGGG GACGCAAGAG AAGATCCAGA TGTATCGGAG TGTCCCTGCA GAGCCTGCCT
             841 AGATTCATTC TGATGGAACT GATGGCCGGA CGGAACCTGA AGTCCTTCCT GAGAGAAACC
             901 CGGGGACGCA AACGCAGAAG CGCCAGAGAT ATTGCCTGCG GCTGTCAGTA CCTGGAAGAG
             961 AACCACTGCA TCCACCGGGA TATCGCCGCC AGAAACTGCC TGCTGACATG CCCTCGGGGA
             1021 AGAAAAAGAC GGTCCCTCAT GGAAGCACTT ATCATTAGCA AGTTCAATCA CCAGAATATC
             1081 CTCCGCTGCA TTGGCGTCAG CCTGCAGTCT CTGCCTCGCT TTATCCTGCG CGGTAGAAAA
             1141 CGGCGCAGCC CCAGATTCAT CCTCCTCGAA CTTATGGCAG GCGGCGACCT GAAGTACTTT
             1201 CTGCGCGAGA CTCGGCCCAG ACCTAGCCAG CCAAGTTCTC TGCGTGGACG GAAGCGGAGA
             1261 AGCAATTGTC TGCTGACCTG TCCTGGACCT GGCAGAGTGG CCAAGATCGC CGATTTTGGC
             1321 ATGGCCCAGG ACATCTACAG AGCCAGCTAC TACAGACGCG GACGGAAGAG GCGGAGCTAC
             1381 AGAAGAAAGC ACCAAGAGCT GCAGGCAATG CAAATGGAAC TGCAGTCCCC TGAGTACAAG
             1441 CTGAGCAAGC TGCGGACCAG CACCATCATG ACCGACTACA ACCCCAACTA CTGCTTCGCC
             1501 GGCAAGACCA GCAGCATCTC CGATCTGAAA GAGGTGCCCC GGAAGAACAT CACCCTGATC
             1561 TGGGATCTTG GACATGGCGC CTTCGGAGAG GTGTACGAGG GCCAAGTGTC CCGGATGCCT
             1621 AACGACCCAT CTCCAATGAA GGTGGCCGTC AAGACTCTGC CCGAAGTGTG CTCTGAACAA
             1681 GATGAGCTGG ATTTTCTTAT GGAAGCACTG ATTATCTCCA AGTTCAACCA TCAAAACATT
             1741 GTCCGCTGTA TTGGGGTGTC CCTCCAGTCC ATGCCACGGT TTATTCTGCT CGAGCTGATG
             1801 GTCGGAGGCG ACCTCAAAAG CTTCCTGCGG GAAACCAGAC CTCGGCCAAG CCAGCCATCA
             1861 TCTCTGGCCA TGCTGGATCT GCTGCACGTG GCCCTGGATA TCGCTTGTGG CTGCCAGTAT
             1921 CTCGAGAAGA ATCACTTCAT CCACAGAGAC ATTGCCGCTC GGAATTGCCT GCTCACTTGC
             1981 CCAGGACCTG GACGCGTGGC CAAAATTGGA GACTTCGGCA TGGCTCGCGA TATCTACCGG
             2041 GTGTCCTACT ACCGGAAACG CGGCTGTGCC ATGCTGCCCA TCAAATGGAT GCCTCCAGAG
             2101 GCCTTTATGG AAGGCATCTT CACCAGCAAG ACAGACACCC TGAGCTTCGG CGTGCTGCTG
             2161 TGGGAGATCT TTAGCGTGGG CTACATGCCC TATCCTAGCA AGAGCAATCA AGAGGTGCTG
             2221 GAATTCGTGA CCAGCGGCGG CAGAATGGAC CCTCCTAAGA ATTGTCTGGG CCCCGTGTAC
             2281 CGGATCATGA CCCAGTGTTG GCAGCACCAG CCTGAGGACA GGCCCAACTT TGCCATCATC
             2341 CTCGAGCGGA TCGAGTACTG CACACAGGAC CCCGACGTGA TCAACACAGC CCTGCCTATC
             2401 GAGTACGGCC CTCTGGTGGA AGAGGAAGAG AAAGTCCCCG TCAGACCCAA GAATCCCGAA
             2461 GGCGTTCCAC CTCTGCTGGT GTCCCAGCAG GCCAAGAGAG AAGAGGAACG CTCTCCTGCT
             2521 GTGCCTCCTC CACTGCCTAC AACAAGCTCT GGAAAGGCCG CCAAGAAGCC TACAGCCGCC
             2581 GAAATTAGCG TGCGGGTGCC AAGAGGACCT GCTGTGGAAG GCGGACATGT GAACATGGCC
             2641 TTCAGCCAGA GCAACCCTCC ACTCGAGCTG CACAGAGTGC ACCGGTTCAG AAACAAGCCT
             2701 ACCAGCCTGT GGAACCCTAT GTACGGCAGC TGGTTCACCG AGAAGCCCAC CAAGAAGAAC
             2761 AACCCTATCG CCAAGAAAGA GCCCCACGAC AGAGGCAATC TGGGCCTCGA GGGAAGCTGT
             2821 ACCGTGCCTC CTAATGTGGC CACTGGTAGA CTGCCAGGCG CTAGCCTTCT GCTGGAACCC
             2881 TCTCTGCTGA CAGCCAACAT GAAGAAGGTG CCCCTGTTCC GGCTGAGACA CTTCCCCTGT
             2941 GGCAACGTGA ACTACAGCTA TCAGCAGCAG GGACTGCCTC TGGAAGCCGC TACAGCTCCT
             3001 GGCGCTGGAC ACTACGAGGA CACCATCCTG AAGTCTAAGA ACAGCATGAA CCAGCCTGGG
             3061 CCT
NSCLC        Protein Sequence*
construct    1 MLTSTVQLIM QLMPFGSILD YVREHKDNIG SQYRGRKRRS RTLRRLLQER ELVEPVTPSG
insert       61 EAPNQALLRI LRGRKRRSPS GEAPNQALLR ILKKTEFKKI KVLGSGAFGR GRKRRSEDRR
encoding     121 LVHRDLAARN VVVKTPQHVK ITDFGLARGR KRRSLLRILK ETEFKKIKVQ GSGAFSTVYK
EGFR and     181 GLWIPRGRKR RSDPSPLQVA VKTTLPEVYS EQDELDFLME ALIIRGRKRR SCSEQDELDF
ALK          241 LMEALINSKL NHQNIVRCIG VSRGRKRRSR CIGVSLQSLP RFILMELMAG RNLKSFLRET
acquired     301 RGRKRRSARD IACGCQYLEE NHCIHRDIAA RNCLLTCPRG RKRRSLMEAL IISKFNHQNI
mutations    361 LRCIGVSLQS LPRFILRGRK RRSPRFILLE LMAGGDLKYF LRETRPRPSQ PSSLRGRKRR
and modified 421 SNCLLTCPGP GRVAKIADFG MAQDIYRASY YRRGRKRRSY RRKHQELQAM QMELQSPEYK
ALKIC        481 LSKLRTSTIM TDYNPNYCFA GKTSSISDLK EVPRKNITLI WDLGHGAFGE VYEGQVSRMP
(SEQ ID NO:  541 NDPSPMKVAV KTLPEVCSEQ DELDFLMEAL IISKFNHQNI VRCIGVSLQS MPRFILLELM
94)          601 VGGDLKSFLR ETRPRPSQPS SLAMLDLLHV ALDIACGCQY LEKNHFIHRD IAARNCLLTC
             661 PGPGRVAKIG DFGMARDIYR VSYYRKRGCA MLPIKWMPPE AFMEGIFTSK TDTLSFGVLL
             721 WEIFSVGYMP YPSKSNQEVL EFVTSGGRMD PPKNCLGPVY RIMTQCWQHQ PEDRPNFAII
             781 LERIEYCTQD PDVINTALPI EYGPLVEEEE KVPVRPKNPE GVPPLLVSQQ AKREEERSPA
             841 VPPPLPTTSS GKAAKKPTAA EISVRVPRGP AVEGGHVNMA FSQSNPPLEL HRVHRFRNKP
             901 TSLWNPMYGS WFTEKPTKKN NPIAKKEPHD RGNLGLEGSC TVPPNVATGR LPGASLLLEP
             961 SLLTANMKKV PLFRLRHFPC GNVNYSYQQQ GLPLEAATAP GAGHYEDTIL KSKNSMNQPG
             1021 P
NSCLC        DNA SEQUENCE
modALK-IC    1 TACAGAAGAA AGCACCAAGA GCTGCAGGCA ATGCAAATGG AACTGCAGTC CCCTGAGTAC
(SEQ ID NO:  61 AAGCTGAGCA AGCTGCGGAC CAGCACCATC ATGACCGACT ACAACCCCAA CTACTGCTTC
95)          121 GCCGGCAAGA CCAGCAGCAT CTCCGATCTG AAAGAGGTGC CCCGGAAGAA CATCACCCTG
             181 ATCTGGGATC TTGGACATGG CGCCTTCGGA GAGGTGTACG AGGGCCAAGT GTCCCGGATG
             241 CCTAACGACC CATCTCCAAT GAAGGTGGCC GTCAAGACTC TGCCCGAAGT GTGCTCTGAA
             301 CAAGATGAGC TGGATTTTCT TATGGAAGCA CTGATTATCT CCAAGTTCAA CCATCAAAAC
             361 ATTGTCCGCT GTATTGGGGT GTCCCTCCAG TCCATGCCAC GGTTTATTCT GCTCGAGCTG
             421 ATGGTCGGAG GCGACCTCAA AAGCTTCCTG CGGGAAACCA GACCTCGGCC AAGCCAGCCA
             481 TCATCTCTGG CCATGCTGGA TCTGCTGCAC GTGGCCCTGG ATATCGCTTG TGGCTGCCAG
             541 TATCTCGAGA AGAATCACTT CATCCACAGA GACATTGCCG CTCGGAATTG CCTGCTCACT
             601 TGCCCAGGAC CTGGACGCGT GGCCAAAATT GGAGACTTCG GCATGGCTCG CGATATCTAC
             661 CGGGTGTCCT ACTACCGGAA ACGCGGCTGT GCCATGCTGC CCATCAAATG GATGCCTCCA
             721 GAGGCCTTTA TGGAAGGCAT CTTCACCAGC AAGACAGACA CCCTGAGCTT CGGCGTGCTG
             781 CTGTGGGAGA TCTTTAGCGT GGGCTACATG CCCTATCCTA GCAAGAGCAA TCAAGAGGTG
             841 CTGGAATTCG TGACCAGCGG CGGCAGAATG GACCCTCCTA AGAATTGTCT GGGCCCCGTG
             901 TACCGGATCA TGACCCAGTG TTGGCAGCAC CAGCCTGAGG ACAGGCCCAA CTTTGCCATC
             961 ATCCTCGAGC GGATCGAGTA CTGCACACAG GACCCCGACG TGATCAACAC AGCCCTGCCT
             1021 ATCGAGTACG GCCCTCTGGT GGAAGAGGAA GAGAAAGTCC CCGTCAGACC CAAGAATCCC
             1081 GAAGGCGTTC CACCTCTGCT GGTGTCCCAG CAGGCCAAGA GAGAAGAGGA ACGCTCTCCT
             1141 GCTGTGCCTC CTCCACTGCC TACAACAAGC TCTGGAAAGG CCGCCAAGAA GCCTACAGCC
             1201 GCCGAAATTA GCGTGCGGGT GCCAAGAGGA CCTGCTGTGG AAGGCGGACA TGTGAACATG
             1261 GCCTTCAGCC AGAGCAACCC TCCACTCGAG CTGCACAGAG TGCACCGGTT CAGAAACAAG
             1321 CCTACCAGCC TGTGGAACCC TATGTACGGC AGCTGGTTCA CCGAGAAGCC CACCAAGAAG
             1381 AACAACCCTA TCGCCAAGAA AGAGCCCCAC GACAGAGGCA ATCTGGGCCT CGAGGGAAGC
             1441 TGTACCGTGC CTCCTAATGT GGCCACTGGT AGACTGCCAG GCGCTAGCCT TCTGCTGGAA
             1501 CCCTCTCTGC TGACAGCCAA CATGAAGAAG GTGCCCCTGT TCCGGCTGAG ACACTTCCCC
             1561 TGTGGCAACG TGAACTACAG CTATCAGCAG CAGGGACTGC CTCTGGAAGC CGCTACAGCT
             1621 CCTGGCGCTG GACACTACGA GGACACCATC CTGAAGTCTA AGAACAGCAT GAACCAGCCT
             1681 GGGCCT
NSCLC        Protein Sequence
modALK-IC    1 YRRKHQELQA MQMELQSPEY KLSKLRTSTI MTDYNPNYCF AGKTSSISDL KEVPRKNITL
(SEQ ID NO:  61 IWDLGHGAFG EVYEGQVSRM PNDPSPMKVA VKTLPEVCSE QDELDFLMEA LIISKFNHQN
96)          121 IVRCIGVSLQ SMPRFILLEL MVGGDLKSFL RETRPRPSQP SSLAMLDLLH VALDIACGCQ
             181 YLEKNHFIHR DIAARNCLLT CPGPGRVAKI GDFGMARDIY RVSYYRKRGC AMLPIKWMPP
             241 EAFMEGIFTS KTDTLSFGVL LWEIFSVGYM PYPSKSNQEV LEFVTSGGRM DPPKNCLGPV
             301 YRIMTQCWQH QPEDRPNFAI ILERIEYCTQ DPDVINTALP IEYGPLVEEE EKVPVRPKNP
             361 EGVPPLLVSQ QAKREEERSP AVPPPLPTTS SGKAAKKPTA AEISVRVPRG PAVEGGHVNM
             421 AFSQSNPPLE LHRVHRFRNK PTSLWNPMYG SWFTEKPTKK NNPIAKKEPH DRGNLGLEGS
             481 CTVPPNVATG RLPGASLLLE PSLLTANMKK VPLFRLRHFP CGNVNYSYQQ QGLPLEAATA
             541 PGAGHYEDTI LKSKNSMNQP GP
*Acquired resistance mutation is highlighted in bold.
The furin cleavage sequence is underlined

Immune Responses to EGFR and ALK Acquired TKI Resistance Mutations and ALK-IC Induced by the NSCLC Vaccine-B NCI-H23 Cell Line

The NSCLC vaccine-B NCI-H23 cell line modified to reduce expression of CD276, reduce secretion of TGFβ1 and TGFβ2, and to express GM-CSF, membrane bound CD40L, IL-12, and modMSLN was transduced with lentiviral particles expressing eight EGFR acquired TKI resistance mutations encoded by five peptide sequences, and twelve ALK acquired TKI resistance mutations and modALK-IC encoded by seven peptide sequences separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37) as described above.

Immune responses to the inserted EGFR and ALK acquired TKI resistance mutations and modALK-IC were evaluated by IFNγ ELISpot. Specifically, 1.5×10⁶ of unmodified NCI-H23 or the NSCLC vaccine-B NCI-H23 modified to express EGFR and ALK acquired TKI mutations and modALK-IC were co-cultured with 1.5×10⁶ iDCs from eight HLA diverse donors. HLA-A, HLA-B, and HLA-C alleles for each donor are in Table 4-10. CD14-PBMCs were isolated from co-culture with DCs on day 6 and stimulated with peptide pools, 15-mers overlapping by 9 amino acids (Thermo Scientific Custom Peptide Service) for 24 hours prior to detection of IFNγ producing cells. Peptides, 15-mers overlapping by 9 amino acids, were designed to cover the full amino acid sequences for the individual peptides encoding the EGFR and ALK acquired TKI resistance mutations and modALK-IC, excluding the furin cleavage sequences. Only the 15-mer peptides containing the mutations and spanning the entire length of modALK-IC were used to stimulate PBMCs in the IFNγ ELISpot assay.

FIG. 13 demonstrates immune responses to all five EGFR acquired TKI resistance mutation encoding peptides inserted into the NSCLC vaccine-B NCI-H23 cell line by at least four of eight HLA-diverse donors by IFNγ ELISpot. NSCLC vaccine-B NCI-H23 induced IFNγ responses against EGFR acquired TKI resistance mutations that were greater in magnitude compared to the unmodified NCI-H23 cell line (Table 4-45). The magnitude of IFNγ responses induced by the NSCLC vaccine-B NCI-H23 cell line against the peptide encoding L718Q and G724S EGFR mutations were significantly greater (p=0.039) compared to the unmodified NCI-H23 cell line. Statistical significance was determined using the Mann-Whitney U test.

FIG. 14 demonstrates the NSCLC vaccine-B NCI-H23 cell line induces immune responses to inserted ALK acquired TKI resistance mutations and modALK-IC by at least one of eight HLA-diverse donors by IFNγ ELISpot. The average magnitude of IFNγ responses elicited by the modified NSCLC vaccine-B NCI-H23 cell line increased relative to unmodified NCI-H23 for all inserted ALK mutations and modALK-IC (Table 4-46). Statistical significance was determined using the Mann-Whitney U test.

TABLE 4-45
Immune responses to EGFR acquired TKI resistance mutations
NSCLC EGFR	T790M C797S
Mutation	L798I	L692V	E709K	L844V	L718Q G724S
Unmodified NCI-H23 (SFU ± SEM)
Donor 1	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 2	193 ± 119	293 ± 147	160 ± 92	200 ± 110	0 ± 0
Donor 3	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 4	160 ± 135	80 ± 57	190 ± 112	0 ± 0	0 ± 0
Donor 5	170 ± 131	165 ± 57	180 ± 96	0 ± 0	110 ± 85
Donor 6	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 7	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 8	130 ± 70	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Average	83 ± 32	67 ± 39	66 ± 32	25 ± 25	14 ± 14
Modified NCI-H23 (SFU ± SEM)
Donor 1	445 ± 257	2,690 ± 803	3,110 ± 1,270	1,990 ± 633	2,790 ± 1,083
Donor 2	0 ± 0	0 ± 0	0 ± 0	570 ± 410	0 ± 0
Donor 3	140 ± 115	230 ± 85	605 ± 385	570 ± 254	290 ± 132
Donor 4	380 ± 255	0 ± 0	970 ± 561	1,028 ± 516	800 ± 355
Donor 5	1,910 ± 688	910 ± 326	1,520 ± 520	1,900 ± 862	1,670 ± 1,015
Donor 6	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 7	100 ± 66	265 ± 155	260 ± 150	0 ± 0	0 ± 0
Donor 8	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Average	372 ± 228	512 ± 330	808 ± 381	757 ± 289	694 ± 365

TABLE 4-46
Immune responses to ALK acquired TKI resistance mutations and modALK IC
NSCLC			L1196M					modALK
ALK	1151Tins	I1171N	G1202R				G1269A	intracellular
Mutation	C1156Y	F1174L	D1203N	F1245C	V1180L	S1206Y	R1275Q	domain
Unmodified NCI-H23 (SFU ± SEM)
Donor 1	0 ± 0	210 ± 82	0 ± 0	0 ± 0	60 ± 48	0 ± 0	100 ± 71	210 ± 151
Donor 2	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	493 ± 247
Donor 3	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 4	0 ± 0	70 ± 57	0 ± 0	0 ± 0	130 ± 94	130 ± 85	0 ± 0	240 ± 65
Donor 5	270 ± 133	0 ± 0	0 ± 0	195 ± 131	0 ± 0	50 ± 30	135 ± 113	180 ± 74
Donor 6	0 ± 0	115 ± 68	125 ± 99	300 ± 141	250 ± 170	268 ± 145	1,530 ± 1,156	170 ± 93
Donor 7	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 8	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	1,822 ± 1,507
Average	34 ± 34	49 ± 49	16 ± 16	62 ± 42	61 ± 31	56 ± 34	221 ± 188	397 ± 210
Modified NCI-H23 (SFU ± SEM)
Donor 1	1,800 ± 503	0 ± 0	553 ± 390	500 ± 305	1,070 ± 773	975 ± 566	965 ± 566	0 ± 0
Donor 2	0 ± 0	0 ± 0	0 ± 0	2,070 ± 786	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 3	260 ± 154	0 ± 0	0 ± 0	200 ± 69	490 ± 398	300 ± 101	180 ± 112	4,430 ± 4,232
Donor 4	1,140 ± 481	0 ± 0	140 ± 82	1,205 ± 560	1,230 ± 475	2,740 ± 1,875	1,370 ± 509	60 ± 26
Donor 5	0 ± 0	740 ± 430	630 ± 473	0 ± 0	4,610 ± 3,262	0 ± 0	0 ± 0	1,580 ± 993
Donor 6	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 7	0 ± 0	0 ± 0	480 ± 335	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 8	0 ± 0	0 ± 0	1,280 ± 763	0 ± 0	0 ± 0	0 ± 0	0 ± 0	3,120 ± 1,569
Average	400 ± 244	93 ± 93	385 ± 158	497 ± 269	925 ± 556	502 ± 342	314 ± 191	1,149 ± 617

Genetic modifications completed for NSCLC vaccine-A and NSCLC-B cell lines are described in Table 4-47, below and herein. The CD276 gene was knocked out (KO) by electroporation of zinc-finger nucleases (ZFN) (SEQ ID NO: 52) as described above. All other genetic modifications were completed by lentiviral transduction.

NSCLC Vaccine-A

NCI-H460 was modified to reduce expression of CD276 (SEQ ID NO: 52), knockdown (KD) secretion of transforming growth factor-beta 1 (TGFβ1) (SEQ ID NO: 54) and transforming growth factor-beta 2 (TGFβ2) (SEQ ID NO: 55), and to express granulocyte macrophage-colony stimulating factor (GM-CSF) (SEQ ID NO: 7, SEQ ID NO: 8), membrane-bound CD40L (mCD40L) (SEQ ID NO: 2, SEQ ID NO: 3), interleukin 12 p70 (IL-12) (SEQ ID NO: 9, SEQ ID NO: 10), modBORIS ((SEQ ID NO: 19, SEQ ID NO: 20), peptide sequences encoding TP53 driver mutations R110L, C141Y, G154V, V157F, R158L, R175H, C176F, H214R, Y220C, Y234C, M237I, G245V, R249M, I251F, R273L, R337L, PIK3CA driver mutations E542K and H1047R, and KRAS driver mutations G12A and G13C as (SEQ ID NO: 78, SEQ ID NO: 79).

NCI-H520 was modified reduce expression of CD276 (SEQ ID NO: 52), to reduce secretion of TGFβ1 (SEQ ID NO: 54) and TGFβ2 (SEQ ID NO: 55), and to express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8) and membrane bound CD40L (SEQ ID NO: 2, SEQ ID NO: 3).

A549 was modified to reduce expression of CD276 (SEQ ID NO: 52), reduce secretion of TGFβ1 (SEQ ID NO: 54) and TGFβ2 (SEQ ID NO: 55), and to express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), membrane bound CD40L (SEQ ID NO: 2, SEQ ID NO: 3), IL-12 (SEQ ID NO: 9, SEQ ID NO: 10), modWT1 (SEQ ID NO: 17, SEQ ID NO: 18) and modTBXT (SEQ ID NO: 17, SEQ ID NO: 18), and peptides encoding the KRAS driver mutations G12D (SEQ ID NO: 23, SEQ ID NO: 24) and G12V (SEQ ID NO: 25, SEQ ID NO: 26), and EGFR activating mutations D761 E762insEAFQ, A763 Y764insFQEA, A767 S768insSVA, S768 V769insVAS, V769 D770insASV, D770 N771insSVD, N771repGF, P772 H773insPR, H773 V774insH, V774 C775insHV, G719A, L858R and L861Q (SEQ ID NO: 81, SEQ ID NO: 82).

NSCLC Vaccine-B

NCI-H23 was modified to reduce expression of CD276 (SEQ ID NO: 52), reduce secretion of TGFβ1 (SEQ ID NO: 54) and TGFβ2 (SEQ ID NO: 55), and to express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), membrane bound CD40L (SEQ ID NO: 2, SEQ ID NO: 3), IL-12 (SEQ ID NO: 9, SEQ ID NO: 10), modMSLN (SEQ ID NO: 21, SEQ ID NO: 22), EGFR tyrosine kinase inhibitor (TKI) acquired resistance mutations L692V, E709K, L718Q, G724S, T790M, C797S, L798I and L844V (SEQ ID NO: 93, SEQ ID NO: 94), ALK TKI acquired resistance mutations 1151Tins C1156Y, I1171N F1174L, V1180L, L1196M, G1202R, D1203N, S1206Y, F1245C, G1269A and R1275Q (SEQ ID NO: 93, SEQ ID NO: 94) and modALK-IC (SEQ ID NO: 93, SEQ ID NO: 94).

LK-2 was modified to reduce expression of CD276 (SEQ ID NO: 52), reduce secretion of TGFβ1 (SEQ ID NO: 54) and TGFβ2 (SEQ ID NO: 55) and to express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8) and membrane bound CD40L (SEQ ID NO: 2, SEQ ID NO: 3).

DMS 53 cell line was modified to reduce expression of CD276 (SEQ ID NO: 52), reduce secretion of TGFβ1 (SEQ ID NO: 54) and TGFβ2 (SEQ ID NO: 55), and to express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), membrane bound CD40L (SEQ ID NO: 2, SEQ ID NO: 3) and IL-12 (SEQ ID NO: 9, SEQ ID NO: 10).

TABLE 4-47
NSCLC Vaccine cell line nomenclature and modifications
											TKI
										EGFR	acquired
	Cell	TGF β1	TGFβ2	CD276	GM-				Driver	activating	resistance
Cocktail	Line	KD	KD	KO	CSF	mCD40L	IL-12	TAA(s)	Mutations	mutations	mutations
A	NCI-	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	modBORIS	TP53	—	—
	H460	NO: 54	NO: 55	NO: 52	NO: 8	NO: 3	NO: 10	(SEQ ID	PIK3CA
								NO: 20)	KRAS
									(SEQ ID
									NO: 79)
A	A549	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	modTBXT	KRAS	SEQ ID	—
		NO: 54	NO: 55	NO: 52	NO: 8	NO: 3	NO: 10	modWT1	(SEQ ID	NO: 82
								(SEQ ID	NO: 24,
								NO: 18)	SEQ ID
									NO: 26)
A	NCI-	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	—	—	—	—	—
	H520	NO: 54	NO: 55	NO: 52	NO: 8	NO: 3
B	NCI-	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	modMSLN	—	—	EGFR, ALK,
	H23	NO: 54	NO: 55	NO: 52	NO: 8	NO: 3	NO: 10	(SEQ ID			modALK-IC
								NO: 22)			(SEQ ID
											NO: 94)
B	LK-2	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	—	—	—	—	—
		NO: 54	NO: 55	NO: 52	NO: 8	NO: 3
B	DMS 53*	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	—	—	—	—
		NO: 54	NO: 55	NO: 52	NO: 8	NO: 3	NO: 10
— = Not done/not required/will not be completed.
*Cell lines identified as CSC-like cells. mCD40L, membrane bound CD40L.

Example 5: Preparation of Colorectal Cancer (CRC) Vaccines

Example 5 demonstrates reduction of TGFβ1, TGFβ2, and CD276 expression with concurrent introduction of GM-CSF, membrane bound CD40L, and IL-12 expression in a vaccine composition of two cocktails, each cocktail composed of three cell lines for a total of six cell lines, significantly increased the magnitude of cellular immune responses against at least nine CRC-associated antigens in an HLA-diverse population. Example 5 also describes the process for identification, selection, and design of driver mutations expressed by CRC patient tumors. As described here in, expression of peptides encoding these mutations in certain cell lines of the of the CRC vaccine, described above and herein, also generate potent immune responses in an HLA diverse population.

As described herein, the first cocktail, CRC vaccine-A, is composed of cell line HCT-15, cell line HuTu-80 also modified to express modPSMA and peptides encoding one TP53 driver mutation, one PIK3CA driver mutation, one FBXW7 driver mutation, one SMAD4 driver mutation, one GNAS driver mutation and one ATM driver mutation, and cell line LS411N.

The second cocktail, CRC vaccine-B, is composed of cell line HCT-116 also modified to express modTBXT, modWT1 and peptides encoding two KRAS driver mutations, cell line RKO also modified to express peptides encoding three TP53 driver mutations, one KRAS driver mutation, three PIK3CA driver mutations, two FBXW7 driver mutations, one CTNNB1 driver mutation and one ERBB3 driver mutation, and cell line DMS 53.

The six component cell lines collectively express at least twenty full-length antigens and twenty driver mutations that can provide an anti-CRC tumor response. Table 5-23, below, provides a summary of each cell line and the modifications associated with each cell line.

CRC Vaccine Components

Example 30 of WO/2021/113328 first described selection of the cell lines comprising the CRC vaccine described herein. CRC vaccine cell lines were selected to express a wide array of TAAs, including those known to be important specifically for CRC antitumor responses, such as CEA, and TAAs known to be important for targets for CRC and other solid tumors, such as TERT. Expression of TAAs by vaccine cell lines was determined using RNA expression data sourced from the Broad Institute Cancer Cell Line Encyclopedia (CCLE). The HGNC gene symbol was included in the CCLE search and mRNA expression was downloaded for each TAA. Expression of a TAA by a cell line was considered positive if the RNA-seq value was >0.5. The six component cell lines expressed twelve to eighteen TAAs (FIG. 15A).

As shown herein, to further enhance antigenic breadth, HuTu80 was transduced with a gene encoding modPSMA and HCT-116 was transduced with genes encoding modTBXT, modWT1, and two 28 amino acid peptides spanning KRAS mutations G12D and G12V. Identification and design of antigen sequences inserted by lentiviral transduction into the CRC vaccine is described in Example 40 of WO/2021/113328 and herein. Identification, selection, and design of driver mutations was completed as described in Example 1 and herein.

RNA abundance of twenty prioritized CRC TAAs, identified as described in Example 40 of WO/2021/113328, was evaluated in 365 CRC patient samples Fourteen of the prioritized CRC TAAs were expressed by 100% of samples, 15 TAAs were expressed by 94.5% of samples, 16 TAAs were expressed by 65.8% of samples, 17 TAAs were expressed by 42.2% of samples, 18 TAAs were expressed by 25.8% of samples, 19 TAAs were expressed by 11.5% of samples and 20 TAAs were expressed by 1.4% samples (FIG. 15B).

Expression of lentiviral transduced antigens modPSMA (FIG. 16A) (SEQ ID NO: 29; SEQ ID NO: 30) by HuTu80, modTBXT (FIG. 16B) (SEQ ID NO: 17; SEQ ID NO: 18) and modWT1 (FIG. 16C) (SEQ ID NO: 17; SEQ ID NO: 18) by HCT-116 was detected by flow cytometry described herein. Expression of the genes encoding KRAS G12D (FIG. 16D, 16E) (SEQ ID NO: 23; SEQ ID NO: 24) and G12V (FIG. 16D, 16E) (SEQ ID NO: 25; SEQ ID NO: 26) peptides were detected by RT-PCR as described herein. Genes encoding modTBXT, modWT1, KRAS G12D and KRAS G12V were subcloned into the same lentiviral transfer vector separated by furin cleavage sequences (SEQ ID NO: 37). PSMA was endogenously expressed in one of the six component cell lines at >0.5 FPKM as described below. TBXT and WT1 were not expressed endogenously >0.5 FPKM by any of the six component CRC vaccine components (FIG. 15A). Endogenous expression of KRAS driver mutations is described herein.

Provided herein are two compositions comprising, three cancer cell lines, wherein the combination of the cell lines express at least 14 TAAs associated with a subset of CRC cancer subjects intended to receive said composition. To maintain maximal heterogeneity of antigens and clonal subpopulations of each cell line, the modified cell lines utilized in the present vaccine have been established using antibiotic selection and flow cytometry and not through limiting dilution subcloning. The cell lines identified in Table 5-1 comprise the present CRC vaccine.

TABLE 5-1
CRC vaccine cell lines and histology
	Cocktail	Cell Line Name	Histology
	A	HCT-15	Colorectal Adenocarcinoma
	A	HuTu-80	Duodenum Adenocarcinoma
	A	LS411N	Colorectal Adenocarcinoma
	B	HCT-116	Colorectal Carcinoma
	B	RKO	Colorectal Carcinoma
	B	DMS 53	Lung Small Cell Carcinoma

Reduction of CD276 Expression

Unmodified, parental HCT-15, HuTu-80, LS411N, HCT-116, RKO and DMS 53 component cell lines expressed CD276. Expression of CD276 was knocked out by electroporation with a zinc finger nuclease (ZFN) pair specific for CD276 targeting the genomic DNA sequence: GGCAGCCCTGGCATGggtgtgCATGTGGGTGCAGCC (SEQ ID NO: 52). Following ZFN-mediated knockout of CD276, the cell lines were surface stained with PE α-human CD276 antibody (BioLegend, clone DCN.70) and full allelic knockout cells were enriched by cell sorting (BioRad S3e Cell Sorter). Sorted cells were plated in an appropriately sized vessel, based on the number of recovered cells, and expanded in culture. After cell enrichment for full allelic knockouts, cells were passaged 2-5 times and CD276 knockout percentage determined by flow cytometry. Expression of CD276 was determined by extracellular staining of CD276 modified and unmodified parental cell lines with PE α-human CD276 (BioLegend, clone DCN.70). Unstained cells and isotype control PE α-mouse IgG1 (BioLegend, clone MOPC-21) stained parental and CD276 KO cells served as controls. To determine the percent reduction of CD276 expression in the modified cell line, the MFI of the isotype control was subtracted from recorded MFI values of both the parental and modified cell lines. Percent reduction of CD276 expression is expressed as: (1−(MFI of the CD276KO cell line/MFI of the parental))×100). Reduction of CD276 expression by component cell lines is described in Table 5-2. These data demonstrate that gene editing of CD276 with ZFN resulted in greater than 96.9% knockout of CD276 in the six NSCLC vaccine component cell lines.

TABLE 5-2
Reduction of CD276 expression
	Unmodified Cell	Modified Cell	% Reduction
Cell line	Line MFI	Line MFI	CD276
HCT-15	6,737	26	99.6
HuTu-80	10,389	0	100.0
LS411N	34,278	4	100.0
HCT-116	12,782	0	100.0
RKO	3,632	0	100.0
DMS 53	4,479	0	100.0
MFI is reported with isotype controls subtracted

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12

Cell lines were X-ray irradiated at 100 Gy prior to plating in 6-well plates at 2 cell densities (5.0e5 and 7.5e5) in duplicate. The following day, cells were washed with PBS and the media was changed to Secretion Assay Media (Base Media+5% CTS). After 48 hours, media was collected for ELISAs. The number of cells per well was counted using the Luna cell counter (Logos Biosystems). Total cell count and viable cell count were recorded. The secretion of cytokines in the media, as determined by ELISA, was normalized to the average number of cells plated in the assay for all replicates.

TGFβ1 secretion was determined by ELISA according to manufacturers instructions (Human TGFβ1 Quantikine ELISA, R&D Systems #SB100B). Four dilutions were plated in duplicate for each supernatant sample. If the results of the ELISA assay were below the LLD, the percentage decrease relative to parental cell lines was estimated by the number of cells recovered from the assay and the lower limit of detection, 15.4 pg/mL. If TGFβ1 was detected in >2 samples or dilutions the average of the positive values was reported with the n of samples run.

TGFβ2 secretion was determined by ELISA according to manufacturers instructions (Human TGFβ2 Quantikine ELISA, R&D Systems # SB250). Four dilutions were plated in duplicate for each supernatant sample. If the results of the ELISA assay were below the LLD, the percentage decrease relative to parental cell lines was estimated by the number of cells recovered from the assay and the lower limit of detection, 7.0 pg/mL. If TGFβ2 was detected in >2 samples or dilutions the average of the positive values was reported with the n of samples run.

GM-CSF secretion was determined by ELISA according to manufacturers instructions (GM-CSF Quantikine ELISA, R&D Systems #SGM00). Four dilutions were plated in duplicate for each supernatant sample. If the results of the ELISA assay were below the LLD, the percentage increase relative to parental cell lines was estimated by the number of cells recovered from the assay and the lower limit of detection, 3.0 pg/mL. If GM-CSF was detected in >2 samples or dilutions the average of the positive values was reported with the n of samples run.

IL-12 secretion was determined by ELISA according to manufacturer's instructions (LEGEND MAX Human IL-12 (p70) ELISA, Biolegend #431707). Four dilutions were plated in duplicate for each supernatant sample. If the results of the ELISA assay were below the LLD, the percentage increase was estimated by the number of cells recovered from the assay and the lower limit of detection, 1.2 pg/mL. If IL-12 was detected in >2 samples or dilutions the average of the positive values was reported with the n of samples run.

shRNA Downregulates TGF-β Secretion

Following knockout of CD276, TGFβ1 and/or TGFβ2 secretion levels were reduced using shRNA and resulting secretion levels determined as described above. All unmodified CRC vaccine-A components secreted measurable levels of TGFβ1. HuTu80 also secreted detectable levels of TGFβ2. CRC vaccine-B cell lines HCT-116 and RKO secreted measurable levels of TGFβ1 but not TGFβ2 and DMS 53 secreted measurable levels of TGFβ1 and TGFβ2.

The five CRC-derived vaccine cell lines were transduced with the lentiviral particles encoding both TGFβ1 shRNA (shTGFβ1, mature antisense sequence: TTTCCACCATTAGCACGCGGG (SEQ ID NO: 54)) and the gene for expression of membrane bound CD40L (SEQ ID NO: 3) under the control of a different promoter. This allowed for simultaneous reduction of TGFβ1 and introduction of expression of membrane bound CD40L. Cell lines genetically modified to reduce TGFβ1, and not TGFβ2, are described by the clonal designation DK2.

HuTu80 was subsequently transduced with lentiviral particles encoding both TGFβ2 shRNA (mature antisense sequence: AATCTGATATAGCTCAATCCG (SEQ ID NO: 55) and GM-CSF (SEQ ID NO: 8) under the control of a different promoter. This allowed for simultaneous reduction of TGFβ2 and introduction of expression of GM-CSF. DMS 53 was concurrently transduced with both lentiviral particles encoding TGFβ1 shRNA and membrane bound CD40L with lentiviral particles encoding TGFβ2 shRNA and GM-CSF. This allowed for simultaneous reduction of TGFβ1 and TGFβ2 secretion and expression of GM-CSF. Cell lines genetically modified to decrease secretion of TGFβ1 and TGFβ2 are described by the clonal designation DK6.

Table 5-3 shows the percent reduction of TGFβ1 and/or TGFβ2 secretion by gene modified component cell lines compared to matched, unmodified cell lines. Gene modification resulted in at least 49% reduction of TGFβ1 secretion. Gene modification of TGFβ2 resulted in at least 97% reduction in secretion of TGFβ2.

TABLE 5-3
TGF-β Secretion (pg/106 cells/24 hr) in Component Cell Lines
Cell Line	Cocktail	Clone	TGFβ1	TGFβ2
HCT-15	A	Wild type	369	21
HCT-15	A	DK2	189	NA
HCT-15	A	Percent reduction	49%	NA
HuTu-80	A	Wild type	2,529	4,299
HuTu-80	A	DK6	327	115
HuTu-80	A	Percent reduction	87%	97%
LS411N	A	Wild type	413	*≤9
LS411N	A	DK2	89	NA
LS411N	A	Percent reduction	78%	NA
HCT-116	B	Wild type	2,400	*≤8
HCT-116	B	DK2	990	NA
HCT-116	B	Percent reduction	59%	NA
RKO	B	Wild type	971	*≤6
RKO	B	DK2	206	NA
RKO	B	Percent reduction	79%	NA
DMS 53	B	Wild type	205	806
DMS 53	B	DK6	*≤14	*≤16
DMS 53	B	Percent reduction	93%	99%
DK6: TGFβ1/TGFβ2 double knockdown;
DK2: TGFβ1 single knockdown;
*estimated using LLD, not detected;
NA = not applicable

Based on a dose of 5×10⁵ of each component cell line, the total TGFβ1 and TGFβ2 secretion by CRC vaccine-A and CRC vaccine-B and respective unmodified parental controls are shown in Table 5-4. Secretion of TGFβ1 by CRC vaccine-A was reduced by 82% and TGFβ2 by 97% pg/dose/24 hr. Secretion of TGFβ1 by CRC vaccine-B was reduced by 69% and TGFβ2 by 98% pg/dose/24 hr.

TABLE 5-4
TGF-β Secretion (pg/dose/24
hr) by CRC vaccine-A and CRC vaccine-B
	Cocktail	Clones	TGFβ1	TGFβ2
	A	Unmodified	1,656	2,165
		DK2/DK6	303	58
		Percent Reduction	82%	97%
	B	Unmodified	1,788	410
		DK2/DK6	605	8
		Percent Reduction	66%	98%

Membrane Bound CD40L (CD154) Expression

All CRC vaccine cell lines were transduced with lentiviral particles to reduced TGFβ1 secretion and to express membrane bound CD40L as described above and herein. Cells were analyzed for cell surface expression CD40L expression by flow cytometry. Unmodified and modified cells were stained with PE-conjugated human α-CD40L (BD Biosciences, clone TRAP1) or Isotype Control PE α-mouse IgG1 (BioLegend, clone MOPC-21). The MFI of the isotype control was subtracted from the CD40L MFI of both the unmodified and modified cell lines. If subtraction of the MFI of the isotype control resulted in a negative value, an MFI of 1.0 was used to calculate the fold increase in expression of CD40L by the modified component cell line relative to the unmodified cell line. Expression of membrane bound CD40L by all six vaccine component cell lines is described in Table 5-5. The results described below demonstrate CD40L membrane expression was substantially increased by all six cell CRC vaccine cell lines.

TABLE 5-5
Increase in membrane-bound CD40L (mCD40L) expression
	Unmodified Cell	Modified Cell	Fold Increase
Cell line	Line MFI	Line MFI	mCD40L
HCT-15	0	669	669
HuTu80	5	5,890	1,178
LS411N	0	4,703	4,703
HCT-116	0	21,549	21,549
RKO	0	7,107	7,107
DMS 53	0	4,317	4,317
MFI is reported with isotype controls subtracted

GM-CSF Expression

HuTu80 and DMS 53 were transduced with lentiviral particles encoding both TGFβ2 shRNA and the gene to express GM-CSF (SEQ ID NO: 8) under the control of a different promoter. The HCT-15, LS411N, HCT-116 and RKO cell lines were transduced with lentiviral particles to only express GM-CSF (SEQ ID NO: 8). GM-CSF expression level by the CRC vaccine cell lines are described in Error! Reference source not found. 5-6 and herein.

TABLE 5-6
GM-CSF Secretion in Component Cell Lines
		GM-CSF	GM-CSF
	Cell Line	(ng/106 cells/24 hr)	(ng/dose/24 hr)
	HCT-15	59	30
	HuTu80	101	51
	LS411N	145	73
	Cocktail A Total	305	154
	HCT-116	342	171
	RKO	131	66
	DMS 53	30	15
	Cocktail B Total	503	252

Based on a dose of 5×10⁵ of each component cell line, the total GM-CSF secretion for CRC vaccine-A was 154 ng per dose per 24 hours. The total GM-CSF secretion for CRC vaccine-B was 252 ng per dose per 24 hours. The total GM-CSF secretion per dose was therefore 406 ng per 24 hours.

IL-12 Expression

All vaccine cell lines were transduced with the lentivirus particles resulting in stable expression of IL-12 p70. Expression of IL-12 by components cell lines was determined as described above and the results are shown in Table 5-7.

TABLE 5-7
IL-12 expression by CRC vaccine-A and CRC vaccine-B
		IL-12	IL-12
	Cell Line	(ng/106 cells/24 hr)	(ng/dose/24 hr)
	HCT-15	27	14
	HuTu80	51	26
	LS411N	26	13
	Cocktail A Total	104	52
	HCT-116	186	93
	RKO	43	22
	DMS 53	28	14
	Cocktail B Total	257	129

Based on a dose of 5×10⁵ of each component cell line per cocktail IL-12 secretion by CRC vaccine-A was 52 ng per dose per 24 hours and 129 ng per dose per 24 hours by CRC vaccine-B. Total IL-12 secretion per unit dose 181 ng per 24 hours.

Stable Expression of modPSMA (SEQ ID NO: 30) by the HuTu80 Cell Line

CRC vaccine-A cell line HuTu80 modified to reduce expression of CD276, secretion of TGFβ1 and TGFβ2, and express GM-CSF, membrane bound CD40L, and IL-12 was transduced with lentiviral particles expressing the gene encoding modPSMA. Expression of PSMA was characterized by flow cytometry. Unmodified and antigen modified cells were stained intracellularly with 0.06 μg/test anti-mouse IgG1 anti-PSMA antibody (AbCam ab268061, Clone FOLH1/3734) followed by 0.125 ug/test AF647-conjugated goat anti-mouse IgG1 antibody (Biolegend #405322). The MFI of isotype control stained modPSMA transduced and antigen unmodified cells was subtracted from the MFI of cells stained for PSMA. Fold increase in antigen expression was calculated as: (background subtracted modified MFI/background subtracted parental MFI). Expression of PSMA increased by the antigen modified cell line (756,908 MFI) 9.1-fold over that of the cell line not modified to express modPSMA (82,993 MFI) (FIG. 16A).

Stable Expression of modTBXT, modWT1, KRAS G12D and KRAS G12V (SEQ ID NO: 18) by the HCT-116 Cell Line

CRC vaccine-B cell line HCT-116 modified to reduce the expression of CD276, reduce secretion of TGFβ1, and express GM-CSF, membrane bound CD40L, and IL-12 was transduced with lentiviral particles to express the genes encoding modTBXT, modWT1, and peptides encoding KRAS driver mutations G12D and G12V. Expression of TBXT and WT1 were confirmed by flow cytometry. Unmodified and antigen modified cells were stained intracellularly to detect the expression of each antigen as follows. For detection of modTBXT, cells were stained with rabbit anti-human TBXT antibody (Abcam ab209665, Clone EPR18113) (0.06 μg/test) or Rabbit Polyclonal Isotype Control (Biolegend 910801) followed by AF647-conjugated donkey anti-rabbit IgG antibody (Biolegend 406414) (0.125 μg/test). For detection of modWT1, cells were stained with rabbit anti-human WT1 antibody (AbCam ab89901, Clone CAN-R9) (0.06 μg/test) or Rabbit Polyclonal Isotype Control (Biolegend 910801) followed by AF647-conjugated donkey anti-rabbit IgG antibody (Biolegend 406414) (0.125 μg/test). The MFI of cells stained with the isotype control was subtracted from the MFI of the cells stained for TBXT or WT1. Fold increase in antigen expression was calculated as: (background subtracted modified MFI/background subtracted parental MFI). Expression of modTBXT increased by the antigen modified cell line (356,691 MFI) 356,691-fold over that of the antigen unmodified cell line (0 MFI) (FIG. 16B). Subtraction of the MFI of the isotype control from the MFI of the TBXT stained unmodified cell line resulted in negative value and fold increase of modTBXT expression by the antigen modified HCT-116 cell line was calculated using 1 MFI. Expression of modTBXT increased by TBXT expression (356,691 MFI) 356,691-fold over that of the antigen unmodified cell line (0 MFI) (FIG. 16B). Expression of modWT1 by increased WT-1 expression (362,698 MFI) 69.3-fold over the that of the antigen unmodified cell line (5,235 MFI) (FIG. 16C).

Expression of peptides encoding KRAS driver mutations G12D and G12V by HCT-116 was confirmed by RT-PCR. For KRAS G12D, the forward primer designed to anneal at the 2786-2807 base pair (bp) position of the transgene (GAAGCCCTTCAGCTGTAGATGG (SEQ ID NO: 97) and reverse primer designed to anneal at 2966-2984 bp position in the transgene (CTGAATTGTCAGGGCGCTC (SEQ ID NO: 98) and yield 199 bp product. For KRAS G12V, the forward primer was designed to anneal at the 2861-2882 bp location in the transgene (CATGCACCAGAGGAACATGACC (SEQ ID NO: 99) and reverse primer designed to anneal at the 3071-3094 bp location in the transgene (GAGTTGGATGGTCAGGGCAGAT (SEQ ID NO: 100) and yield 238 bp product. β-tubulin primers that anneal to variant 1, exon 1 (TGTCTAGGGGAAGGGTGTGG (SEQ ID NO: 101)) and exon 4 (TGCCCCAGACTGACCAAATAC (SEQ ID NO: 102)) were used as a control. PCR products were imaged using ChemiDoc Imaging System (BioRAD, #17001401) and relative quantification to the β-tubulin gene calculated using Image Lab Software v6.0 (BioRAD). Gene products for both KRAS G12D and KRAS G12V were detected at the expected size, 199 bp and 238 bp, respectively (FIG. 16D). KRAS G12D mRNA increased 3,127-fold and KRAS G12V mRNA increased 4,095-fold relative to parental controls (FIG. 16E).

Immune Responses to PSMA (SEQ ID NO: 30) by CRC-Vaccine A

IFNγ responses to PSMA were evaluated in the context of the CRC-vaccine A for six HLA diverse donors (Table 5-8). Specifically, 5×10⁵ of unmodified or CRC vaccine-A HCT-15, HuTu80 and LS411N cell lines, a total of 1.5×10⁶ total modified cells, were co-cultured with 1.5×10⁶ iDCs from six HLA diverse donors (n=4/donor). CD14-PBMCs were isolated from co-culture with DCs on day 6 and stimulated with peptide pools, 15-mers overlapping by 9 amino acids, spanning the native PSMA protein (Thermo Scientific Custom Peptide Service) the IFNγ ELISpot assay for 24 hours prior to detection of IFNγ producing cells. CRC vaccine-A (6,204±1,744 SFU) induced significantly stronger PSMA specific IFNγ responses compared to unmodified CRC vaccine-A (69±36 SFU) (p=0.006, Mann-Whitney U test) (FIG. 16F).

TABLE 5-8
Healthy Donor MHC-I characteristics
	Donor #	HLA-A	HLA-B	HLA-C
	1	*02:01 *11:01	*07:02 *37:02	*06:02 *07:02
	2	*03:01 *25:01	*15:01 *44:02	*03:03 *05:01
	3	*02:01 *24:01	*08:01 *44:02	*05:01 *07:01
	4	*29:01 *29:02	*44:03 *50:01	*06:02 *16:01
	5	*11:01 *29:02	*18:01 *44:03	*07:01 *11:01
	6	*02:01 *03:01	*07:02 *41:02	*07:02 *17:01

Immune Responses to TBXT, WT1, and KRAS Mutations (SEQ ID NO: 18) by CRC-Vaccine B

IFNγ responses to TBXT, WT1, KRAS G12D and KRAS G12V antigens were evaluated in the context of the CRC-vaccine B for six HLA diverse donors (n=4/donor) (Table 5-8). Specifically, 5×10⁵ of unmodified or CRC vaccine-B HCT-116, RKO and DMS 53 cell lines, a total of 1.5×10⁶ total modified cells, were co-cultured with 1.5×10⁶ iDCs from six HLA diverse donors. CD14-PBMCs were isolated from co-culture with DCs on day 6 and stimulated with peptide pools of 15-mer peptides, overlapping by 11 amino acids covering for the full-length protein sequences of TBXT (JPT, PM-BRAC) or WT1 (JPT, PM-WT1). KRAS G12D and G12V 15-mers overlapping by 9 amino acids, were purchased from Thermo Scientific Custom Peptide Service. IFNγ responses to TBXT increased by modified CRC vaccine-B (2,257±538 SFU) compared to unmodified CRC vaccine-B (121±35 SFU) (p=0.003) (FIG. 16G). WT1 specific IFNγ responses were significantly increased by modified CRC vaccine-B (2,910±794 SFU) compared unmodified CRC vaccine-B (277±78 SFU) (p=0.007) (FIG. 16I). KRAS G12D specific IFNγ responses significantly increased with modified CRC vaccine-B (2,302±771 SFU) compared unmodified CRC vaccine-B (123±30 SFU) (p=0.017) (FIG. 16I). KRAS G12V specific IFNγ responses significantly increased with modified CRC vaccine-B (2,246±612 SFU) compared unmodified CRC vaccine-B (273±37 SFU) (p=0.008) (FIG. 16J). Statistical significance was determined by Student's T test.

Cocktails Induce Immune Responses Against Prioritized TAAs

IFNγ responses generated by CRC vaccine-A and CRC vaccine-B against nine prioritized CRC antigens was measured by ELISpot as described above and herein. CD14-PBMCs from six HLA-diverse healthy donors (Table 5-8) were co-cultured with autologous DCs loaded with unmodified control cocktails, CRC vaccine-A or CRC vaccine-B for 6 days prior to stimulation with TAA-specific specific peptide pools designed to cover the full-length native antigen protein. Antigen specific IFNγ responses against PSMA, WT1, TBXT, KRAS G12D and KRAS G12V were evaluated in ELISpot by stimulating primed CD14-PBMCs with peptide pools described above. Additional peptide pools were sourced as follows: Survivin (thinkpeptides, 7769_001-011), PRAME (Miltenyi Biotech, 130-097-286), STEAP (PM-STEAP1), TERT (JPT, PM-TERT), MUC1 (JPT, PM-MUC1), and CEACAM (CEA) (JPT, PM-CEA).

FIG. 17 demonstrates the CRC vaccine can induce antigen specific IFNγ responses in six HLA-diverse donors significantly more robust (59,976±13,542 SFU) compared to unmodified parental controls (6,247±2,891 SFU) (p=0.004) (FIG. 17A). CRC vaccine-A and CRC vaccine-B independently demonstrated antigen specific responses significantly greater compared to parental controls. Specifically, CRC vaccine-A elicited 31,489±7,103 SFU compared to the unmodified controls (1,931±1,333 SFU) (p=0.004) (FIG. 17B). CRC vaccine-B significantly increased antigen specific IFNγ ELISpot (28,487±7,156 SFU) compared to parental controls (4,316±1,645 SFU) (p=0.004) (FIG. 17C). Immune responses by individual donors is described in FIG. 4 and Table 5-9). Statistical significance was determined by the Mann-Whitney U test.

TABLE 5-9
IFNγ Responses to TAAs induced by the unmodified and modified CRC vaccine
	Unmodified (SFU ± SEM)	Modified (SFU ± SEM)
Donor	CRC	CRC	CRC	CRC	CRC	CRC
(n = 4)	vaccine-A	vaccine-B	vaccine	vaccine-A	vaccine-B	vaccine
1	135 ± 8	370 ± 18	505 ± 23	6,753 ± 129	6,993 ± 134	13,745 ± 242
2	1,150 ± 44	3,258 ± 78	4,408 ± 114	32,930 ± 333	37,335 ± 460	70,265 ± 734
3	630 ± 22	1,050 ± 25	1,680 ± 36	14,193 ± 244	14,715 ± 253	28,908 ± 469
4	1,150 ± 27	4,328 ± 92	5,478 ± 107	42,350 ± 646	47,860 ± 755	90,210 ± 1,376
5	0 ± 0	5,308 ± 221	5,308 ± 221	50,855 ± 677	46,260 ± 830	97,115 ± 1,461
6	8,520 ± 396	11,583 ± 581	20,103 ± 963	41,855 ± 982	17,758 ± 617	59,613 ± 1,562
Ave.	1,931 ± 1,333	4,316 ± 1,645	6,247 ± 2,891	31,489 ± 7,103	28,487 ± 7,156	59,976 ± 13,542

Identification of Frequently Mutated Oncogenes in Colorectal Cancer (CRC)

Driver mutations for CRC were identified, selected and constructs designed as described as described in Example 1 and herein. As described herein, expression of selected driver mutations by CRC vaccine-A cell line Hutu80 and CRC vaccine-B cell lines HCT-116 and RKO can generate a CRC anti-tumor response in an HLA diverse population. Table 5-10 describes oncogenes that exhibit greater than 5% mutation frequency (percentage of samples with one or more mutations) in 1363 profiled CRC patient samples.

TABLE 5-10
Oncogenes in CRC with greater than 5% mutation frequency
		Number of samples		Percentage of samples
	Total number	with one or more	Profiled	with one or more	Is Cancer Gene
Gene	of mutations	mutations	Samples	mutations	(source: OncoKB)
APC	1385	902	1363	66.20%	Yes
TP53	835	785	1363	57.60%	Yes
KRAS	514	504	1363	37.00%	Yes
PIK3CA	382	328	1363	24.10%	Yes
FAT4	409	250	1363	18.30%	Yes
LRP1B	357	207	1363	15.20%	Yes
FBXW7	242	203	1363	14.90%	Yes
BRAF	214	201	1363	14.70%	Yes
SMAD4	198	176	1363	12.90%	Yes
PCLO	261	171	1363	12.50%	Yes
KMT2C	209	159	1363	11.70%	Yes
KMT2D	203	155	1363	11.40%	Yes
ATM	212	150	1363	11.00%	Yes
RNF213	174	143	1363	10.50%	Yes
ZFHX3	164	138	1363	10.10%	Yes
AMER1	143	135	1363	9.90%	Yes
TRRAP	173	132	1363	9.70%	Yes
ARID1A	150	130	1363	9.50%	Yes
FAT1	191	129	1363	9.50%	Yes
EP400	157	129	1363	9.50%	Yes
SOX9	145	128	1363	9.40%	Yes
RNF43	162	126	1363	9.20%	Yes
MKI67	146	119	1363	8.70%	Yes
RELN	172	119	1363	8.70%	Yes
PTPRS	133	116	1363	8.50%	Yes
PDE4DIP	157	114	1363	8.40%	Yes
CHD4	138	111	1363	8.10%	Yes
PTPRT	126	109	1363	8.00%	Yes
ANKRD11	131	108	1363	7.90%	Yes
ROBO1	128	107	1363	7.90%	Yes
MTOR	118	103	1363	7.60%	Yes
CREBBP	122	102	1363	7.50%	Yes
LRRK2	144	102	1363	7.50%	Yes
TCF7L2	105	100	1363	7.30%	Yes
KMT2B	126	100	1363	7.30%	Yes
PRKDC	146	99	1363	7.30%	Yes
UBR5	121	99	1363	7.30%	Yes
ACVR2A	110	98	1363	7.20%	Yes
ERBB4	114	98	1363	7.20%	Yes
PREX2	127	98	1363	7.20%	Yes
CARD11	107	97	1363	7.10%	Yes
NOTCH1	106	94	1363	6.90%	Yes
PTEN	119	92	1363	6.70%	Yes
NCOR2	108	92	1363	6.70%	Yes
GRIN2A	110	91	1363	6.70%	Yes
KMT2A	124	91	1363	6.70%	Yes
ATRX	126	90	1363	6.60%	Yes
CACNA1D	121	90	1363	6.60%	Yes
ALK	101	89	1363	6.50%	Yes
MYH9	112	89	1363	6.50%	Yes
NOTCH3	105	89	1363	6.50%	Yes
POLE	113	89	1363	6.50%	Yes
BCORL1	105	89	1363	6.50%	Yes
SPEN	119	88	1363	6.50%	Yes
BCL9L	101	88	1363	6.50%	Yes
BRCA2	137	86	1363	6.30%	Yes
CUX1	97	86	1363	6.30%	Yes
ARID1B	100	85	1363	6.20%	Yes
CTNNB1	101	84	1363	6.20%	Yes
MYH11	107	84	1363	6.20%	Yes
SMARCA4	94	84	1363	6.20%	Yes
NF1	100	82	1363	6.00%	Yes
PIK3CG	95	82	1363	6.00%	Yes
PLCG2	92	82	1363	6.00%	Yes
AXIN2	96	82	1363	6.00%	Yes
MGA	104	81	1363	5.90%	Yes
SLX4	92	81	1363	5.90%	Yes
FLT4	88	80	1363	5.90%	Yes
ERBB3	85	79	1363	5.80%	Yes
POLQ	107	79	1363	5.80%	Yes
ASXL1	83	79	1363	5.80%	Yes
CAD	87	78	1363	5.70%	Yes
PTPRK	92	78	1363	5.70%	Yes
ARID2	106	78	1363	5.70%	Yes
CIC	84	77	1363	5.60%	Yes
EP300	89	76	1363	5.60%	Yes
EPHA5	86	76	1363	5.60%	Yes
NUMA1	87	76	1363	5.60%	Yes
CAMTA1	84	76	1363	5.60%	Yes
GNAS	79	75	1363	5.50%	Yes
LRP5	84	75	1363	5.50%	Yes
BCL9	87	74	1363	5.40%	Yes
PTPRD	94	74	1363	5.40%	Yes
RANBP2	96	74	1363	5.40%	Yes
IRS1	83	73	1363	5.40%	Yes
MYO5A	84	73	1363	5.40%	Yes
ROS1	113	73	1363	5.40%	Yes
IRS4	86	73	1363	5.40%	Yes
SETD1A	87	73	1363	5.40%	Yes
PIK3R1	87	72	1363	5.30%	Yes
PTPRC	90	72	1363	5.30%	Yes
COL1A1	75	71	1363	5.20%	Yes
TP53BP1	96	71	1363	5.20%	Yes
DICER1	88	71	1363	5.20%	Yes
SETBP1	90	71	1363	5.20%	Yes
ZBTB20	77	71	1363	5.20%	Yes
KDM2B	78	71	1363	5.20%	Yes
B2M	104	70	1363	5.10%	Yes
AFDN	88	70	1363	5.10%	Yes
ZNF521	85	69	1363	5.10%	Yes
LARP4B	77	68	1363	5.00%	Yes

The CRC driver mutations in TP53, KRAS, PIK3CA, FBXW7, BRAF, SMAD4, ATM, CTNNB, ERBB3 and GNAS occurring in ≥0.5% of profiled patient samples are shown in Table 5-11. There were no missense mutations occurring in ≥0.5% of profiled patient samples for the rest of CRC oncogenes listed in Table 5-10.

TABLE 5-11
Identification of driver mutations in selected CRC oncogenes
	Driver	Number of samples	Total number of	Fre-
Gene	mutation	with mutation	samples	quency
TP53	G245S	15	1363	1.1%
	R273H	31	1363	2.3%
	R248W	34	1363	2.5%
	R273C	37	1363	2.7%
	R248Q	41	1363	3.0%
	R282W	41	1363	3.0%
	R175H	93	1363	6.8%
KRAS	G12S	16	1363	1.2%
	G12A	21	1363	1.5%
	A146T	27	1363	2.0%
	G12C	44	1363	3.2%
	G12V	97	1363	7.1%
	G13D	99	1363	7.3%
	G12D	142	1363	10.4%
PIK3CA	M1043I	7	1363	0.5%
	H1047Y	7	1363	0.5%
	C420R	9	1363	0.7%
	E546K	11	1363	0.8%
	R88Q	26	1363	1.9%
	E542K	37	1363	2.7%
	H1047R	43	1363	3.2%
	E545K	64	1363	4.7%
FBXW7	S582L	8	1363	0.6%
	R505C	11	1363	0.8%
	R465H	23	1363	1.7%
	R465C	31	1363	2.3%
BRAF	V600E	165	1363	12.1%
SMAD4	R361C	11	1363	0.8%
	R361H	20	1363	1.5%
ATM	R337C	7	1363	0.5%
CTNNB1	S45F	8	1363	0.6%
ERBB3	V104M	8	1363	0.6%
GNAS	R201H	14	1363	1.0%

Prioritization and Selection of Identified CRC Driver Mutations

HLA-A and HLA-B supertype-restricted 9-mer CD8 epitopes analysis was performed as described in Example 1. Based on the CD8 epitope analysis result and the frequency (%) of each mutation, a list of mutations was selected to be either included in the final constructs or obtain further CD4 epitope analysis. The results are shown in Table 5-12.

TABLE 5-12
Prioritization and selection of identified CRC driver mutations
based on CD8 epitope analysis and frequency of each mutation
		Number of
		total CD8
	Driver	epitopes	Frequency	Included
Gene	mutation	(SB + WB)	(%)	Yes (Y) or No (N)
TP53	R175H	2	6.8	Y
	G245S	3	1.1	N
	R248W	3	2.5	N
	R248Q	0	3	N
	G245S	3	3.6	Y
	R248W
	R273C	1	2.7	Y
	R273H	1	2.3	N
	R282W	0	3	N
KRAS	G12S	1	1.2	N
	G12A	2	1.5	CD4 analysis
	G12C	1	3.2	CD4 analysis
	G12V	3	7.1	Y
	G12D	1	10.4	Y
	G13D	0	7.3	N
	A146T	0	2	N
PIK3CA	R88Q	6	1.9	Y
	C420R	0	0.7	N
	E542K	1	2.7	Y
	E545K	0	4.7	N
	Q546K	0	0.9	N
	M1043I	1	0.5	N
	H1047Y	4	0.5	CD4 analysis
	M1043I	4	1	CD4 analysis
	H1047Y
	H1047R	2	3.2	RKO and HCT116
FBXW7	R465H	3	1.7	CD4 analysis
	R465C	2	2.3	CD4 analysis
	R505C	3	0.8	Y
	S582L	5	0.6	Y
BRAF	V600E	0	12.1	N
SMAD4	R361C	0	0.8	N
	R361H	1	1.5	Y
ATM	R337C	2	0.5	Y
CTNNB1	S45F	3	0.6	Y
ERBB3	V104M	7	0.6	Y
GNAS	R201H	2	1	Y

CD4 epitopes analysis was performed as described in Example 1 to complete the final selection of CRC driver mutations described in Table 5-13.

Among the identified mutations, PIK3CA H1047R was endogenously expressed by CRC vaccine component cell lines RKO and HCT-116, and therefore was excluded from the final driver mutation insert design. KRAS G12D and KRAS G12V, the two most frequently occurring KRAS mutations, were excluded from the final driver mutation insert design because these driver mutations were previously inserted into the CRC vaccine component cell line HCT-116 as described above and herein. If KRAS G12D and KRAS G12V were not inserted into HCT-116 they would be included in the current insert.

Taken together, as shown in Table 5-13, 17 CRC driver mutations encoded by 15 peptide sequences were selected and included as driver mutation vaccine targets.

TABLE 5-13
Final selection of identified CRC driver mutations based
on CD4 epitope analysis and frequency of each mutation
		Number of
		total CD4
	Driver	epitopes	Frequency	Included
Gene	mutation	(SB + WB)	(%)	Yes (Y) or No (N)
TP53	R175H	0	6.8	Y
	G245S	28	3.6	Y
	R248W
	R273C	0	2.7	Y
KRAS	G12A	0	1.5	N
	G12C	0	3.2	Y
	G12V	7	7.1	Y
	G12D	11	10.4	Y
PIK3CA	R88Q	21	1.9	Y
	E542K	0	2.7	Y
	H1047Y	47	0.5	N
	M1043I	80	1	Y
	H1047Y
	H1047R	8	3.2	RKO and HCT116
FBXW7	R465H	0	1.7	Y
	R465C	0	2.3	N
	R505C	0	0.8	Y
	S582L	6	0.6	Y
SMAD4	R361H	0	1.5	Y
ATM	R337C	0	0.5	Y
CTNNB1	S45F	45	0.6	Y
ERBB3	V104M	2	0.6	Y
GNAS	R201H	0	1	Y

The total number of CD8 epitopes for each HLA-A and HLA-B supertype introduced by 17 selected CRC driver mutations encoded by 15 peptide sequences was determined as described in Example 1. Results of the epitope prediction analysis are shown in Table 5-14.

TABLE 5-14
CD8 epitopes introduced by 17 selected CRC driver
mutations encoded by 15 peptide sequences
		HLA-A	HLA-B	Total number
	Driver	Supertypes	Supertypes	of CD8
Gene	mutation	(n = 5)	(n = 7)	epitopes
TP53	R175H	1	1	2
	G245S	1	2	3
	R248W
	R273C	0	1	1
KRAS	G12C	1	0	1
PIK3CA	R88Q	1	5	6
	E542K	1	0	1
	M1043I	2	2	4
	H1047Y
FBXW7	R465H	2	1	3
	R505C	1	2	3
	S582L	2	3	5
SMAD4	R361H	1	0	1
ATM	R337C	2	0	2
CTNNB1	S45F	2	1	3
ERBB3	V104M	1	6	7
GNAS	R201H	0	2	2

The total number of CD4 epitopes for Class II locus DRB1, DRB 3/4/5, DQA1/DQB1 and DPB1 introduced by 17 selected CRC driver mutations encoded by 15 peptide sequences was determined as described in Example 1 and the results are shown in Table 5-15.

TABLE 5-15
CD4 epitopes introduced by 17 selected CRC driver
mutations encoded by 15 peptide sequences
						Total number
	Driver	DRB1	DRB3/4/5	DQA1/DQB1	DPB1	of CD4
Gene	mutation	(n = 26)	(n = 6)	(n = 8)	(n = 6)	epitopes
TP53	R175H	0	0	0	0	0
	G245S R248W	10	8	1	9	28
	R273C	0	0	0	0	0
KRAS	G12C	0	0	0	0	0
PIK3CA	R88Q	16	1	0	4	21
	E542K	0	0	0	0	0
	M1043I H1047Y	34	12	1	33	80
FBXW7	R465H	0	0	0	0	0
	R505C	0	0	0	0	0
	S582L	0	0	0	6	6
SMAD4	R361H	0	0	0	0	0
ATM	R337C	0	0	0	0	0
CTNNB1	S45F	10	8	0	27	45
ERBB3	V104M	0	0	0	2	2
GNAS	R201H	0	0	0	0	0

CRC Patient Sample Coverage by Selected Driver Mutations

As shown in Table 5-16, the 17 selected CRC driver mutations were assembled into two construct inserts. Once two construct inserts were assembled, the analysis of CRC patient sample coverage by each insert was performed. The results indicated that the CRC patient sample coverage by construct encoded driver mutations was 36.2% (Table 5-17). When the driver mutations endogenously expressed by the CRC vaccine component cell lines were also included, the total CRC patient sample coverage was 37.5% (Table 5-18).

TABLE 5-16
Generation of two constructs encoding 17 selected CRC driver mutations
				Total	Total	Total CD4
		Driver	Frequency	CD8	CD4	and CD8
	Gene	mutation	(%)	epitopes	epitopes	epitopes
CRC	TP53	R175H	6.8	2	0	2
Construct 1	TP53	G245S R248W	3.6	3	28	31
Insert	KRAS	G12C	3.2	1	0	1
	PIK3CA	R88Q	1.9	6	21	27
	FBXW7	R465H	1.7	3	0	3
	PIK3CA	M1043I H1047Y	1	4	80	84
	FBXW7	S582L	0.6	5	6	11
	CTNNB1	S45F	0.6	3	45	48
	ERBB3	V104M	0.6	7	2	9
CRC	TP53	R273C	2.7	1	0	1
Construct 2	PIK3CA	E542K	2.7	1	0	1
Insert	SMAD4	R361H	1.5	1	0	1
	GNAS	R201H	1	2	0	2
	FBXW7	R505C	0.8	3	0	3
	ATM	R337C	0.5	2	0	2

TABLE 5-17
CRC patient sample coverage by the construct encoded driver mutations
Coverage
(Construct
Insert
Only)			% of
Sample	Driver Mutation Target Gene		patients
Description	TP53	KRAS	PIK3CA	FBXW7	SMAD4	ATM	CTNNB1	ERBB3	GNAS	Total	(n = 3056)
Samples with one	271	470	60	47	20	8	4	15	11	906	29.6
driver mutation
Samples with ≥2 DMs	2	2	1	0	0	0	0	0	0	5	0.2
from same antigen
Samples with ≥2 DMs		194	6.3
from different antigens
Total	1105	36.2

TABLE 5-18
CRC patient sample coverage by construct and cell encoded driver mutations
Coverage
(Construct
Insert, RKO,
HCT-116)			% of
Sample	Driver Mutation Target Gene		patients
Description	TP53	KRAS	PIK3CA	FBXW7	SMAD4	ATM	CTNNB1	ERBB3	GNAS	Total	(n = 3056)
Samples with one	267	450	100	47	20	8	4	13	11	906	30.1
driver mutation
Samples with ≥2 DMs	2	2	3	0	0	0	0	0	0	6	0.2
from same antigen
Samples with ≥2 DMs		220	7.2
from different antigens
Total	1105	37.5

Oncogene Sequences and Insert Sequences of the CRC Driver Mutation Constructs

Native DNA and protein sequences of FBXW7, CTNNB1, ERBB3, SMAD4, GNAS and ATM oncogenes and inserts encoding driver mutations are included in Table 5-19. Native DNA and protein sequences TP53 and PIK3CA (Table 2-10) and for KRAS (SEQ ID NO: 77) are describe above and herein.

The CRC driver mutation Construct 1 (SEQ ID NO: 115 and SEQ ID NO: 116; encoding driver mutation sequences from oncogenes TP53, KRAS, PIK3CA, FBXW7, CTNNB1 and ERBB3) insert gene encodes 333 amino acids containing the gene encoding driver mutation peptides separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37). The CRC driver mutation Construct 2 (SEQ ID NO: 117 and SEQ ID NO: 118; encoding driver mutation sequences from oncogenes TP53, PIK3CA, SMAD4, GNAS, FBXW7 and ATM) insert gene encodes 222 amino acids containing the gene encoding driver mutation peptides separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37).

TABLE 5-19
Oncogene sequences and insert sequences for CRC driver
mutation Construct s 1 and Construct 2
FBXW7       DNA Sequence
(SEQ ID     1 ATGAATCAGG AACTGCTCTC TGTGGGCAGC AAAAGACGAC GAACTGGAGG CTCTCTGAGA
NO: 103)    61 GGTAACCCTT CCTCAAGCCA GGTAGATGAA GAACAGATGA ATCGTGTGGT AGAGGAGGAA
            121 CAGCAACAGC AACTCAGACA ACAAGAGGAG GAGCACACTG CAAGGAATGG TGAAGTTGTT
            181 GGAGTAGAAC CTAGACCTGG AGGCCAAAAT GATTCCCAGC AAGGACAGTT GGAAGAAAAC
            241 AATAATAGAT TTATTTCGGT AGATGAGGAC TCCTCAGGAA ACCAAGAAGA ACAAGAGGAA
            301 GATGAAGAAC ATGCTGGTGA ACAAGATGAG GAGGATGAGG AGGAGGAGGA GATGGACCAG
            361 GAGAGTGACG ATTTTGATCA GTCTGATGAT AGTAGCAGAG AAGATGAACA TACACATACT
            421 AACAGTGTCA CGAACTCCAG TAGTATTGTG GACCTGCCCG TTCACCAACT CTCCTCCCCA
            481 TTCTATACAA AAACAACAAA AATGAAAAGA AAGTTGGACC ATGGTTCTGA GGTCCGCTCT
            541 TTTTCTTTGG GAAAGAAACC ATGCAAAGTC TCAGAATATA CAAGTACCAC TGGGCTTGTA
            601 CCATGTTCAG CAACACCAAC AACTTTTGGG GACCTCAGAG CAGCCAATGG CCAAGGGCAA
            661 CAACGACGCC GAATTACATC TGTCCAGCCA CCTACAGGCC TCCAGGAATG GCTAAAAATG
            721 TTTCAGAGCT GGAGTGGACC AGAGAAATTG CTTGCTTTAG ATGAACTCAT TGATAGTTGT
            781 GAACCAACAC AAGTAAAACA TATGATGCAA GTGATAGAAC CCCAGTTTCA ACGAGACTTC
            841 ATTTCATTGC TCCCTAAAGA GTTGGCACTC TATGTGCTTT CATTCCTGGA ACCCAAAGAC
            901 CTGCTACAAG CAGCTCAGAC ATGTCGCTAC TGGAGAATTT TGGCTGAAGA CAACCTTCTC
            961 TGGAGAGAGA AATGCAAAGA AGAGGGGATT GATGAACCAT TGCACATCAA GAGAAGAAAA
            1021 GTAATAAAAC CAGGTTTCAT ACACAGTCCA TGGAAAAGTG CATACATCAG ACAGCACAGA
            1081 ATTGATACTA ACTGGAGGCG AGGAGAACTC AAATCTCCTA AGGTGCTGAA AGGACATGAT
            1141 GATCATGTGA TCACATGCTT ACAGTTTTGT GGTAACCGAA TAGTTAGTGG TTCTGATGAC
            1201 AACACTTTAA AAGTTTGGTC AGCAGTCACA GGCAAATGTC TGAGAACATT AGTGGGACAT
            1261 ACAGGTGGAG TATGGTCATC ACAAATGAGA GACAACATCA TCATTAGTGG ATCTACAGAT
            1321 CGGACACTCA AAGTGTGGAA TGCAGAGACT GGAGAATGTA TACACACCTT ATATGGGCAT
            1381 ACTTCCACTG TGCGTTGTAT GCATCTTCAT GAAAAAAGAG TTGTTAGCGG TTCTCGAGAT
            1441 GCCACTCTTA GGGTTTGGGA TATTGAGACA GGCCAGTGTT TACATGTTTT GATGGGTCAT
            1501 GTTGCAGCAG TCCGCTGTGT TCAATATGAT GGCAGGAGGG TTGTTAGTGG AGCATATGAT
            1561 TTTATGGTAA AGGTGTGGGA TCCAGAGACT GAAACCTGTC TACACACGTT GCAGGGGCAT
            1621 ACTAATAGAG TCTATTCATT ACAGTTTGAT GGTATCCATG TGGTGAGTGG ATCTCTTGAT
            1681 ACATCAATCC GTGTTTGGGA TGTGGAGACA GGGAATTGCA TTCACACGTT AACAGGGCAC
            1741 CAGTCGTTAA CAAGTGGAAT GGAACTCAAA GACAATATTC TTGTCTCTGG GAATGCAGAT
            1801 TCTACAGTTA AAATCTGGGA TATCAAAACA GGACAGTGTT TACAAACATT GCAAGGTCCC
            1861 AACAAGCATC AGAGTGCTGT GACCTGTTTA CAGTTCAACA AGAACTTTGT AATTACCAGC
            1921 TCAGATGATG GAACTGTAAA ACTATGGGAC TTGAAAACGG GTGAATTTAT TCGAAACCTA
            1981 GTCACATTGG AGAGTGGGGG GAGTGGGGGA GTTGTGTGGC GGATCAGAGC CTCAAACACA
            2041 AAGCTGGTGT GTGCAGTTGG GAGTCGGAAT GGGACTGAAG AAACCAAGCT GCTGGTGCTG
            2101 GACTTTGATG TGGACATGAA GTGA
FBXW7       Protein Sequence
(SEQ ID     1 MNQELLSVGS KRRRTGGSLR GNPSSSQVDE EQMNRVVEEE QQQQLRQQEE EHTARNGEVV
NO: 104)    61 GVEPRPGGQN DSQQGQLEEN NNRFISVDED SSGNQEEQEE DEEHAGEQDE EDEEEEEMDQ
            121 ESDDFDQSDD SSREDEHTHT NSVTNSSSIV DLPVHQLSSP FYTKTTKMKR KLDHGSEVRS
            181 FSLGKKPCKV SEYTSTTGLV PCSATPTTFG DLRAANGQGQ QRRRITSVQP PTGLQEWLKM
            241 FQSWSGPEKL LALDELIDSC EPTQVKHMMQ VIEPQFQRDF ISLLPKELAL YVLSFLEPKD
            301 LLQAAQTCRY WRILAEDNLL WREKCKEEGI DEPLHIKRRK VIKPGFIHSP WKSAYIRQHR
            361 IDTNWRRGEL KSPKVLKGHD DHVITCLQFC GNRIVSGSDD NTLKVWSAVT GKCLRTLVGH
            421 TGGVWSSQMR DNIIISGSTD RTLKVWNAET GECIHTLYGH TSTVRCMHLH EKRVVSGSRD
            481 ATLRVWDIET GQCLHVLMGH VAAVRCVQYD GRRVVSGAYD FMVKVWDPET ETCLHTLQGH
            541 TNRVYSLQFD GIHVVSGSLD TSIRVWDVET GNCIHTLTGH QSLTSGMELK DNILVSGNAD
            601 STVKIWDIKT GQCLQTLQGP NKHQSAVTCL QFNKNFVITS SDDGTVKLWD LKTGEFIRNL
            661 VTLESGGSGG VVWRIRASNT KLVCAVGSRN GTEETKLLVL DFDVDMK
SMAD4       DNA Sequence
(SEQ ID     1 ATGGACAATA TGTCTATTAC GAATACACCA ACAAGTAATG ATGCCTGTCT GAGCATTGTG
NO: 105)    61 CATAGTTTGA TGTGCCATAG ACAAGGTGGA GAGAGTGAAA CATTTGCAAA AAGAGCAATT
            121 GAAAGTTTGG TAAAGAAGCT GAAGGAGAAA AAAGATGAAT TGGATTCTTT AATAACAGCT
            181 ATAACTACAA ATGGAGCTCA TCCTAGTAAA TGTGTTACCA TACAGAGAAC ATTGGATGGG
            241 AGGCTTCAGG TGGCTGGTCG GAAAGGATTT CCTCATGTGA TCTATGCCCG TCTCTGGAGG
            301 TGGCCTGATC TTCACAAAAA TGAACTAAAA CATGTTAAAT ATTGTCAGTA TGCGTTTGAC
            361 TTAAAATGTG ATAGTGTCTG TGTGAATCCA TATCACTACG AACGAGTTGT ATCACCTGGA
            421 ATTGATCTCT CAGGATTAAC ACTGCAGAGT AATGCTCCAT CAAGTATGAT GGTGAAGGAT
            481 GAATATGTGC ATGACTTTGA GGGACAGCCA TCGTTGTCCA CTGAAGGACA TTCAATTCAA
            541 ACCATCCAGC ATCCACCAAG TAATCGTGCA TCGACAGAGA CATACAGCAC CCCAGCTCTG
            601 TTAGCCCCAT CTGAGTCTAA TGCTACCAGC ACTGCCAACT TTCCCAACAT TCCTGTGGCT
            661 TCCACAAGTC AGCCTGCCAG TATACTGGGG GGCAGCCATA GTGAAGGACT GTTGCAGATA
            721 GCATCAGGGC CTCAGCCAGG ACAGCAGCAG AATGGATTTA CTGGTCAGCC AGCTACTTAC
            781 CATCATAACA GCACTACCAC CTGGACTGGA AGTAGGACTG CACCATACAC ACCTAATTTG
            841 CCTCACCACC AAAACGGCCA TCTTCAGCAC CACCCGCCTA TGCCGCCCCA TCCCGGACAT
            901 TACTGGCCTG TTCACAATGA GCTTGCATTC CAGCCTCCCA TTTCCAATCA TCCTGCTCCT
            961 GAGTATTGGT GTTCCATTGC TTACTTTGAA ATGGATGTTC AGGTAGGAGA GACATTTAAG
            1021 GTTCCTTCAA GCTGCCCTAT TGTTACTGTT GATGGATACG TGGACCCTTC TGGAGGAGAT
            1081 CGCTTTTGTT TGGGTCAACT CTCCAATGTC CACAGGACAG AAGCCATTGA GAGAGCAAGG
            1141 TTGCACATAG GCAAAGGTGT GCAGTTGGAA TGTAAAGGTG AAGGTGATGT TTGGGTCAGG
            1201 TGCCTTAGTG ACCACGCGGT CTTTGTACAG AGTTACTACT TAGACAGAGA AGCTGGGCGT
            1261 GCACCTGGAG ATGCTGTTCA TAAGATCTAC CCAAGTGCAT ATATAAAGGT CTTTGATTTG
            1321 CGTCAGTGTC ATCGACAGAT GCAGCAGCAG GCGGCTACTG CACAAGCTGC AGCAGCTGCC
            1381 CAGGCAGCAG CCGTGGCAGG AAACATCCCT GGCCCAGGAT CAGTAGGTGG AATAGCTCCA
            1441 GCTATCAGTC TGTCAGCTGC TGCTGGAATT GGTGTTGATG ACCTTCGTCG CTTATGCATA
            1501 CTCAGGATGA GTTTTGTGAA AGGCTGGGGA CCGGATTACC CAAGACAGAG CATCAAAGAA
            1561 ACACCTTGCT GGATTGAAAT TCACTTACAC CGGGCCCTCC AGCTCCTAGA CGAAGTACTT
            1621 CATACCATGC CGATTGCAGA CCCACAACCT TTAGACTGA
SMAD4       Protein Sequence
(SEQ ID     1 MDNMSITNTP TSNDACLSIV HSLMCHRQGG ESETFAKRAI ESLVKKLKEK KDELDSLITA
NO: 106)    61 ITTNGAHPSK CVTIQRTLDG RLQVAGRKGF PHVIYARLWR WPDLHKNELK HVKYCQYAFD
            121 LKCDSVCVNP YHYERVVSPG IDLSGLTLQS NAPSSMMVKD EYVHDFEGQP SLSTEGHSIQ
            181 TIQHPPSNRA STETYSTPAL LAPSESNATS TANFPNIPVA STSQPASILG GSHSEGLLQI
            241 ASGPQPGQQQ NGFTGQPATY HHNSTTTWTG SRTAPYTPNL PHHQNGHLQH HPPMPPHPGH
            301 YWPVHNELAF QPPISNHPAP EYWCSIAYFE MDVQVGETFK VPSSCPIVTV DGYVDPSGGD
            361 RFCLGQLSNV HRTEAIERAR LHIGKGVQLE CKGEGDVWVR CLSDHAVFVQ SYYLDREAGR
            421 APGDAVHKIY PSAYIKVFDL RQCHRQMQQQ AATAQAAAAA QAAAVAGNIP GPGSVGGIAP
            481 AISLSAAAGI GVDDLRRLCI LRMSFVKGWG PDYPRQSIKE TPCWIEIHLH RALQLLDEVL
            541 HTMPIADPQP LD
ATM         DNA Sequence
(SEQ ID     1 ATGAGTCTAG TACTTAATGA TCTGCTTATC TGCTGCCGTC AACTAGAACA TGATAGAGCT
NO: 107)    61 ACAGAACGAA AGAAAGAAGT TGAGAAATTT AAGCGCCTGA TTCGAGATCC TGAAACAATT
            121 AAACATCTAG ATCGGCATTC AGATTCCAAA CAAGGAAAAT ATTTGAATTG GGATGCTGTT
            181 TTTAGATTTT TACAGAAATA TATTCAGAAA GAAACAGAAT GTCTGAGAAT AGCAAAACCA
            241 AATGTATCAG CCTCAACACA AGCCTCCAGG CAGAAAAAGA TGCAGGAAAT CAGTAGTTTG
            301 GTCAAATACT TCATCAAATG TGCAAACAGA AGAGCACCTA GGCTAAAATG TCAAGAACTC
            361 TTAAATTATA TCATGGATAC AGTGAAAGAT TCATCTAATG GTGCTATTTA CGGAGCTGAT
            421 TGTAGCAACA TACTACTCAA AGACATTCTT TCTGTGAGAA AATACTGGTG TGAAATATCT
            481 CAGCAACAGT GGTTAGAATT GTTCTCTGTG TACTTCAGGC TCTATCTGAA ACCTTCACAA
            541 GATGTTCATA GAGTTTTAGT GGCTAGAATA ATTCATGCTG TTACCAAAGG ATGCTGTTCT
            601 CAGACTGACG GATTAAATTC CAAATTTTTG GACTTTTTTT CCAAGGCTAT TCAGTGTGCG
            661 AGACAAGAAA AGAGCTCTTC AGGTCTAAAT CATATCTTAG CAGCTCTTAC TATCTTCCTC
            721 AAGACTTTGG CTGTCAACTT TCGAATTCGA GTGTGTGAAT TAGGAGATGA AATTCTTCCC
            781 ACTTTGCTTT ATATTTGGAC TCAACATAGG CTTAATGATT CTTTAAAAGA AGTCATTATT
            841 GAATTATTTC AACTGCAAAT TTATATCCAT CATCCGAAAG GAGCCAAAAC CCAAGAAAAA
            901 GGTGCTTATG AATCAACAAA ATGGAGAAGT ATTTTATACA ACTTATATGA TCTGCTAGTG
            961 AATGAGATAA GTCATATAGG AAGTAGAGGA AAGTATTCTT CAGGATTTCG TAATATTGCC
            1021 GTCAAAGAAA ATTTGATTGA ATTGATGGCA GATATCTGTC ACCAGGTTTT TAATGAAGAT
            1081 ACCAGATCCT TGGAGATTTC TCAATCTTAC ACTACTACAC AAAGAGAATC TAGTGATTAC
            1141 AGTGTCCCTT GCAAAAGGAA GAAAATAGAA CTAGGCTGGG AAGTAATAAA AGATCACCTT
            1201 CAGAAGTCAC AGAATGATTT TGATCTTGTG CCTTGGCTAC AGATTGCAAC CCAATTAATA
            1261 TCAAAGTATC CTGCAAGTTT ACCTAACTGT GAGCTGTCTC CATTACTGAT GATACTATCT
            1321 CAGCTTCTAC CCCAACAGCG ACATGGGGAA CGTACACCAT ATGTGTTACG ATGCCTTACG
            1381 GAAGTTGCAT TGTGTCAAGA CAAGAGGTCA AACCTAGAAA GCTCACAAAA GTCAGATTTA
            1441 TTAAAACTCT GGAATAAAAT TTGGTGTATT ACCTTTCGTG GTATAAGTTC TGAGCAAATA
            1501 CAAGCTGAAA ACTTTGGCTT ACTTGGAGCC ATAATTCAGG GTAGTTTAGT TGAGGTTGAC
            1561 AGAGAATTCT GGAAGTTATT TACTGGGTCA GCCTGCAGAC CTTCATGTCC TGCAGTATGC
            1621 TGTTTGACTT TGGCACTGAC CACCAGTATA GTTCCAGGAA CGGTAAAAAT GGGAATAGAG
            1681 CAAAATATGT GTGAAGTAAA TAGAAGCTTT TCTTTAAAGG AATCAATAAT GAAATGGCTC
            1741 TTATTCTATC AGTTAGAGGG TGACTTAGAA AATAGCACAG AAGTGCCTCC AATTCTTCAC
            1801 AGTAATTTTC CTCATCTTGT ACTGGAGAAA ATTCTTGTGA GTCTCACTAT GAAAAACTGT
            1861 AAAGCTGCAA TGAATTTTTT CCAAAGCGTG CCAGAATGTG AACACCACCA AAAAGATAAA
            1921 GAAGAACTTT CATTCTCAGA AGTAGAAGAA CTATTTCTTC AGACAACTTT TGACAAGATG
            1981 GACTTTTTAA CCATTGTGAG AGAATGTGGT ATAGAAAAGC ACCAGTCCAG TATTGGCTTC
            2041 TCTGTCCACC AGAATCTCAA GGAATCACTG GATCGCTGTC TTCTGGGATT ATCAGAACAG
            2101 CTTCTGAATA ATTACTCATC TGAGATTACA AATTCAGAAA CTCTTGTCCG GTGTTCACGT
            2161 CTTTTGGTGG GTGTCCTTGG CTGCTACTGT TACATGGGTG TAATAGCTGA AGAGGAAGCA
            2221 TATAAGTCAG AATTATTCCA GAAAGCCAAG TCTCTAATGC AATGTGCAGG AGAAAGTATC
            2281 ACTCTGTTTA AAAATAAGAC AAATGAGGAA TTCAGAATTG GTTCCTTGAG AAATATGATG
            2341 CAGCTATGTA CACGTTGCTT GAGCAACTGT ACCAAGAAGA GTCCAAATAA GATTGCATCT
            2401 GGCTTTTTCC TGCGATTGTT AACATCAAAG CTAATGAATG ACATTGCAGA TATTTGTAAA
            2461 AGTTTAGCAT CCTTCATCAA AAAGCCATTT GACCGTGGAG AAGTAGAATC AATGGAAGAT
            2521 GATACTAATG GAAATCTAAT GGAGGTGGAG GATCAGTCAT CCATGAATCT ATTTAACGAT
            2581 TACCCTGATA GTAGTGTTAG TGATGCAAAC GAACCTGGAG AGAGCCAAAG TACCATAGGT
            2641 GCCATTAATC CTTTAGCTGA AGAATATCTG TCAAAGCAAG ATCTACTTTT CTTAGACATG
            2701 CTCAAGTTCT TGTGTTTGTG TGTAACTACT GCTCAGACCA ATACTGTGTC CTTTAGGGCA
            2761 GCTGATATTC GGAGGAAATT GTTAATGTTA ATTGATTCTA GCACGCTAGA ACCTACCAAA
            2821 TCCCTCCACC TGCATATGTA TCTAATGCTT TTAAAGGAGC TTCCTGGAGA AGAGTACCCC
            2881 TTGCCAATGG AAGATGTTCT TGAACTTCTG AAACCACTAT CCAATGTGTG TTCTTTGTAT
            2941 CGTCGTGACC AAGATGTTTG TAAAACTATT TTAAACCATG TCCTTCATGT AGTGAAAAAC
            3001 CTAGGTCAAA GCAATATGGA CTCTGAGAAC ACAAGGGATG CTCAAGGACA GTTTCTTACA
            3061 GTAATTGGAG CATTTTGGCA TCTAACAAAG GAGAGGAAAT ATATATTCTC TGTAAGAATG
            3121 GCCCTAGTAA ATTGCCTTAA AACTTTGCTT GAGGCTGATC CTTATTCAAA ATGGGCCATT
            3181 CTTAATGTAA TGGGAAAAGA CTTTCCTGTA AATGAAGTAT TTACACAATT TCTTGCTGAC
            3241 AATCATCACC AAGTTCGCAT GTTGGCTGCA GAGTCAATCA ATAGATTGTT CCAGGACACG
            3301 AAGGGAGATT CTTCCAGGTT ACTGAAAGCA CTTCCTTTGA AGCTTCAGCA AACAGCTTTT
            3361 GAAAATGCAT ACTTGAAAGC TCAGGAAGGA ATGAGAGAAA TGTCCCATAG TGCTGAGAAC
            3421 CCTGAAACTT TGGATGAAAT TTATAATAGA AAATCTGTTT TACTGACGTT GATAGCTGTG
            3481 GTTTTATCCT GTAGCCCTAT CTGCGAAAAA CAGGCTTTGT TTGCCCTGTG TAAATCTGTG
            3541 AAAGAGAATG GATTAGAACC TCACCTTGTG AAAAAGGTTT TAGAGAAAGT TTCTGAAACT
            3601 TTTGGATATA GACGTTTAGA AGACTTTATG GCATCTCATT TAGATTATCT GGTTTTGGAA
            3661 TGGCTAAATC TTCAAGATAC TGAATACAAC TTATCTTCTT TTCCTTTTAT TTTATTAAAC
            3721 TACACAAATA TTGAGGATTT CTATAGATCT TGTTATAAGG TTTTGATTCC ACATCTGGTG
            3781 ATTAGAAGTC ATTTTGATGA GGTGAAGTCC ATTGCTAATC AGATTCAAGA GGACTGGAAA
            3841 AGTCTTCTAA CAGACTGCTT TCCAAAGATT CTTGTAAATA TTCTTCCTTA TTTTGCCTAT
            3901 GAGGGTACCA GAGACAGTGG GATGGCACAG CAAAGAGAGA CTGCTACCAA GGTCTATGAT
            3961 ATGCTTAAAA GTGAAAACTT ATTGGGAAAA CAGATTGATC ACTTATTCAT TAGTAATTTA
            4021 CCAGAGATTG TGGTGGAGTT ATTGATGACG TTACATGAGC CAGCAAATTC TAGTGCCAGT
            4081 CAGAGCACTG ACCTCTGTGA CTTTTCAGGG GATTTGGATC CTGCTCCTAA TCCACCTCAT
            4141 TTTCCATCGC ATGTGATTAA AGCAACATTT GCCTATATCA GCAATTGTCA TAAAACCAAG
            4201 TTAAAAAGCA TTTTAGAAAT TCTTTCCAAA AGCCCTGATT CCTATCAGAA AATTCTTCTT
            4261 GCCATATGTG AGCAAGCAGC TGAAACAAAT AATGTTTATA AGAAGCACAG AATTCTTAAA
            4321 ATATATCACC TGTTTGTTAG TTTATTACTG AAAGATATAA AAAGTGGCTT AGGAGGAGCT
            4381 TGGGCCTTTG TTCTTCGAGA CGTTATTTAT ACTTTGATTC ACTATATCAA CCAAAGGCCT
            4441 TCTTGTATCA TGGATGTGTC ATTACGTAGC TTCTCCCTTT GTTGTGACTT ATTAAGTCAG
            4501 GTTTGCCAGA CAGCCGTGAC TTACTGTAAG GATGCTCTAG AAAACCATCT TCATGTTATT
            4561 GTTGGTACAC TTATACCCCT TGTGTATGAG CAGGTGGAGG TTCAGAAACA GGTATTGGAC
            4621 TTGTTGAAAT ACTTAGTGAT AGATAACAAG GATAATGAAA ACCTCTATAT CACGATTAAG
            4681 CTTTTAGATC CTTTTCCTGA CCATGTTGTT TTTAAGGATT TGCGTATTAC TCAGCAAAAA
            4741 ATCAAATACA GTAGAGGACC CTTTTCACTC TTGGAGGAAA TTAACCATTT TCTCTCAGTA
            4801 AGTGTTTATG ATGCACTTCC ATTGACAAGA CTTGAAGGAC TAAAGGATCT TCGAAGACAA
            4861 CTGGAACTAC ATAAAGATCA GATGGTGGAC ATTATGAGAG CTTCTCAGGA TAATCCGCAA
            4921 GATGGGATTA TGGTGAAACT AGTTGTCAAT TTGTTGCAGT TATCCAAGAT GGCAATAAAC
            4981 CACACTGGTG AAAAAGAAGT TCTAGAGGCT GTTGGAAGCT GCTTGGGAGA AGTGGGTCCT
            5041 ATAGATTTCT CTACCATAGC TATACAACAT AGTAAAGATG CATCTTATAC CAAGGCCCTT
            5101 AAGTTATTTG AAGATAAAGA ACTTCAGTGG ACCTTCATAA TGCTGACCTA CCTGAATAAC
            5161 ACACTGGTAG AAGATTGTGT CAAAGTTCGA TCAGCAGCTG TTACCTGTTT GAAAAACATT
            5221 TTAGCCACAA AGACTGGACA TAGTTTCTGG GAGATTTATA AGATGACAAC AGATCCAATG
            5281 CTGGCCTATC TACAGCCTTT TAGAACATCA AGAAAAAAGT TTTTAGAAGT ACCCAGATTT
            5341 GACAAAGAAA ACCCTTTTGA AGGCCTGGAT GATATAAATC TGTGGATTCC TCTAAGTGAA
            5401 AATCATGACA TTTGGATAAA GACACTGACT TGTGCTTTTT TGGACAGTGG AGGCACAAAA
            5461 TGTGAAATTC TTCAATTATT AAAGCCAATG TGTGAAGTGA AAACTGACTT TTGTCAGACT
            5521 GTACTTCCAT ACTTGATTCA TGATATTTTA CTCCAAGATA CAAATGAATC ATGGAGAAAT
            5581 CTGCTTTCTA CACATGTTCA GGGATTTTTC ACCAGCTGTC TTCGACACTT CTCGCAAACG
            5641 AGCCGATCCA CAACCCCTGC AAACTTGGAT TCAGAGTCAG AGCACTTTTT CCGATGCTGT
            5701 TTGGATAAAA AATCACAAAG AACAATGCTT GCTGTTGTGG ACTACATGAG AAGACAAAAG
            5761 AGACCTTCTT CAGGAACAAT TTTTAATGAT GCTTTCTGGC TGGATTTAAA TTATCTAGAA
            5821 GTTGCCAAGG TAGCTCAGTC TTGTGCTGCT CACTTTACAG CTTTACTCTA TGCAGAAATC
            5881 TATGCAGATA AGAAAAGTAT GGATGATCAA GAGAAAAGAA GTCTTGCATT TGAAGAAGGA
            5941 AGCCAGAGTA CAACTATTTC TAGCTTGAGT GAAAAAAGTA AAGAAGAAAC TGGAATAAGT
            6001 TTACAGGATC TTCTCTTAGA AATCTACAGA AGTATAGGGG AGCCAGATAG TTTGTATGGC
            6061 TGTGGTGGAG GGAAGATGTT ACAACCCATT ACTAGACTAC GAACATATGA ACACGAAGCA
            6121 ATGTGGGGCA AAGCCCTAGT AACATATGAC CTCGAAACAG CAATCCCCTC ATCAACACGC
            6181 CAGGCAGGAA TCATTCAGGC CTTGCAGAAT TTGGGACTCT GCCATATTCT TTCCGTCTAT
            6241 TTAAAAGGAT TGGATTATGA AAATAAAGAC TGGTGTCCTG AACTAGAAGA ACTTCATTAC
            6301 CAAGCAGCAT GGAGGAATAT GCAGTGGGAC CATTGCACTT CCGTCAGCAA AGAAGTAGAA
            6361 GGAACCAGTT ACCATGAATC ATTGTACAAT GCTCTACAAT CTCTAAGAGA CAGAGAATTC
            6421 TCTACATTTT ATGAAAGTCT CAAATATGCC AGAGTAAAAG AAGTGGAAGA GATGTGTAAG
            6481 CGCAGCCTTG AGTCTGTGTA TTCGCTCTAT CCCACACTTA GCAGGTTGCA GGCCATTGGA
            6541 GAGCTGGAAA GCATTGGGGA GCTTTTCTCA AGATCAGTCA CACATAGACA ACTCTCTGAA
            6601 GTATATATTA AGTGGCAGAA ACACTCCCAG CTTCTCAAGG ACAGTGATTT TAGTTTTCAG
            6661 GAGCCTATCA TGGCTCTACG CACAGTCATT TTGGAGATCC TGATGGAAAA GGAAATGGAC
            6721 AACTCACAAA GAGAATGTAT TAAGGACATT CTCACCAAAC ACCTTGTAGA ACTCTCTATA
            6781 CTGGCCAGAA CTTTCAAGAA CACTCAGCTC CCTGAAAGGG CAATATTTCA AATTAAACAG
            6841 TACAATTCAG TTAGCTGTGG AGTCTCTGAG TGGCAGCTGG AAGAAGCACA AGTATTCTGG
            6901 GCAAAAAAGG AGCAGAGTCT TGCCCTGAGT ATTCTCAAGC AAATGATCAA GAAGTTGGAT
            6961 GCCAGCTGTG CAGCGAACAA TCCCAGCCTA AAACTTACAT ACACAGAATG TCTGAGGGTT
            7021 TGTGGCAACT GGTTAGCAGA AACGTGCTTA GAAAATCCTG CGGTCATCAT GCAGACCTAT
            7081 CTAGAAAAGG CAGTAGAAGT TGCTGGAAAT TATGATGGAG AAAGTAGTGA TGAGCTAAGA
            7141 AATGGAAAAA TGAAGGCATT TCTCTCATTA GCCCGGTTTT CAGATACTCA ATACCAAAGA
            7201 ATTGAAAACT ACATGAAATC ATCGGAATTT GAAAACAAGC AAGCTCTCCT GAAAAGAGCC
            7261 AAAGAGGAAG TAGGTCTCCT TAGGGAACAT AAAATTCAGA CAAACAGATA CACAGTAAAG
            7321 GTTCAGCGAG AGCTGGAGTT GGATGAATTA GCCCTGCGTG CACTGAAAGA GGATCGTAAA
            7381 CGCTTCTTAT GTAAAGCAGT TGAAAATTAT ATCAACTGCT TATTAAGTGG AGAAGAACAT
            7441 GATATGTGGG TATTCCGACT TTGTTCCCTC TGGCTTGAAA ATTCTGGAGT TTCTGAAGTC
            7501 AATGGCATGA TGAAGAGAGA CGGAATGAAG ATTCCAACAT ATAAATTTTT GCCTCTTATG
            7561 TACCAATTGG CTGCTAGAAT GGGGACCAAG ATGATGGGAG GCCTAGGATT TCATGAAGTC
            7621 CTCAATAATC TAATCTCTAG AATTTCAATG GATCACCCCC ATCACACTTT GTTTATTATA
            7681 CTGGCCTTAG CAAATGCAAA CAGAGATGAA TTTCTGACTA AACCAGAGGT AGCCAGAAGA
            7741 AGCAGAATAA CTAAAAATGT GCCTAAACAA AGCTCTCAGC TTGATGAGGA TCGAACAGAG
            7801 GCTGCAAATA GAATAATATG TACTATCAGA AGTAGGAGAC CTCAGATGGT CAGAAGTGTT
            7861 GAGGCACTTT GTGATGCTTA TATTATATTA GCAAACTTAG ATGCCACTCA GTGGAAGACT
            7921 CAGAGAAAAG GCATAAATAT TCCAGCAGAC CAGCCAATTA CTAAACTTAA GAATTTAGAA
            7981 GATGTTGTTG TCCCTACTAT GGAAATTAAG GTGGACCACA CAGGAGAATA TGGAAATCTG
            8041 GTGACTATAC AGTCATTTAA AGCAGAATTT CGCTTAGCAG GAGGTGTAAA TTTACCAAAA
            8101 ATAATAGATT GTGTAGGTTC CGATGGCAAG GAGAGGAGAC AGCTTGTTAA GGGCCGTGAT
            8161 GACCTGAGAC AAGATGCTGT CATGCAACAG GTCTTCCAGA TGTGTAATAC ATTACTGCAG
            8221 AGAAACACGG AAACTAGGAA GAGGAAATTA ACTATCTGTA CTTATAAGGT GGTTCCCCTC
            8281 TCTCAGCGAA GTGGTGTTCT TGAATGGTGC ACAGGAACTG TCCCCATTGG TGAATTTCTT
            8341 GTTAACAATG AAGATGGTGC TCATAAAAGA TACAGGCCAA ATGATTTCAG TGCCTTTCAG
            8401 TGCCAAAAGA AAATGATGGA GGTGCAAAAA AAGTCTTTTG AAGAGAAATA TGAAGTCTTC
            8461 ATGGATGTTT GCCAAAATTT TCAACCAGTT TTCCGTTACT TCTGCATGGA AAAATTCTTG
            8521 GATCCAGCTA TTTGGTTTGA GAAGCGATTG GCTTATACGC GCAGTGTAGC TACTTCTTCT
            8581 ATTGTTGGTT ACATACTTGG ACTTGGTGAT AGACATGTAC AGAATATCTT GATAAATGAG
            8641 CAGTCAGCAG AACTTGTACA TATAGATCTA GGTGTTGCTT TTGAACAGGG CAAAATCCTT
            8701 CCTACTCCTG AGACAGTTCC TTTTAGACTC ACCAGAGATA TTGTGGATGG CATGGGCATT
            8761 ACGGGTGTTG AAGGTGTCTT CAGAAGATGC TGTGAGAAAA CCATGGAAGT GATGAGAAAC
            8821 TCTCAGGAAA CTCTGTTAAC CATTGTAGAG GTCCTTCTAT ATGATCCACT CTTTGACTGG
            8881 ACCATGAATC CTTTGAAAGC TTTGTATTTA CAGCAGAGGC CGGAAGATGA AACTGAGCTT
            8941 CACCCTACTC TGAATGCAGA TGACCAAGAA TGCAAACGAA ATCTCAGTGA TATTGACCAG
            9001 AGTTTCAACA AAGTAGCTGA ACGTGTCTTA ATGAGACTAC AAGAGAAACT GAAAGGAGTG
            9061 GAAGAAGGCA CTGTGCTCAG TGTTGGTGGA CAAGTGAATT TGCTCATACA GCAGGCCATA
            9121 GACCCCAAAA ATCTCAGCCG ACTTTTCCCA GGATGGAAAG CTTGGGTGTG A
ATM         Protein Sequence
(SEQ ID     1 MSLVLNDLLI CCRQLEHDRA TERKKEVEKF KRLIRDPETI KHLDRHSDSK QGKYLNWDAV
NO: 108)    61 FRFLQKYIQK ETECLRIAKP NVSASTQASR QKKMQEISSL VKYFIKCANR RAPRLKCQEL
            121 LNYIMDTVKD SSNGAIYGAD CSNILLKDIL SVRKYWCEIS QQQWLELFSV YFRLYLKPSQ
            181 DVHRVLVARI IHAVTKGCCS QTDGLNSKFL DFFSKAIQCA RQEKSSSGLN HILAALTIFL
            241 KTLAVNFRIR VCELGDEILP TLLYIWTQHR LNDSLKEVII ELFQLQIYIH HPKGAKTQEK
            301 GAYESTKWRS ILYNLYDLLV NEISHIGSRG KYSSGFRNIA VKENLIELMA DICHQVFNED
            361 TRSLEISQSY TTTQRESSDY SVPCKRKKIE LGWEVIKDHL QKSQNDFDLV PWLQIATQLI
            421 SKYPASLPNC ELSPLLMILS QLLPQQRHGE RTPYVLRCLT EVALCQDKRS NLESSQKSDL
            481 LKLWNKIWCI TFRGISSEQI QAENFGLLGA IIQGSLVEVD REFWKLFTGS ACRPSCPAVC
            541 CLTLALTTSI VPGTVKMGIE QNMCEVNRSF SLKESIMKWL LFYQLEGDLE NSTEVPPILH
            601 SNFPHLVLEK ILVSLTMKNC KAAMNFFQSV PECEHHQKDK EELSFSEVEE LFLQTTFDKM
            661 DFLTIVRECG IEKHQSSIGF SVHQNLKESL DRCLLGLSEQ LLNNYSSEIT NSETLVRCSR
            721 LLVGVLGCYC YMGVIAEEEA YKSELFQKAK SLMQCAGESI TLFKNKTNEE FRIGSLRNMM
            781 QLCTRCLSNC TKKSPNKIAS GFFLRLLTSK LMNDIADICK SLASFIKKPF DRGEVESMED
            841 DTNGNLMEVE DQSSMNLFND YPDSSVSDAN EPGESQSTIG AINPLAEEYL SKQDLLFLDM
            901 LKFLCLCVTT AQTNTVSFRA ADIRRKLLML IDSSTLEPTK SLHLHMYLML LKELPGEEYP
            961 LPMEDVLELL KPLSNVCSLY RRDQDVCKTI LNHVLHVVKN LGQSNMDSEN TRDAQGQFLT
            1021 VIGAFWHLTK ERKYIFSVRM ALVNCLKTLL EADPYSKWAI LNVMGKDFPV NEVFTQFLAD
            1081 NHHQVRMLAA ESINRLFQDT KGDSSRLLKA LPLKLQQTAF ENAYLKAQEG MREMSHSAEN
            1141 PETLDEIYNR KSVLLTLIAV VLSCSPICEK QALFALCKSV KENGLEPHLV KKVLEKVSET
            1201 FGYRRLEDFM ASHLDYLVLE WLNLQDTEYN LSSFPFILLN YTNIEDFYRS CYKVLIPHLV
            1261 IRSHFDEVKS IANQIQEDWK SLLTDCFPKI LVNILPYFAY EGTRDSGMAQ QRETATKVYD
            1321 MLKSENLLGK QIDHLFISNL PEIVVELLMT LHEPANSSAS QSTDLCDFSG DLDPAPNPPH
            1381 FPSHVIKATF AYISNCHKTK LKSILEILSK SPDSYQKILL AICEQAAETN NVYKKHRILK
            1441 IYHLFVSLLL KDIKSGLGGA WAFVLRDVIY TLIHYINQRP SCIMDVSLRS FSLCCDLLSQ
            1501 VCQTAVTYCK DALENHLHVI VGTLIPLVYE QVEVQKQVLD LLKYLVIDNK DNENLYITIK
            1561 LLDPFPDHVV FKDLRITQQK IKYSRGPFSL LEEINHFLSV SVYDALPLTR LEGLKDLRRQ
            1621 LELHKDQMVD IMRASQDNPQ DGIMVKLVVN LLQLSKMAIN HTGEKEVLEA VGSCLGEVGP
            1681 IDFSTIAIQH SKDASYTKAL KLFEDKELQW TFIMLTYLNN TLVEDCVKVR SAAVTCLKNI
            1741 LATKTGHSFW EIYKMTTDPM LAYLQPFRTS RKKFLEVPRF DKENPFEGLD DINLWIPLSE
            1801 NHDIWIKTLT CAFLDSGGTK CEILQLLKPM CEVKTDFCQT VLPYLIHDIL LQDTNESWRN
            1861 LLSTHVQGFF TSCLRHFSQT SRSTTPANLD SESEHFFRCC LDKKSQRTML AVVDYMRRQK
            1921 RPSSGTIFND AFWLDLNYLE VAKVAQSCAA HFTALLYAEI YADKKSMDDQ EKRSLAFEEG
            1981 SQSTTISSLS EKSKEETGIS LQDLLLEIYR SIGEPDSLYG CGGGKMLQPI TRLRTYEHEA
            2041 MWGKALVTYD LETAIPSSTR QAGIIQALQN LGLCHILSVY LKGLDYENKD WCPELEELHY
            2101 QAAWRNMQWD HCTSVSKEVE GTSYHESLYN ALQSLRDREF STFYESLKYA RVKEVEEMCK
            2161 RSLESVYSLY PTLSRLQAIG ELESIGELFS RSVTHRQLSE VYIKWQKHSQ LLKDSDFSFQ
            2221 EPIMALRTVI LEILMEKEMD NSQRECIKDI LTKHLVELSI LARTFKNTQL PERAIFQIKQ
            2281 YNSVSCGVSE WQLEEAQVFW AKKEQSLALS ILKQMIKKLD ASCAANNPSL KLTYTECLRV
            2341 CGNWLAETCL ENPAVIMQTY LEKAVEVAGN YDGESSDELR NGKMKAFLSL ARFSDTQYQR
            2401 IENYMKSSEF ENKQALLKRA KEEVGLLREH KIQTNRYTVK VQRELELDEL ALRALKEDRK
            2461 RFLCKAVENY INCLLSGEEH DMWVFRLCSL WLENSGVSEV NGMMKRDGMK IPTYKFLPLM
            2521 YQLAARMGTK MMGGLGFHEV LNNLISRISM DHPHHTLFII LALANANRDE FLTKPEVARR
            2581 SRITKNVPKQ SSQLDEDRTE AANRIICTIR SRRPQMVRSV EALCDAYIIL ANLDATQWKT
            2641 QRKGINIPAD QPITKLKNLE DVVVPTMEIK VDHTGEYGNL VTIQSFKAEF RLAGGVNLPK
            2701 IIDCVGSDGK ERRQLVKGRD DLRQDAVMQQ VFQMCNTLLQ RNTETRKRKL TICTYKVVPL
            2761 SQRSGVLEWC TGTVPIGEFL VNNEDGAHKR YRPNDFSAFQ CQKKMMEVQK KSFEEKYEVF
            2821 MDVCQNFQPV FRYFCMEKFL DPAIWFEKRL AYTRSVATSS IVGYILGLGD RHVQNILINE
            2881 QSAELVHIDL GVAFEQGKIL PTPETVPFRL TRDIVDGMGI TGVEGVFRRC CEKTMEVMRN
            2941 SQETLLTIVE VLLYDPLFDW TMNPLKALYL QQRPEDETEL HPTLNADDQE CKRNLSDIDQ
            3001 SFNKVAERVL MRLQEKLKGV EEGTVLSVGG QVNLLIQQAI DPKNLSRLFP GWKAWV
CTNNB1      DNA Sequence
(SEQ ID     1 ATGGCTACTC AAGCTGATTT GATGGAGTTG GACATGGCCA TGGAACCAGA CAGAAAAGCG
NO:109)     61 GCTGTTAGTC ACTGGCAGCA ACAGTCTTAC CTGGACTCTG GAATCCATTC TGGTGCCACT
            121 ACCACAGCTC CTTCTCTGAG TGGTAAAGGC AATCCTGAGG AAGAGGATGT GGATACCTCC
            181 CAAGTCCTGT ATGAGTGGGA ACAGGGATTT TCTCAGTCCT TCACTCAAGA ACAAGTAGCT
            241 GATATTGATG GACAGTATGC AATGACTCGA GCTCAGAGGG TACGAGCTGC TATGTTCCCT
            301 GAGACATTAG ATGAGGGCAT GCAGATCCCA TCTACACAGT TTGATGCTGC TCATCCCACT
            361 AATGTCCAGC GTTTGGCTGA ACCATCACAG ATGCTGAAAC ATGCAGTTGT AAACTTGATT
            421 AACTATCAAG ATGATGCAGA ACTTGCCACA CGTGCAATCC CTGAACTGAC AAAACTGCTA
            481 AATGACGAGG ACCAGGTGGT GGTTAATAAG GCTGCAGTTA TGGTCCATCA GCTTTCTAAA
            541 AAGGAAGCTT CCAGACACGC TATCATGCGT TCTCCTCAGA TGGTGTCTGC TATTGTACGT
            601 ACCATGCAGA ATACAAATGA TGTAGAAACA GCTCGTTGTA CCGCTGGGAC CTTGCATAAC
            661 CTTTCCCATC ATCGTGAGGG CTTACTGGCC ATCTTTAAGT CTGGAGGCAT TCCTGCCCTG
            721 GTGAAAATGC TTGGTTCACC AGTGGATTCT GTGTTGTTTT ATGCCATTAC AACTCTCCAC
            781 AACCTTTTAT TACATCAAGA AGGAGCTAAA ATGGCAGTGC GTTTAGCTGG TGGGCTGCAG
            841 AAAATGGTTG CCTTGCTCAA CAAAACAAAT GTTAAATTCT TGGCTATTAC GACAGACTGC
            901 CTTCAAATTT TAGCTTATGG CAACCAAGAA AGCAAGCTCA TCATACTGGC TAGTGGTGGA
            961 CCCCAAGCTT TAGTAAATAT AATGAGGACC TATACTTACG AAAAACTACT GTGGACCACA
            1021 AGCAGAGTGC TGAAGGTGCT ATCTGTCTGC TCTAGTAATA AGCCGGCTAT TGTAGAAGCT
            1081 GGTGGAATGC AAGCTTTAGG ACTTCACCTG ACAGATCCAA GTCAACGTCT TGTTCAGAAC
            1141 TGTCTTTGGA CTCTCAGGAA TCTTTCAGAT GCTGCAACTA AACAGGAAGG GATGGAAGGT
            1201 CTCCTTGGGA CTCTTGTTCA GCTTCTGGGT TCAGATGATA TAAATGTGGT CACCTGTGCA
            1261 GCTGGAATTC TTTCTAACCT CACTTGCAAT AATTATAAGA ACAAGATGAT GGTCTGCCAA
            1321 GTGGGTGGTA TAGAGGCTCT TGTGCGTACT GTCCTTCGGG CTGGTGACAG GGAAGACATC
            1381 ACTGAGCCTG CCATCTGTGC TCTTCGTCAT CTGACCAGCC GACACCAAGA AGCAGAGATG
            1441 GCCCAGAATG CAGTTCGCCT TCACTATGGA CTACCAGTTG TGGTTAAGCT CTTACACCCA
            1501 CCATCCCACT GGCCTCTGAT AAAGGCTACT GTTGGATTGA TTCGAAATCT TGCCCTTTGT
            1561 CCCGCAAATC ATGCACCTTT GCGTGAGCAG GGTGCCATTC CACGACTAGT TCAGTTGCTT
            1621 GTTCGTGCAC ATCAGGATAC CCAGCGCCGT ACGTCCATGG GTGGGACACA GCAGCAATTT
            1681 GTGGAGGGGG TCCGCATGGA AGAAATAGTT GAAGGTTGTA CCGGAGCCCT TCACATCCTA
            1741 GCTCGGGATG TTCACAACCG AATTGTTATC AGAGGACTAA ATACCATTCC ATTGTTTGTG
            1801 CAGCTGCTTT ATTCTCCCAT TGAAAACATC CAAAGAGTAG CTGCAGGGGT CCTCTGTGAA
            1861 CTTGCTCAGG ACAAGGAAGC TGCAGAAGCT ATTGAAGCTG AGGGAGCCAC AGCTCCTCTG
            1921 ACAGAGTTAC TTCACTCTAG GAATGAAGGT GTGGCGACAT ATGCAGCTGC TGTTTTGTTC
            1981 CGAATGTCTG AGGACAAGCC ACAAGATTAC AAGAAACGGC TTTCAGTTGA GCTGACCAGC
            2041 TCTCTCTTCA GAACAGAGCC AATGGCTTGG AATGAGACTG CTGATCTTGG ACTTGATATT
            2101 GGTGCCCAGG GAGAACCCCT TGGATATCGC CAGGATGATC CTAGCTATCG TTCTTTTCAC
            2161 TCTGGTGGAT ATGGCCAGGA TGCCTTGGGT ATGGACCCCA TGATGGAACA TGAGATGGGT
            2221 GGCCACCACC CTGGTGCTGA CTATCCAGTT GATGGGCTGC CAGATCTGGG GCATGCCCAG
            2281 GACCTCATGG ATGGGCTGCC TCCAGGTGAC AGCAATCAGC TGGCCTGGTT TGATACTGAC
            2341 CTGTAA
CTNNB1      Protein Sequence
(SEQ ID     1 MATQADLMEL DMAMEPDRKA AVSHWQQQSY LDSGIHSGAT TTAPSLSGKG NPEEEDVDTS
NO:110)     61 QVLYEWEQGF SQSFTQEQVA DIDGQYAMTR AQRVRAAMFP ETLDEGMQIP STQFDAAHPT
            121 NVQRLAEPSQ MLKHAVVNLI NYQDDAELAT RAIPELTKLL NDEDQVVVNK AAVMVHQLSK
            181 KEASRHAIMR SPQMVSAIVR TMQNTNDVET ARCTAGTLHN LSHHREGLLA IFKSGGIPAL
            241 VKMLGSPVDS VLFYAITTLH NLLLHQEGAK MAVRLAGGLQ KMVALLNKTN VKFLAITTDC
            301 LQILAYGNQE SKLIILASGG PQALVNIMRT YTYEKLLWTT SRVLKVLSVC SSNKPAIVEA
            361 GGMQALGLHL TDPSQRLVQN CLWTLRNLSD AATKQEGMEG LLGTLVQLLG SDDINVVTCA
            421 AGILSNLTCN NYKNKMMVCQ VGGIEALVRT VLRAGDREDI TEPAICALRH LTSRHQEAEM
            481 AQNAVRLHYG LPVVVKLLHP PSHWPLIKAT VGLIRNLALC PANHAPLREQ GAIPRLVQLL
            541 VRAHQDTQRR TSMGGTQQQF VEGVRMEEIV EGCTGALHIL ARDVHNRIVI RGLNTIPLFV
            601 QLLYSPIENI QRVAAGVLCE LAQDKEAAEA IEAEGATAPL TELLHSRNEG VATYAAAVLF
            661 RMSEDKPQDY KKRLSVELTS SLFRTEPMAW NETADLGLDI GAQGEPLGYR QDDPSYRSFH
            721 SGGYGQDALG MDPMMEHEMG GHHPGADYPV DGLPDLGHAQ DLMDGLPPGD SNQLAWFDTD
            781 L
ERBB3       DNA Sequence
(SEQ ID     1 ATGAGGGCGA ACGACGCTCT GCAGGTGCTG GGCTTGCTTT TCAGCCTGGC CCGGGGCTCC
NO: 111)    61 GAGGTGGGCA ACTCTCAGGC AGTGTGTCCT GGGACTCTGA ATGGCCTGAG TGTGACCGGC
            121 GATGCTGAGA ACCAATACCA GACACTGTAC AAGCTCTACG AGAGGTGTGA GGTGGTGATG
            181 GGGAACCTTG AGATTGTGCT CACGGGACAC AATGCCGACC TCTCCTTCCT GCAGTGGATT
            241 CGAGAAGTGA CAGGCTATGT CCTCGTGGCC ATGAATGAAT TCTCTACTCT ACCATTGCCC
            301 AACCTCCGCG TGGTGCGAGG GACCCAGGTC TACGATGGGA AGTTTGCCAT CTTCGTCATG
            361 TTGAACTATA ACACCAACTC CAGCCACGCT CTGCGCCAGC TCCGCTTGAC TCAGCTCACC
            421 GAGATTCTGT CAGGGGGTGT TTATATTGAG AAGAACGATA AGCTTTGTCA CATGGACACA
            481 ATTGACTGGA GGGACATCGT GAGGGACCGA GATGCTGAGA TAGTGGTGAA GGACAATGGC
            541 AGAAGCTGTC CCCCCTGTCA TGAGGTTTGC AAGGGGCGAT GCTGGGGTCC TGGATCAGAA
            601 GACTGCCAGA CATTGACCAA GACCATCTGT GCTCCTCAGT GTAATGGTCA CTGCTTTGGG
            661 CCCAACCCCA ACCAGTGCTG CCATGATGAG TGTGCCGGGG GCTGCTCAGG CCCTCAGGAC
            721 ACAGACTGCT TTGCCTGCCG GCACTTCAAT GACAGTGGAG CCTGTGTACC TCGCTGTCCA
            781 CAGCCTCTTG TCTACAACAA GCTAACTTTC CAGCTGGAAC CCAATCCCCA CACCAAGTAT
            841 CAGTATGGAG GAGTTTGTGT AGCCAGCTGT CCCCATAACT TTGTGGTGGA TCAAACATCC
            901 TGTGTCAGGG CCTGTCCTCC TGACAAGATG GAAGTAGATA AAAATGGGCT CAAGATGTGT
            961 GAGCCTTGTG GGGGACTATG TCCCAAAGCC TGTGAGGGAA CAGGCTCTGG GAGCCGCTTC
            1021 CAGACTGTGG ACTCGAGCAA CATTGATGGA TTTGTGAACT GCACCAAGAT CCTGGGCAAC
            1081 CTGGACTTTC TGATCACCGG CCTCAATGGA GACCCCTGGC ACAAGATCCC TGCCCTGGAC
            1141 CCAGAGAAGC TCAATGTCTT CCGGACAGTA CGGGAGATCA CAGGTTACCT GAACATCCAG
            1201 TCCTGGCCGC CCCACATGCA CAACTTCAGT GTTTTTTCCA ATTTGACAAC CATTGGAGGC
            1261 AGAAGCCTCT ACAACCGGGG CTTCTCATTG TTGATCATGA AGAACTTGAA TGTCACATCT
            1321 CTGGGCTTCC GATCCCTGAA GGAAATTAGT GCTGGGCGTA TCTATATAAG TGCCAATAGG
            1381 CAGCTCTGCT ACCACCACTC TTTGAACTGG ACCAAGGTGC TTCGGGGGCC TACGGAAGAG
            1441 CGACTAGACA TCAAGCATAA TCGGCCGCGC AGAGACTGCG TGGCAGAGGG CAAAGTGTGT
            1501 GACCCACTGT GCTCCTCTGG GGGATGCTGG GGCCCAGGCC CTGGTCAGTG CTTGTCCTGT
            1561 CGAAATTATA GCCGAGGAGG TGTCTGTGTG ACCCACTGCA ACTTTCTGAA TGGGGAGCCT
            1621 CGAGAATTTG CCCATGAGGC CGAATGCTTC TCCTGCCACC CGGAATGCCA ACCCATGGAG
            1681 GGCACTGCCA CATGCAATGG CTCGGGCTCT GATACTTGTG CTCAATGTGC CCATTTTCGA
            1741 GATGGGCCCC ACTGTGTGAG CAGCTGCCCC CATGGAGTCC TAGGTGCCAA GGGCCCAATC
            1801 TACAAGTACC CAGATGTTCA GAATGAATGT CGGCCCTGCC ATGAGAACTG CACCCAGGGG
            1861 TGTAAAGGAC CAGAGCTTCA AGACTGTTTA GGACAAACAC TGGTGCTGAT CGGCAAAACC
            1921 CATCTGACAA TGGCTTTGAC AGTGATAGCA GGATTGGTAG TGATTTTCAT GATGCTGGGC
            1981 GGCACTTTTC TCTACTGGCG TGGGCGCCGG ATTCAGAATA AAAGGGCTAT GAGGCGATAC
            2041 TTGGAACGGG GTGAGAGCAT AGAGCCTCTG GACCCCAGTG AGAAGGCTAA CAAAGTCTTG
            2101 GCCAGAATCT TCAAAGAGAC AGAGCTAAGG AAGCTTAAAG TGCTTGGCTC GGGTGTCTTT
            2161 GGAACTGTGC ACAAAGGAGT GTGGATCCCT GAGGGTGAAT CAATCAAGAT TCCAGTCTGC
            2221 ATTAAAGTCA TTGAGGACAA GAGTGGACGG CAGAGTTTTC AAGCTGTGAC AGATCATATG
            2281 CTGGCCATTG GCAGCCTGGA CCATGCCCAC ATTGTAAGGC TGCTGGGACT ATGCCCAGGG
            2341 TCATCTCTGC AGCTTGTCAC TCAATATTTG CCTCTGGGTT CTCTGCTGGA TCATGTGAGA
            2401 CAACACCGGG GGGCACTGGG GCCACAGCTG CTGCTCAACT GGGGAGTACA AATTGCCAAG
            2461 GGAATGTACT ACCTTGAGGA ACATGGTATG GTGCATAGAA ACCTGGCTGC CCGAAACGTG
            2521 CTACTCAAGT CACCCAGTCA GGTTCAGGTG GCAGATTTTG GTGTGGCTGA CCTGCTGCCT
            2581 CCTGATGATA AGCAGCTGCT ATACAGTGAG GCCAAGACTC CAATTAAGTG GATGGCCCTT
            2641 GAGAGTATCC ACTTTGGGAA ATACACACAC CAGAGTGATG TCTGGAGCTA TGGTGTGACA
            2701 GTTTGGGAGT TGATGACCTT CGGGGCAGAG CCCTATGCAG GGCTACGATT GGCTGAAGTA
            2761 CCAGACCTGC TAGAGAAGGG GGAGCGGTTG GCACAGCCCC AGATCTGCAC AATTGATGTC
            2821 TACATGGTGA TGGTCAAGTG TTGGATGATT GATGAGAACA TTCGCCCAAC CTTTAAAGAA
            2881 CTAGCCAATG AGTTCACCAG GATGGCCCGA GACCCACCAC GGTATCTGGT CATAAAGAGA
            2941 GAGAGTGGGC CTGGAATAGC CCCTGGGCCA GAGCCCCATG GTCTGACAAA CAAGAAGCTA
            3001 GAGGAAGTAG AGCTGGAGCC AGAACTAGAC CTAGACCTAG ACTTGGAAGC AGAGGAGGAC
            3061 AACCTGGCAA CCACCACACT GGGCTCCGCC CTCAGCCTAC CAGTTGGAAC ACTTAATCGG
            3121 CCACGTGGGA GCCAGAGCCT TTTAAGTCCA TCATCTGGAT ACATGCCCAT GAACCAGGGT
            3181 AATCTTGGGG AGTCTTGCCA GGAGTCTGCA GTTTCTGGGA GCAGTGAACG GTGCCCCCGT
            3241 CCAGTCTCTC TACACCCAAT GCCACGGGGA TGCCTGGCAT CAGAGTCATC AGAGGGGCAT
            3301 GTAACAGGCT CTGAGGCTGA GCTCCAGGAG AAAGTGTCAA TGTGTAGGAG CCGGAGCAGG
            3361 AGCCGGAGCC CACGGCCACG CGGAGATAGC GCCTACCATT CCCAGCGCCA CAGTCTGCTG
            3421 ACTCCTGTTA CCCCACTCTC CCCACCCGGG TTAGAGGAAG AGGATGTCAA CGGTTATGTC
            3481 ATGCCAGATA CACACCTCAA AGGTACTCCC TCCTCCCGGG AAGGCACCCT TTCTTCAGTG
            3541 GGTCTCAGTT CTGTCCTGGG TACTGAAGAA GAAGATGAAG ATGAGGAGTA TGAATACATG
            3601 AACCGGAGGA GAAGGCACAG TCCACCTCAT CCCCCTAGGC CAAGTTCCCT TGAGGAGCTG
            3661 GGTTATGAGT ACATGGATGT GGGGTCAGAC CTCAGTGCCT CTCTGGGCAG CACACAGAGT
            3721 TGCCCACTCC ACCCTGTACC CATCATGCCC ACTGCAGGCA CAACTCCAGA TGAAGACTAT
            3781 GAATATATGA ATCGGCAACG AGATGGAGGT GGTCCTGGGG GTGATTATGC AGCCATGGGG
            3841 GCCTGCCCAG CATCTGAGCA AGGGTATGAA GAGATGAGAG CTTTTCAGGG GCCTGGACAT
            3901 CAGGCCCCCC ATGTCCATTA TGCCCGCCTA AAAACTCTAC GTAGCTTAGA GGCTACAGAC
            3961 TCTGCCTTTG ATAACCCTGA TTACTGGCAT AGCAGGCTTT TCCCCAAGGC TAATGCCCAG
            4021 AGAACGTAA
ERBB3       Protein Sequence
(SEQ ID     1 MRANDALQVL GLLFSLARGS EVGNSQAVCP GTLNGLSVTG DAENQYQTLY KLYERCEVVM
NO: 112)    61 GNLEIVLTGH NADLSFLQWI REVTGYVLVA MNEFSTLPLP NLRVVRGTQV YDGKFAIFVM
            121 LNYNTNSSHA LRQLRLTQLT EILSGGVYIE KNDKLCHMDT IDWRDIVRDR DAEIVVKDNG
            181 RSCPPCHEVC KGRCWGPGSE DCQTLTKTIC APQCNGHCFG PNPNQCCHDE CAGGCSGPQD
            241 TDCFACRHFN DSGACVPRCP QPLVYNKLTF QLEPNPHTKY QYGGVCVASC PHNFVVDQTS
            301 CVRACPPDKM EVDKNGLKMC EPCGGLCPKA CEGTGSGSRF QTVDSSNIDG FVNCTKILGN
            361 LDFLITGLNG DPWHKIPALD PEKLNVFRTV REITGYLNIQ SWPPHMHNFS VFSNLTTIGG
            421 RSLYNRGFSL LIMKNLNVTS LGFRSLKEIS AGRIYISANR QLCYHHSLNW TKVLRGPTEE
            481 RLDIKHNRPR RDCVAEGKVC DPLCSSGGCW GPGPGQCLSC RNYSRGGVCV THCNFLNGEP
            541 REFAHEAECF SCHPECQPME GTATCNGSGS DTCAQCAHFR DGPHCVSSCP HGVLGAKGPI
            601 YKYPDVQNEC RPCHENCTQG CKGPELQDCL GQTLVLIGKT HLTMALTVIA GLVVIFMMLG
            661 GTFLYWRGRR IQNKRAMRRY LERGESIEPL DPSEKANKVL ARIFKETELR KLKVLGSGVF
            721 GTVHKGVWIP EGESIKIPVC IKVIEDKSGR QSFQAVTDHM LAIGSLDHAH IVRLLGLCPG
            781 SSLQLVTQYL PLGSLLDHVR QHRGALGPQL LLNWGVQIAK GMYYLEEHGM VHRNLAARNV
            841 LLKSPSQVQV ADFGVADLLP PDDKQLLYSE AKTPIKWMAL ESIHFGKYTH QSDVWSYGVT
            901 VWELMTFGAE PYAGLRLAEV PDLLEKGERL AQPQICTIDV YMVMVKCWMI DENIRPTFKE
            961 LANEFTRMAR DPPRYLVIKR ESGPGIAPGP EPHGLTNKKL EEVELEPELD LDLDLEAEED
            1021 NLATTTLGSA LSLPVGTLNR PRGSQSLLSP SSGYMPMNQG NLGESCQESA VSGSSERCPR
            1081 PVSLHPMPRG CLASESSEGH VTGSEAELQE KVSMCRSRSR SRSPRPRGDS AYHSQRHSLL
            1141 TPVTPLSPPG LEEEDVNGYV MPDTHLKGTP SSREGTLSSV GLSSVLGTEE EDEDEEYEYM
            1201 NRRRRHSPPH PPRPSSLEEL GYEYMDVGSD LSASLGSTQS CPLHPVPIMP TAGTTPDEDY
            1261 EYMNRQRDGG GPGGDYAAMG ACPASEQGYE EMRAFQGPGH QAPHVHYARL KTLRSLEATD
            1321 SAFDNPDYWH SRLFPKANAQ RT
GNAS        DNA Sequence
(SEQ ID     1 ATGGGCTGCC TCGGGAACAG TAAGACCGAG GACCAGCGCA ACGAGGAGAA GGCGCAGCGT
NO: 113)    61 GAGGCCAACA AAAAGATCGA GAAGCAGCTG CAGAAGGACA AGCAGGTCTA CCGGGCCACG
            121 CACCGCCTGC TGCTGCTGGG TGCTGGAGAA TCTGGTAAAA GCACCATTGT GAAGCAGATG
            181 AGGATCCTGC ATGTTAATGG GTTTAATGGA GAGGGCGGCG AAGAGGACCC GCAGGCTGCA
            241 AGGAGCAACA GCGATGGTGA GAAGGCAACC AAAGTGCAGG ACATCAAAAA CAACCTGAAA
            301 GAGGCGATTG AAACCATTGT GGCCGCCATG AGCAACCTGG TGCCCCCCGT GGAGCTGGCC
            361 AACCCCGAGA ACCAGTTCAG AGTGGACTAC ATCCTGAGTG TGATGAACGT GCCTGACTTT
            421 GACTTCCCTC CCGAATTCTA TGAGCATGCC AAGGCTCTGT GGGAGGATGA AGGAGTGCGT
            481 GCCTGCTACG AACGCTCCAA CGAGTACCAG CTGATTGACT GTGCCCAGTA CTTCCTGGAC
            541 AAGATCGACG TGATCAAGCA GGCTGACTAT GTGCCGAGCG ATCAGGACCT GCTTCGCTGC
            601 CGTGTCCTGA CTTCTGGAAT CTTTGAGACC AAGTTCCAGG TGGACAAAGT CAACTTCCAC
            661 ATGTTTGACG TGGGTGGCCA GCGCGATGAA CGCCGCAAGT GGATCCAGTG CTTCAACGAT
            721 GTGACTGCCA TCATCTTCGT GGTGGCCAGC AGCAGCTACA ACATGGTCAT CCGGGAGGAC
            781 AACCAGACCA ACCGCCTGCA GGAGGCTCTG AACCTCTTCA AGAGCATCTG GAACAACAGA
            841 TGGCTGCGCA CCATCTCTGT GATCCTGTTC CTCAACAAGC AAGATCTGCT CGCTGAGAAA
            901 GTCCTTGCTG GGAAATCGAA GATTGAGGAC TACTTTCCAG AATTTGCTCG CTACACTACT
            961 CCTGAGGATG CTACTCCCGA GCCCGGAGAG GACCCACGCG TGACCCGGGC CAAGTACTTC
            1021 ATTCGAGATG AGTTTCTGAG GATCAGCACT GCCAGTGGAG ATGGGCGTCA CTACTGCTAC
            1081 CCTCATTTCA CCTGCGCTGT GGACACTGAG AACATCCGCC GTGTGTTCAA CGACTGCCGT
            1141 GACATCATTC AGCGCATGCA CCTTCGTCAG TACGAGCTGC TCTAA
GNAS        Protein Sequence
(SEQ ID     1 MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKSTIVKQM
NO: 114)    61 RILHVNGFNG EGGEEDPQAA RSNSDGEKAT KVQDIKNNLK EAIETIVAAM SNLVPPVELA
            121 NPENQFRVDY ILSVMNVPDF DFPPEFYEHA KALWEDEGVR ACYERSNEYQ LIDCAQYFLD
            181 KIDVIKQADY VPSDQDLLRC RVLTSGIFET KFQVDKVNFH MFDVGGQRDE RRKWIQCFND
            241 VTAIIFVVAS SSYNMVIRED NQTNRLQEAL NLFKSIWNNR WLRTISVILF LNKQDLLAEK
            301 VLAGKSKIED YFPEFARYTT PEDATPEPGE DPRVTRAKYF IRDEFLRIST ASGDGRHYCY
            361 PHFTCAVDTE NIRRVFNDCR DIIQRMHLRQ YELL
CRC Erd     DNA Sequence
construct 1 1 ATGACCACCA TCCACTACAA CTACATGTGC AACAGCAGCT GCATGGGCAG CATGAACTGG
insert      61 CGGCCTATCC TGACCATCAT CACCCTGGAA GATAGCCGGG GCAGAAAGCG GAGAAGCGTG
(SEQ ID     121 GCCATGAACG AGTTCAGCAC ACTGCCCCTG CCTAACCTGA GAATGGTTCG AGGCACCCAG
MD: 115)    181 GTGTACGACG GCAAGTTCGC CATCTTTGTG CGCGGCAGAA AGAGGCGGAG CTACCTGGAT
            241 TCTGGCATCC ACTCTGGCGC TACAACAACA GCCCCATTCC TGAGCGGCAA GGGCAACCCC
            301 GAAGAGGAAG ATGTGGATAC CAGCAGAGGC CGGAAGAGAA GATCCGACGT GGAAACCGGC
            361 AACTGCATCC ACACACTGAC AGGCCACCAG CTGCTGACCT CTGGCATGGA ACTGAAGGAC
            421 AACATCCTGG TGTCCGGCAG AGGAAGAAAG CGCAGATCTA CCGGCGAGTG CATTCACACC
            481 CTGTATGGCC ACACCAGCAC CGTGCACTGC ATGCATCTGC ACGAGAAGAG AGTGGTGTCT
            541 GGCAGCAGAG ACAGAGGACG CAAGCGGAGA TCCGAGCAAG AGGCCCTGGA ATACTTTATG
            601 AAGCAGATCA ACGACGCCTA CCACGGCGGC TGGACTACCA AGATGGACTG GATCTTCCAC
            661 ACCATCCGCG GACGCAAGAG AAGAAGCGTG ACACAAGAGG CCGAGCGGGA AGAGTTCTTC
            721 GACGAGACAA GACAGCTGTG CGACCTGCGG CTGTTCCAGC CTTTCCTGAA AGTGATCGAG
            781 CGCGGACGGA AAAGACGGTC CACCGAGTAT AAGCTGGTGG TCGTGGGAGC TTGTGGCGTG
            841 GGAAAAAGCG CCCTGACAAT CCAGCTGATC CAGAACCACT TCGTGCGGGG AAGAAAACGG
            901 CGGAGCATGG CCATCTACAA GCAGAGCCAG CACATGACCG AGGTCGTGCG GCACTGTCCT
            961 CACCACGAGA GATGTAGCGA TAGCGACGGA CTGGCCCCTT GATGA
CRC DM      Protein Sequence*
construct 1 1 MTTIHYNYMC NSSCMGSMNW RPILTIITLE DSRGRKRRSV AMNEFSTLPL PNLRMVRGTQ
insert      61 VYDGKFAIFV RGRKRRSYLD SGIHSGATTT APFLSGKGNP EEEDVDTSRG RKRRSDVETG
(SEQ ID     121 NCIHTLTGHQ LLTSGMELKD NILVSGRGRK RRSTGECIHT LYGHTSTVHC MHLHEKRVVS
NO: 116)    181 GSRDRGRKRR SEQEALEYFM KQINDAYHGG WTTKMDWIFH TIRGRKRRSV TQEAEREEFF
            241 DETRQLCDLR LFQPFLKVIE RGRKRRSTEY KLVVVGACGV GKSALTIQLI QNHFVRGRKR
            301 RSMAIYKQSQ HMTEVVRHCP HHERCSDSDG LAP
CRC DM      DNA Sequence
construct 2 1 ATGGAAGATA GCAGCGGCAA TCTGCTGGGC AGAAACAGCT TCGAAGTGTG CGTGTGTGCC
insert      61 TGTCCTGGCA GAGACAGAAG AACCGAGGAA GAGAACCGGG GCAGAAAGCG GAGAAGCGAC
(SEQ ID     121 AAAGAGCAGC TGAAGGCCAT CAGCACCAGA GATCCTCTGA GCAAGATCAC AGAGCAAGAG
NO: 117)    181 AAGGACTTCC TGTGGTCCCA CCGGCACTAC AGAGGCCGGA AGAGAAGATC TACCGGCCAG
            241 TGTCTGCACG TCCTGATGGG ACATGTGGCC GCCGTGTGTT GCGTGCAGTA CGATGGCAGA
            301 AGAGTGGTTT CCGGCGCCTA CGACAGAGGA AGAAAAAGGC GGTCCCCTAT CGTGACCGTG
            361 GACGGCTATG TTGATCCCTC TGGCGGCGAT CACTTCTGCC TGGGCCAGCT GTCTAACGTG
            421 CACAGAACCG AAGCCATCAG AGGACGGAAG CGGAGATCCG AGATCAGCCA CATCGGCAGC
            481 AGAGGCAAGT ACAGCAGCGG CTTCTGCAAT ATCGCCGTGA AAGAGAACCT GATCGAACTG
            541 ATGGCCGACA TCAGAGGTAG AAAGCGGCGG AGCAAGCAGG CCGATTACGT GCCATCTGAC
            601 CAGGACCTGC TGAGATGCCA CGTGCTGACC AGCGGCATCT TCGAGACAAA GTTCCAGGTG
            661 GACAAGTGAT GA
CRC DM      Protein Sequence*
construct 2 1 MEDSSGNLLG RNSFEVCVCA CPGRDRRTEE ENRGRKRRSD KEQLKAISTR DPLSKITEQE
insert      61 KDFLWSHRHY RGRKRRSTGQ CLHVLMGHVA AVCCVQYDGR RVVSGAYDRG RKRRSPIVTV
(SEQ ID     121 DGYVDPSGGD HFCLGQLSNV HRTEAIRGRK RRSEISHIGS RGKYSSGFCN IAVKENLIEL
NO: 118)    181 MADIRGRKRR SKQADYVPSD QDLLRCHVLT SGIFETKFQV DK
*Driver mutation is highlighted in bold.
The furin cleavage sequence is underlined.

Immune Responses to Driver Mutations Induced by the CRC Vaccine-B RKO Cell Line (CRC Construct 1 SEQ ID NO: 116))

CRC vaccine-B cell line RKO modified to reduce expression of CD276 and TGFβ1, and express GM-CSF, membrane bound CD40L, IL-12 was transduced with lentiviral particles expressing to three TP53 driver mutations, one KRAS driver mutation, three PIK3CA driver mutations, two FBXW7 driver mutations, one CTNNB1 driver mutation and one ERBB3 driver mutations encoded by nine peptide sequences separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37) as described above.

Immune responses to the inserted TP53, KRAS, PIK3CA, FBXW7, CTNNB1 and ERBB3 driver mutations were evaluated by IFNγ ELISpot as described above and herein. Specifically, 1.5×10⁶ of unmodified RKO or RKO modified to express driver mutations peptides were co-cultured with 1.5×10⁶ iDCs from six HLA diverse donors (n=4/donor). HLA-A, HLA-B, and HLA-C alleles for each of the six donors are in Table 5-20. Peptides, 15-mers overlapping by 9 amino acids, were designed to cover the full amino acid sequences of the twelve individual driver mutations peptides. Only the 15-mer peptides containing the mutations were used to stimulate PBMCs in the IFNγ ELISpot assay.

TABLE 5-20
Donor MHC-I HLA Alleles
	Donor #	HLA-A	HLA-B	HLA-C
	1	*02:01 *33:01	*07:02 14:02	*07:02 *08:02
	2	*03:01 *25:01	*15:01 44:02	*03:03 *05:01
	3	*02:01 *25:01	*18:01 *44:03	*12:03 *06:01
	4	*03:01 *11:01	*18:01 *51:01	*06:02 *07:01
	5	*01:01 *03:01	*07:02 *44:02	*05:01 *07:02
	6	*03:01 *31:01	*35:01 *40:01	*04:01 *07:02

FIG. 19 demonstrates immune responses against nine driver mutation encoding peptides expressed by the CRC vaccine-B RKO cell line for six HLA-diverse donors by IFNγ ELISpot. CRC vaccine-B RKO induced IFNγ responses against all inserted driver mutation encoding peptides greater in magnitude relative to the unmodified RKO cell line (Table 5-21). The magnitude of IFNγ responses induced by CRC vaccine-B RKO cell line significantly increased against the inserted driver mutation peptides encoding TP53 G245S R248W (p=0.015), ERBB3 V104M (p=0.035), and FBXW7 R465H (p=0.022) compared to the unmodified RKO cell line. Statistical significance was determined using the Mann-Whitney U test.

TABLE 5-21
Immune responses to TP53, KRAS, PIK3CA, FBXW7, CTNNB1 and ERBB3 driver mutations expressed by the CRC vaccine-B RKO cell line
CRC
Driver		TP53					PIK3CA
Muta-	TP53	G245S	ERBB3	CTNNB1	FBXW7	FBXW7	M1043I	PIK3CA	KRAS
tion	R175H	R248W	V216M	S45F	S582L	R465H	H1047Y	R88Q	G12C
Unmodified RKO (SFU ± SEM)
Donor 1	50 ± 30	80 ± 57	150 ± 53	0 ± 0	90 ± 77	60 ± 35	0 ± 0	100 ± 26	0 ± 0
Donor 2	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 3	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 4	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 5	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 6	0 ± 0	0 ± 0	50 ± 19	0 ± 0	50 ± 38	70 ± 34	70 ± 37	0 ± 0	0 ± 0
Average	8 ± 8	13 ± 13	33 ± 25	0 ± 0	23 ± 16	22 ± 14	12 ± 12	17 ± 17	0 ± 0
Modified RKO (SFU ± SEM)
Donor 1	350 ± 311	1,950 ± 595	1,330 ± 804	0 ± 0	660 ± 491	1,150 ± 461	500 ± 22	840 ± 788	1,160 ± 1,056
Donor 2	0 ± 0	1,413 ± 1,033	0 ± 0	900 ± 520	1,343 ± 696	1,035 ± 922	1,228 ± 583	0 ± 0	0 ± 0
Donor 3	0 ± 0	1,178 ± 609	2,785 ± 932	2,143 ± 1,150	0 ± 0	1,140 ± 661	0 ± 0	0 ± 0	0 ± 0
Donor 4	495 ± 314	785 ± 469	1,610 ± 1,131	0 ± 0	0 ± 0	1,148 ± 446	1,440 ± 833	288 ± 167	0 ± 0
Donor 5	0 ± 0	0 ± 0	85 ± 59	295 ± 210	0 ± 0	0 ± 0	0 ± 0	565 ± 365	315 ± 224
Donor 6	0 ± 0	3,565 ± 1,535	2,790 ± 1,322	2,710 ± 1,204	3,860 ± 1,467	2,480 ± 1,248	2,800 ± 1,232	0 ± 0	0 ± 0
Average	141 ± 91	1,582 ± 492	1,433 ± 503	1,008 ± 474	977 ± 617	1,159 ± 322	995 ± 437	282 ± 145	246 ± 190

Immune Responses to Driver Mutations Induced by the CRC Vaccine-A HuTu80 Cell Line (CRC Construct 2 SEQ ID NO: 118))

Immune responses to six driver mutation encoding peptides expressed by CRC vaccine-A cell line HuTu80 were determined for six HLA-diverse donors (Table 5-20) by IFNγ ELISpot. CRC vaccine-A HuTu80 induced IFNγ responses against all inserted driver mutation encoding peptides greater in magnitude relative to unmodified HuTu80. FIG. 20 describes immune responses against the six driver mutation encoding peptides inserted into CRC vaccine-A cell line HuTu80 induced IFNγ responses against all inserted driver mutation encoding peptides greater in magnitude relative to the unmodified HuTu80 cell line The magnitude of IFNγ responses induced by CRC vaccine-A HuTu80 cell line significantly increased against the inserted driver mutation peptides encoding TP53 R273C (p=0.013) and GNAS R201H (p=0.028) compared to the unmodified HuTu80 cell line (Table 5-22). Statistical significance was determined using the Mann-Whitney U test.

TABLE 5-22
Immune responses to TP53, PIK3CA, FBXW7, SMAD4, ATM and GNAS driver
mutations expressed by the CRC vaccine-A Hutu80 cell line
CRC
Driver
Muta-	TP53	PIK3CA	FBXW7	SMAD4	ATM	GNAS
tion	R273C	E542K	R505C	R361H	R337C	R201H
Unmodified HuTu80 (SFU ± SEM)
Donor 1	0 ± 0	180 ± 74	170 ± 87	170 ± 62	190 ± 164	210 ± 91
Donor 2	0 ± 0	65 ± 38	0 ± 0	0 ± 0	43 ± 17	0 ± 0
Donor 3	275 ± 161	195 ± 123	0 ± 0	488 ± 405	0 ± 0	115 ± 68
Donor 4	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 5	70 ± 41	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 6	310 ± 254	53 ± 24	110 ± 72	190 ± 117	0 ± 0	110 ± 64
Average	109 ± 59	82 ± 35	47 ± 31	141 ± 78	39 ± 31	73 ± 36
Modified HuTu80 (SFU ± SEM)
Donor 1	1,270 ± 579	1,100 ± 385	690 ± 323	700 ± 356	1,700 ± 228	790 ± 335
Donor 2	900 ± 340	155 ± 95	0 ± 0	0 ± 0	0 ± 0	400 ± 288
Donor 3	1,708 ± 623	1,745 ± 1,323	735 ± 437	0 ± 0	0 ± 0	1,133 ± 568
Donor 4	60 ± 42	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Donor 5	1,090 ± 402	910 ± 308	420 ± 247	0 ± 0	270 ± 257	495 ± 422
Donor 6	1,090 ± 180	1,140 ± 239	0 ± 0	640 ± 262	725 ± 446	970 ± 345
Average	1,020 ± 222	842 ± 268	308 ± 144	223 ± 141	449 ± 276	631 ± 169

Genetic modifications completed for CRC vaccine-A and CRC vaccine-B cell lines are described in Table 5-23 below and herein. The CD276 gene was knocked out (KO) by electroporation of zinc-finger nucleases (ZFN) (SEQ ID NO: 52) as described above. All other genetic modifications were completed by lentiviral transduction.

CRC Vaccine-A

HCT-15 (ATCC, CCL-225) is modified to reduce expression of CD276 (SEQ ID NO: 52), knockdown (KD) secretion of transforming growth factor-beta 1 (TGFβ1) (SEQ ID NO: 54), and to express granulocyte macrophage-colony stimulating factor (GM-CSF) (SEQ ID NO: 7, SEQ ID NO: 8), membrane-bound CD40L (mCD40L) (SEQ ID NO: 2, SEQ ID NO: 3), interleukin 12 p70 and (IL-12) (SEQ ID NO: 9, SEQ ID NO: 10);

HuTu80 (ATCC, HTB-40) is modified to reduce expression of CD276 (SEQ ID NO: 52), reduce secretion of TGFβ1 (SEQ ID NO: 54) and transforming growth factor-beta 1 (TGFβ2) (SEQ ID NO: 55), and express GM-CSF (SEQ ID NO: 8), membrane bound CD40L (SEQ ID NO: 2, SEQ ID NO: 3), IL-12 (SEQ ID NO: 9, SEQ ID NO: 10), modPSMA (SEQ ID NO: 29, SEQ ID NO: 30); and express peptides containing TP53 driver mutation R273C, PIK3CA driver mutation E542K, SMAD4 driver mutation R361H, GNAS driver mutation R201H, FBXW7 driver mutation R505C, and ATM driver mutation R337C (SEQ ID NO: 117, SEQ ID NO: 118);

LS411N (ATCC, CRL-2159) is modified to reduce expression of CD276 (SEQ ID NO: 52), reduced secretion of TGFβ1 (SEQ ID NO: 54) and express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), membrane bound CD40L (SEQ ID NO: 3, SEQ ID NO: 4), IL-12 (SEQ ID NO: 9, SEQ ID NO: 10).

CRC Vaccine-B

HCT-116 (ATCC, CCL-247) modified to reduced expression of CD276 (SEQ ID NO: 52), reduce secretion of TGFβ1 (SEQ ID NO: 54), and express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), membrane bound CD40L (SEQ ID NO: 2, SEQ ID NO: 3), IL-12 (SEQ ID NO: 9, SEQ ID NO: 10), modTBXT (SEQ ID NO: 17, SEQ ID NO: 18), modWT1 (SEQ ID NO: 17, SEQ ID NO: 18), and peptides comprising one or more KRAS (SEQ ID NO: 17, SEQ ID NO: 18) driver mutations selected from the group consisting of G12D and G12V;

RKO (ATCC, CRL-2577) modified to reduce expression of CD276 (SEQ ID NO: 52), reduce secretion of TGFβ1 (SEQ ID NO: 54), and express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), membrane bound CD40L (SEQ ID NO: 2, SEQ ID NO: 3), IL-12 (SEQ ID NO: 9, SEQ ID NO: 10), and express peptides containing TP53 driver mutations selected from the group consisting R175H, G245S, and R248W, KRAS driver mutation G12C, PIK3CA driver mutations selected from the group consisting of R88Q, M1043I, and H1047Y, FBXW7 driver mutations selected from the group consisting of S582L and R465H, CTNNB1 driver mutation S45F, and ERBB3 driver mutation V104M (SEQ ID NO: 115, SEQ ID NO: 116);

DMS 53 (ATCC, CRL-2062) modified to reduce expression of CD276 (SEQ ID NO: 52), reduce secretion of TGFβ1 (SEQ ID NO: 54) and TGFβ2 (SEQ ID NO: 55), and to express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), membrane bound CD40L (SEQ ID NO: 2, SEQ ID NO: 3) and IL-12 (SEQ ID NO: 9, SEQ ID NO: 10).

TABLE 5-23
Colorectal cancer vaccine cell line nomenclature and genetic modifications
								Tumor-
								Associated
	Cell	CD276	TGFβ1	TGFβ2				Antigens	Driver
Cocktail	Line	KO	KD	KD	GM-CSF	mCD40L	IL-12	(TAAs)	Mutations
A	HCT-15	SEQ ID	SEQ ID	—	SEQ ID	SEQ ID	SEQ ID	—	—
		NO: 52	NO: 54		NO: 8	NO: 3	NO: 10
A	HuTu80	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	modPSMA	TP53, PIK3CA,
		NO: 52	NO: 54	NO: 55	NO: 8	NO: 3	NO: 10	(SEQ ID	FBXW7, SMAD4,
								NO: 30)	GNAS, ATM
									(SEQ ID
									NO: 118)
A	LS411N	SEQ ID	SEQ ID	—	SEQ ID	SEQ ID	SEQ ID	—	—
		NO: 52	NO: 54		NO: 8	NO: 3	NO: 10
B	HCT-116	SEQ ID	SEQ ID	—	SEQ ID	SEQ ID	SEQ ID	modTBXT	KRAS
		NO: 52	NO: 54		NO: 8	NO: 3	NO: 10	modWT1	(SEQ ID
								(SEQ ID	NO: 24,
								NO: 18)	SEQ ID
									NO: 26 and
									SEQ ID
									NO: 18)
B	RKO	SEQ ID	SEQ ID		SEQ ID	SEQ ID	SEQ ID	—	TP53, KRAS,
		NO: 52	NO: 54		NO: 8	NO: 3	NO: 10		PIK3CA, FBXW7,
									CTNNB1, ERBB3
									(SEQ ID
									NO: 116)
B	DMS 53*	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	—	—
		NO: 52	NO: 54	NO: 55	NO: 8	NO: 3	NO: 10
—, not completed/not required.
*Cell line identified as CSC-like. mCD40L, membrane bound CD40L.

Example 6: Breast Cancer Vaccine (BRC) Preparation

Example 6 demonstrates reduction of TGFβ1, TGFβ2, and CD276 expression with concurrent introduction of GM-CSF, membrane bound CD40L, and IL-12 expression in a vaccine composition of two cocktails, each cocktail composed of three cell lines for a total of 6 cell lines, significantly increased the magnitude of cellular immune responses against at least ten BRC-associated antigens in an HLA-diverse population. Example 6 also describes the process for identification, selection, and design of driver mutations expressed by BRC patient tumors. As described here in, expression of peptides encoding these mutations in certain cell lines of the of the BRCA vaccine also generate potent immune responses in an HLA diverse population.

As described herein, the first cocktail, BRC vaccine-A, is composed of cell line CAMA-1 also modified to express modPSMA, cell line AU565 also modified to express modTERT, and peptides encoding three TP53 driver mutations and four PIK3CA driver mutations, and cell line HS-578T. The second cocktail, BRC vaccine-B, is composed of cell line MCF-7, cell line T47D also modified to express modTBXT and modBORIS, and cell line DMS 53.

The six component cell lines collectively express at least twenty-two full-length antigens and nine driver mutations that can provide an anti-BRC tumor response. Table 6-23, below, provides a summary of each cell line and the modifications associated with each cell line.

Identification of BRC Vaccine Components

Example 36 of WO/2021/113328 first described identification and selection of the cell lines comprising the BRC vaccine described herein. BRC vaccine cell lines were selected to express a wide array of TAAs, including those known to be important specifically for BRC anti-tumor responses, such as mammaglobin A (SCGB2A2) and MUC1, enriched in TNBC, such as TBXT and NY-ESO-1, and TAAs known to be important antigen targets for BRC and other solid tumors, such TERT. Identification of twenty-two BRC prioritized antigens (FIG. 21A) was completed as described in Example 40 of WO/2021/113328. Expression of TAAs by vaccine cell lines was determined using RNA expression data sourced from the Broad Institute Cancer Cell Line Encyclopedia (CCLE). The HGNC gene symbol was included in the CCLE search and mRNA expression was downloaded for each TAA. Expression of a TAA by a cell line was considered positive if the RNA-seq value was >1.0. The six component cell lines endogenously expressed seven to fifteen prioritized TAAs (FIG. 21A).

As shown herein, to further enhance antigenic breadth, BRC vaccine-A cell line CAMA-1 was modified to express modPSMA, BRC vaccine-A cell line AU565 was modified to express modTERT, and BRC vaccine-B cell line T47D was modified to express modTBXT and modBORIS. Identification and design of the antigen sequences inserted by lentiviral transduction into the BRC vaccine was completed as described in Example 40 of WO/2021/113328. TBXT and BORIS were not endogenously expressed in any of the six component cell lines at >1.0 FPKM. TERT and PSMA were endogenously expressed by one of the six component cell lines at >1.0 FPKM (FIG. 21A).

Expression of transduced antigens modPSMA (SEQ ID NO: 29; SEQ ID NO: 30) (FIG. 22A) by CAMA-1, modTERT (SEQ ID NO: 27; SEQ ID NO: 28) (FIG. 22B) by AU565, and modTBXT (SEQ ID NO: 33; SEQ ID NO: 34) (FIG. 22C) and modBORIS (SEQ ID NO: 33; SEQ ID NO: 34) (FIG. 22D) by T47D, were confirmed by flow cytometry or RT-PCR as described in Example 3 and herein. modTBXT and modBORIS are encoded in the same lentiviral transfer vector separated by a furin cleavage site (SEQ ID NO: 37).

The BRC vaccine, after introduction of genes encoding the antigens described above by lentiviral transduction, expresses twenty-two prioritized TAAs capable of inducing a BRC antitumor response. RNA abundance of the twenty-two prioritized BRC TAAs was determined in 1082 non-redundant BRC patient samples with available mRNA expression data downloaded from the publicly available database, cBioPortal (cbioportal.org) (Cerami, E. et al. Cancer Discovery. 2012.; Gao, J. et al. Sci Signal. 2013.). Fifteen BRC TAAs were expressed by 100% of samples, 16 TAAs were expressed by 99.9% of samples, 17 TAAs were expressed by 99.3% of samples, 18 TAAs were expressed by 95.1% of samples, 19 TAAs were expressed by 79.9% of samples, 20 TAAs were expressed by 47.6% of samples, 21 TAAs were expressed by 17.1% of samples, and 22 TAAs were expressed by 3.4% of samples (FIG. 21B).

To maintain maximal heterogeneity of antigens and clonal subpopulations that comprise individual cell lines, gene modified cell lines utilized in the present vaccine were established using lentiviral transduction with antibiotic selection and flow cytometric sorting, and not through limiting dilution subcloning.

Provided herein are two compositions of three cancer cell lines, wherein the combination of the cell lines, a unit dose of six cell lines, that expresses at least 15 TAAs associated with BRC cancer subjects intended to receive said composition. The cell lines in Table 6-1 comprise the BRC vaccine described herein.

TABLE 6-1
Breast vaccine cell lines and histology
	Cell Line
Cocktail	Name	Histology
A	CAMA-1	Breast Luminal A Adenocarcinoma, ER+, PR+,
		Her2−; derived from metastatic site
		(pleural effusion)
A	AU565	Breast Luminal Adenocarcinoma, ER−, PR−,
		Her2+; derived from metastatic site
		(pleural effusion)
A	HS-578T	Breast Triple Negative Ductal Carcinoma,
		ER−, PR−, Her2−
B	MCF-7	Breast Luminal A Adenocarcinoma, ER+, PR+,
		Her2; derived from metastatic site
		(pleural effusion)
B	T47D	Breast Luminal A Ductal Carcinoma, ER+, PR+,
		Her2; derived from metastatic site
		(pleural effusion)
B	DMS 53	Lung Small Cell Carcinoma

Reduction of CD276 Expression

Unmodified parental CAMA-1, AU565, HS-578T, MCF-7, T47D, and DMS 53 cell lines expressed CD276. Expression of CD276 was knocked out by electroporation with a zinc finger nuclease (ZFN) pair specific for CD276 targeting the genomic DNA sequence: GGCAGCCCTGGCATGggtgtgCATGTGGGTGCAGCC. (SEQ ID NO: 52). Following ZFN-mediated knockout of CD276, the cell lines were surface stained with PE α-human CD276 antibody (BioLegend, clone DCN.70) and full allelic knockout cells were enriched by cell sorting (BioRad S3e Cell Sorter). Sorted cells were plated in an appropriately sized vessel, based on the number of recovered cells, and expanded in culture. After cell enrichment for full allelic knockouts, cells were passaged 2-5 times and CD276 knockout percentage determined by flow cytometry. Expression of CD276 was determined by extracellular staining of CD276 modified and unmodified parental cell lines with PE α-human CD276 (BioLegend, clone DCN.70). Unstained cells and isotype control PE α-mouse IgG1 (BioLegend, clone MOPC-21) stained parental and CD276 KO cells served as controls. To determine the percent reduction of CD276 expression in the modified cell line, the MFI of the isotype control was subtracted from recorded MFI values of both the parental and modified cell lines. Percent reduction of CD276 expression is expressed as: (1-(MFI of the CD276KO cell line/MFI of the parental))×100). Reduction of CD276 expression by BRC vaccine cell lines is described in Table 6-2. The data demonstrate gene editing of CD276 with ZFNs resulted in greater than 95.2% CD276-negative cells in all six vaccine component cell lines.

TABLE 6-2
Reduction of CD276 expression
	Unmodified Cell	Modified Cell	% Reduction
Cell line	Line MFI	Line MFI	CD276
CAMA-1	14,699	75	99.5
AU565	4,085	0	100
HS-578T	33,832	234	99.3
MCF-7	25,952	1,243	95.2
T47D	11,737	3	≥99.9
DMS 53	4,479	0	100
MFI is reported with isotype controls subtracted

Cytokine Secretion Assays for TGFβ1, TGFβ2, GM-CSF, and IL-12

Cell lines were X-ray irradiated at 100 Gy prior to plating in 6-well plates at 2 cell densities (5.0e⁵ and 7.5e⁵) in duplicate. The following day, cells were washed with PBS and the media was changed to Secretion Assay Media (Base Media+5% CTS). After 48 hours, media was collected for ELISAs. The number of cells per well was counted using the Luna cell counter (Logos Biosystems). Total cell count and viable cell count were recorded. The secretion of cytokines in the media, as determined by ELISA, was normalized to the average number of cells plated in the assay for all replicates.

TGFβ1 secretion was determined by ELISA according to manufacturers instructions (Human TGFβ1 Quantikine ELISA, R&D Systems #SB100B). Four dilutions were plated in duplicate for each supernatant sample. If the results of the ELISA assay were below the LLD, the percentage decrease relative to parental cell lines was estimated by the number of cells recovered from the assay and the lower limit of detection, 15.4 pg/mL. If TGFβ1 was detected in >2 samples or dilutions the average of the positive values was reported with the n of samples run.

TGFβ2 secretion was determined by ELISA according to manufacturers instructions (Human TGFβ2 Quantikine ELISA, R&D Systems # SB250). Four dilutions were plated in duplicate for each supernatant sample. If the results of the ELISA assay were below the LLD, the percentage decrease relative to parental cell lines was estimated by the number of cells recovered from the assay and the lower limit of detection, 7.0 pg/mL. If TGFβ2 was detected in >2 samples or dilutions the average of the positive values was reported with the n of samples run.

GM-CSF secretion was determined by ELISA according to manufacturers instructions (GM-CSF Quantikine ELISA, R&D Systems #SGM00). Four dilutions were plated in duplicate for each supernatant sample. If the results of the ELISA assay were below the LLD, the percentage increase relative to parental cell lines was estimated by the number of cells recovered from the assay and the lower limit of detection, 3.0 pg/mL. If GM-CSF was detected in >2 samples or dilutions the average of the positive values was reported with the n of samples run.

IL-12 secretion was determined by ELISA according to manufacturer's instructions (LEGEND MAX Human IL-12 (p70) ELISA, Biolegend #431707). Four dilutions were plated in duplicate for each supernatant sample. If the results of the ELISA assay were below the LLD, the percentage increase was estimated by the number of cells recovered from the assay and the lower limit of detection, 1.2 pg/mL. If IL-12 was detected in >2 samples or dilutions the average of the positive values was reported with the n of samples run.

shRNA Downregulates TGF-β Secretion

After reduction of CD276 expression, secretion TGFβ1 and TGFβ2 were reduced by lentiviral transduction of TGFβ1 and/or TGFβ2 shRNA. TGFβ1 and TGFβ2 secretion levels were determined as described above. BRC vaccine-A cell lines AU565 and HS-578T secreted measurable levels of TGFβ1 and TGFβ2. BRC-vaccine-A cell line AU565 secreted relatively low levels of TGFβ1. BRC vaccine-A cell line CAMA-1 secreted detectable levels of TGFβ2 but not TGFβ1. BRC vaccine-B cell lines MCF-7 and DMS 53 secreted measurable levels of TGFβ1 and TGFβ2. T47D did not secret measurable levels of TGFβ1 or TGFβ2 and therefore was not modified to reduce TGFβ1 or TGFβ2.

HS-578T and MCF-7 cell lines were first transduced with the lentiviral particles encoding both TGFβ1 shRNA (shTGFβ1, mature antisense sequence: TTTCCACCATTAGCACGCGGG (SEQ ID NO: 54) and the gene for expression of membrane bound CD40L (SEQ ID NO: 2, SEQ ID NO: 3) under the control of a different promoter. This allowed for simultaneous reduction of TGFβ1 and introduction of expression of membrane bound CD40L. HS-578T and MCF-7 were then transduced with lentiviral particles encoding both TGFβ2 shRNA (mature antisense sequence: AATCTGATATAGCTCAATCCG (SEQ ID NO: 55) and GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8) under the control of a different promoter. This allowed for simultaneous reduction of TGFβ2 and introduction of expression of GM-CSF. DMS 53 was concurrently transduced with both lentiviral particles encoding TGFβ1 shRNA and membrane bound CD40L with lentiviral particles encoding TGFβ2 shRNA and GM-CSF. Cell lines genetically modified to decrease secretion of TGFβ1 and TGFβ2 are described by the clonal designation DK6.

CAMA-1 and AU565 were transduced with lentiviral particles encoding TGFβ2 shRNA, to decrease the secretion of TGFβ2, and concurrently increase expression of GM-CSF as described in above. Cell lines modified to reduce secretion of TGFβ2 and not TGFβ1 are described by the designation DK4.

Table 6-3 describes the percent reduction in TGFβ1 and/or TGFβ2 secretion in gene modified component cell lines compared to parental, unmodified cell lines. Modification with TGFβ1 shRNA resulted in at least a 44% reduction of TGFβ1 secretion. shRNA modification of TGFβ2 resulted in at least 92% reduction in secretion of TGFβ2.

TABLE 6-3
TGF-β Secretion (pg/106 cells/24 hr) in Component Cell Lines
Cell Line	Cocktail	Clone	TGFβ1	TGFβ2
CAMA-1	A	Wild type	*≤20	249
CAMA-1	A	DK4	NA	*≤11
CAMA-1	A	Percent reduction	NA	≥96%
AU565	A	Wild type	325	306
AU565	A	DK4	NA	*≤23
AU565	A	Percent reduction	NA	≥92%
HS-578T	A	Wild type	3,574	615
HS-578T	A	DK6	1,989	118
HS-578T	A	Percent reduction	44%	81%
MCF-7	B	Wild type	1,279	411
MCF-7	B	DK6	306	*≤14
MCF-7	B	Percent reduction	76%	≥97%
T47D	B	Wild type	*≤32	*≤15
T47D	B	NA	NA	NA
T47D	B	Percent reduction	NA	NA
DMS 53	B	Wild type	205	806
DMS 53	B	DK6	*≤14	*<6
DMS 53	B	Percent reduction	≥93%	≥99%
DK6: TGFβ1/TGFβ2 double knockdown;
DK4: TGFβ2 single knockdown;
*estimated using LLD, not detected;
NA = not applicable

Based on a dose of 5×10⁵ of each component cell line, total TGFβ1 and TGFβ2 secretion by BRC vaccine-A, BRC vaccine-B and respective unmodified parental cell lines are shown in Table 6-4. Secretion of TGFβ1 by BRC vaccine-A was reduced by 49% and TGFβ2 by 87% pg/dose/24 hr. Secretion of TGFβ1 by BRC vaccine-B was reduced by 79% and TGFβ2 by 98% pg/dose/24 hr.

TABLE 6-4
Total TGF-β Secretion (pg/dose/24
hr) in BRC vaccine-A and BRC vaccine-B
	Cocktail	Clones	TGFβ1	TGFβ2
	A	Wild type	1,960	585
		DK4/DK6	995	76
		Percent reduction	49%	87%
	B	Wild type	758	616
		DK6	160	10
		Percent reduction	79%	98%

Membrane Bound CD40L (CD154) Expression

BRC vaccine cell lines HS-578T, MCF-7 and DMS were transduced with lentiviral particles to express TGFβ1 shRNA and membrane bound CD40L as described above and herein. CAMA-1, AU565 and TD47 cell lines were modified with lentiviral particles only encoding the gene to express membrane-bound CD40L (SEQ ID NO: 2, SEQ ID NO: 3). Cells were analyzed for cell surface expression CD40L expression by flow cytometry. Unmodified and modified cells were stained with PE-conjugated human α-CD40L (BD Biosciences, clone TRAP1) or Isotype Control PE α-mouse IgG1 (BioLegend, clone MOPC-21). The MFI of the isotype control was subtracted from the CD40L MFI of both the unmodified and modified cell lines. If subtraction of the MFI of the isotype control resulted in a negative value, an MFI of 1.0 was used to calculate the fold increase in expression of CD40L by the modified component cell line relative to the unmodified cell line. Expression of membrane bound CD40L by all six vaccine component cell lines is described in Table 6-5. The results described below demonstrate CD40L membrane expression was substantially increased by all six cell BRC vaccine cell lines.

TABLE 6-5
Increase in membrane-bound CD40L (mCD40L) expression
	Unmodified Cell	Modified Cell	Fold Increase
Cell line	Line MFI	Line MFI	in mCD40L
CAMA-1	0	3,417	3,417
AU565	0	6,527	6,527
HS-578T	0	6,560	6,560
MCF-7	0	5,986	5,986
TD47	0	45,071	45,071
DMS 53	0	4,317	4,317
MFI reported with isotype controls subtracted

GM-CSF Expression

BRC vaccine cell lines CAMA-1, AU565, HS-578T, MCF-7 and DMS 53 cell lines were transduced with lentiviral particles encoding genes to express both TGFβ2 shRNA and the gene to GM-CSF as described above. T47D was transduced with lentiviral particles to only express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8). GM-CSF expression levels by BRC vaccine cell lines is described in Error! Reference source not found. 6-6 and herein.

TABLE 6-6
GM-CSF Secretion in Component Cell Lines
		GM-CSF	GM-CSF
	Cell Line	(ng/106 cells/24 hr)	(ng/dose/24 hr)
	CAMA-1	145	73
	AU565	66	33
	HS-578T	135	68
	Cocktail A Total	346	174
	MCF-7	302	151
	T47D	212	106
	DMS 53	30	15
	Cocktail B Total	544	272

Expression of GM-CSF for all modified BRC vaccine cell lines compared to the unmodified, parental cell lines. Based on a dose of 5×10⁵ of each component cell line, total expression of GM-CSF by BRC vaccine-A was 174 ng per dose per 24 hours and 272 ng per dose per 24 hours. GM-CSF secretion per unit dose of BRC vaccine was 446 ng per 24 hours.

IL-12 Expression

All BRC vaccine cell lines were transduced with lentiviral particles to express IL-12 p70 (SEQ ID NO: 9, SEQ ID NO: 10) as described and resulting expression levels determined as described above. Error! Reference source not found. 6-7 describes IL-12 expression levels by BRC vaccine cell lines.

TABLE 6-7
IL-12 Secretion in Component Cell Lines
		IL-12	IL-12
	Cell Line	(ng/106 cells/24 hr)	(ng/dose/24 hr)
	CAMA-1	62	31
	AU565	25	13
	HS-578T	49	25
	Cocktail A Total	136	69
	MCF-7	19	10
	T47D	86	43
	DMS 53	28	14
	Cocktail B Total	133	67

Based on a dose of 5×10⁵ of each component cell line, total IL-12 secretion by BRC vaccine-A was 69 ng per dose per 24 hours. Total IL-12 secretion by BRC vaccine-B was 67 ng per dose per 24 hours. Total IL-12 secretion per BRC vaccine unit dose was 136 ng per 24 hours.

Stable Expression of modPSMA (SEQ ID NO: 30) by the CAMA-1 Cell Line

BRC vaccine cell CAMA-1 modified to reduce the expression of CD276, reduce secretion of TGFβ2, and to express GM-CSF, membrane bound CD40L and IL-12 was transduced with lentiviral particles encoding the gene to express modPSMA (SEQ ID NO: 29, SEQ ID NO: 30). Expression of modPSMA by CAMA1 was characterized by flow cytometry. Unmodified and antigen modified cells were stained intracellularly with 0.06 μg/test anti-mouse IgG1 anti-PSMA antibody (AbCam ab268061, Clone FOLH1/3734) followed by 0.125 ug/test AF647-conjugated goat anti-mouse IgG1 antibody (Biolegend #405322). The MFI of isotype control stained modPSMA transduced and antigen unmodified cells was subtracted from the MFI of cells stained for PSMA. Fold increase in antigen expression was calculated as: (background subtracted modified MFI/background subtracted parental MFI). Expression of PSMA increased in the modified cell line (77,718 MFI) 17-fold over the parental cell line (4,269 MFI) (FIG. 22A).

Stable Expression of modTERT (SEQ ID NO: 28) by the AU565 Cell Line

BRC vaccine-A cell line AU565 modified to reduce expression of CD276 secretion, reduce secretion of TGFβ2, and express GM-CSF, membrane bound CD40L and IL-12 was transduced with lentiviral particles encoding the gene to express the modTERT antigen (SEQ ID NO: 27, SEQ ID NO: 28). Expression of modTERT by AU565 was characterized by flow cytometry. Unmodified and modTERT transduced cells were stained intracellular with 0.03 μg/test anti-mouse IgG1 anti-TERT antibody (Abcam, ab32020) followed by 0.125 ug/test donkey anti-rabbit IgG1 antibody (BioLegend #406414). The MFI of isotype control stained modTERT transduced and antigen unmodified cells was subtracted from the MFI of cells stained for TERT. Fold increase in antigen expression was calculated as: (background subtracted modified MFI/background subtracted parental MFI). Expression of TERT increased by the modified cell line (957,873 MFI) 31-fold compared to the unmodified cell line (30,743 MFI) (FIG. 22B).

Stable Expression of modTBXT and modBORIS (SEQ ID NO: 34) by the T47D Cell Line

BRC vaccine cell line T47D modified to the reduce expression of CD276 and express GM-CSF, membrane bound CD40L, and IL-12 was transduced with lentiviral particles encoding the genes to express modTBXT and modBORIS (SEQ ID NO: 33, SEQ ID NO: 34). Expression of modTBXT by T47D was characterized by flow cytometry. Unmodified and antigen modified cells were stained intracellular with 0.06 μg/test anti-rabbit IgG1 anti-TBXT antibody (Abcam, ab209665) followed by 0.125 ug/test AF647-conjugated donkey anti-rabbit IgG1 antibody (BioLegend #406414). The MFI of isotype control stained modTBXT transduced and unmodified cells was subtracted from the MFI of cells stained for TBXT. Expression of TBXT increased in by the modified cell line (147,610 MFI) 147,610-fold compared to the unmodified cell line (0 MFI) (FIG. 22C).

Expression of modBORIS by T47D was determined by RT-PCR. 1.0-3.0×10⁶ cell were used for RNA isolation. RNA was isolated using Direct-zol™ RNA MiniPrep kit (ZYMO RESEARCH, catalog number: R2051) per the manufacturers instructions. RNA quantification was performed using NanoDrop™ OneC (Thermo Scientific™ catalogue number 13-400-519). For reverse transcription, 1 pg of RNA was reverse transcribed using qScript cDNA SuperMix (Quantabio, catalogue number: 95048-025) per the manufacturer's instructions to cDNA. After completion of cDNA synthesis, the reaction was diluted two times and 2 μL of cDNA were used for amplification. The forward primer was designed to anneal at the 1119-1138 bp location in the transgene (TTCCAGTGCTGCCAGTGTAG (SEQ ID NO: 119)) and reverse primer designed to anneal at the 1159-1178 bp location in the transgene (AGCACTTGTTGCAGCTCAGA (SEQ ID NO: 120)) yielding a 460 bp product. β-tubulin primers that anneal to variant 1, exon 1 (TGTCTAGGGGAAGGGTGTGG (SEQ ID NO: 101)) and exon 4 (TGCCCCAGACTGACCAAATAC (SEQ ID NO: 102)) were used as a control. PCR products were imaged using ChemiDoc Imaging System (BioRAD, #17001401) and relative quantification to the β-tubulin gene calculated using Image Lab Software v6.0 (BioRAD). The gene product for modBORIS was detected at the expected size (FIG. 22D) and mRNA increased 2,198-fold relative to the parental control.

Immune Responses to PSMA by BRC Vaccine-A

IFNγ responses to PSMA were evaluated in the context of the BRC-vaccine A for eight HLA diverse donors (Table 6-8) by ELISpot. Specifically, 5×10⁵ of unmodified or BRC vaccine-A CAMA-1, AU565 and HS-578T cell lines, a total of 1.5×10⁶ total modified cells, were co-cultured with 1.5×10⁶ iDCs from the eight HLA diverse donors (n=4/donor). CD14-PBMCs were isolated from co-culture with DCs on day 6 and stimulated with peptide pools, 15-mers overlapping by 9 amino acids, spanning the native PSMA protein (Thermo Scientific Custom Peptide Service) in the IFNγ ELISpot assay for 24 hours prior to detection of IFNγ producing cells. BRC vaccine-A (1,631±359 SFU) induced significantly stronger PSMA specific IFNγ responses compared to unmodified BRC vaccine-A (95±60 SFU) (p=0.001) (FIG. 22E). Statistical analysis significance was determined using the Mann-Whitney U test.

TABLE 6-8
Healthy Donor MHC-I characteristics
	Donor #	HLA-A	HLA-B	HLA-C
	1	*01:01 *30:01	*08:01 *13:02	*06:02 *07:01
	2	*02:01 *25:01	*07:02 *18:01	*07:02 *12:03
	3	*03:01 *32:01	*07:02 *15:17	*07:01 *07:02
	4	*03:01 *03:01	*07:02 *18:01	*07:02 *12:03
	5	*03:01 *11:01	*18:01 *57:01	*06:02 *07:01
	6	*02:01 *02:05	*14:02 *57:01	*06:02 *08:02
	7	*02:01 *02:01	*15:01 *44:02	*03:03 *05:01
	8	*02:01 *11:01	*07:02 37:02	*06:02 07:02

Immune Responses to TERT by BRC Vaccine-A

IFNγ responses to TERT were evaluated in the context of BRC vaccine-A as described above, and herein, for eight HLA diverse donors. HLA-A, HLA-B, and HLA-C alleles for each of the eight donors are shown in Table 6-8. Specifically, 5×10⁵ of unmodified or BRC vaccine-A CAMA-1, AU565 and HS-578T cell lines, a total of 1.5×10⁶ total modified cells, were co-cultured with 1.5×10⁶ iDCs from the eight HLA diverse donors (n=4/donor). CD14-PBMCs were isolated from co-culture with DCs on day 6 and stimulated with peptide pools, 15-mers overlapping by 11 amino acids, spanning the native TERT protein (JPT, PM-TERT) in the IFNγ ELISpot assay for 24 hours prior to detection of IFNγ producing cells. IFNγ responses to TERT significantly increased when priming donor CD14-PBMCs modified with BRC vaccine-A (1,723±226 SFU) compared to the unmodified BRC vaccine-A (715±456) SFU (p=0.010) (FIG. 22F). Statistical significance was determined using the Mann-Whitney U test.

Immune responses to TBXT and BORIS in BRC vaccine-B

IFNγ responses to TBXT and BORIS were evaluated in the context of BRC-vaccine B as described herein for eight HLA diverse donors (n=4/donor). HLA-A, HLA-B, and HLA-C alleles for each of the eight donors are shown in Table 6-8. Specifically, 5×10⁵ of unmodified or modified BRC vaccine-B MCF-7, T47D and DMS 53 cell lines, a total of 1.5×10⁶ total modified cells, were co-cultured with 1.5×10⁶ iDCs from eight donors. CD14-PBMCs were isolated from co-culture with DCs on day 6 and stimulated with peptide pools, 15-mers overlapping by 11 amino acids, spanning the native TBXT protein (JPT, PM-BRAC) or peptide pools, 15-mers overlapping by 9 amino acids, spanning the native BORIS protein (Thermo Scientific Custom Peptide Service) in the IFNγ ELISpot assay for 24 hours prior to detection of IFNγ producing cells. TBXT specific IFNγ responses significantly increased when priming donor CD14-PBMCs modified with BRC vaccine-B (1,210±387 SFU) compared unmodified BRC vaccine-B (140±88 SFU) (p=0.030) (FIG. 22G). BORIS specific IFNγ responses were also significantly increased by BRC vaccine-B (2,251±751 SFU) compared to the unmodified control BRC vaccine-B (171±71 SFU) (p=0.002) (FIG. 22H). Statistical analysis was completed using the Mann-Whitney U test.

BRC Vaccine Cocktails Induce Immune Responses Against Prioritized TAAs

IFNγ production generated by BRC vaccine-A and BRC vaccine-B against ten prioritized BRC antigens was measured by ELISpot. CD14-PBMCs from eight HLA-diverse healthy donors (Table 6-8) were co-cultured with autologous DCs loaded with unmodified BRC vaccine-A, modified BRC vaccine-A, unmodified BRC vaccine-B or modified BRC vaccine-B for 6 days prior to stimulation with TAA-specific specific peptide pools containing known MHC-I restricted epitopes. Peptides for stimulation of CD14-PBMCs to detect IFNγ responses to PSMA, TERT, TBXT and BORIS are described above. Additional 15-mer peptide pools, overlapping by 11 amino acids, were sourced as follows: STEAP1 (PM-STEAP1), PRAME (JPT, PM-01P4), SCGB2A2 (Mammaglobin-A) (JPT, PM-MamA), Survivin (thinkpeptides, 7769_001-011), MUC1 (JPT, PM-MUC1) and MMP11 (JPT, PM-MMP11).

FIG. 23 demonstrates the BRC vaccine induced antigen specific IFNγ responses in eight HLA-diverse donors to ten prioritized BRC antigens that are 4.9-fold more robust (20,600±2,724 SFU) compared to the unmodified parental control (4,205±1,754 SFU) (p<0.001) (FIG. 23A) (Table 6-9). BRC vaccine-A and BRC vaccine-B independently demonstrated 5.5-fold and 4.4-fold increases in antigen specific responses compared to parental controls, respectively. BRC vaccine-A significantly increased antigen specific response (10,661±1,415 SFU) compared to the unmodified controls (1,925±989 SFU) (p<0.001) (FIG. 23B) (Table 6-9). BRC vaccine-B also elicited significantly stronger antigen specific IFNγ production (9,939±2,214 SFU) compared to parental controls (2,280±800 SFU) (p<0.001) (FIG. 23C) (Table 6-9). IFNγ responses generated by BRC vaccine-B compared to unmodified control cocktails for the eight individual donors are shown in FIG. 24. Statistical significance was determined using the Mann-Whitney U test.

TABLE 6-9
Antigen specific IFNγ responses generated by the BRC vaccine
	Unmodified (SFU ± SEM)	Modified (SFU ± SEM)
Donor #	BRC	BRC	BRC	BRC	BRC	BRC
(n = 4)	vaccine-A	vaccine-B	vaccine	vaccine-A	vaccine-B	vaccine
1	435 ± 17	490 ± 17	92 ± 9	5,810 ± 104	10,890 ± 287	16,700 ± 325
2	688 ± 27	1,160 ± 47	185 ± 18	12,838 ± 418	8,710 ± 317	21,548 ± 712
3	258 ± 14	70 ± 7	33 ± 5	4,385 ± 414	5,380 ± 205	9,765 ± 287
4	1,190 ± 46	1,575 ± 51	277 ± 25	14,510 ± 207	9,865 ± 318	24,375 ± 455
5	1,565 ± 51	1,740 ± 75	331 ± 38	11,195 ± 381	2,988 ± 114	14,183 ± 483
6	1,915 ± 78	2,778 ± 106	470 ± 47	15,933 ± 296	8,103 ± 241	24,036 ± 484
7	648 ± 26	3,190 ± 84	384 ± 33	9,503 ± 177	9,515 ± 162	19,018 ± 329
8	8,700 ± 395	7,240 ± 270	1594 ± 143	11,113 ± 393	24,060 ± 748	35,173 ± 1,123
Average	1,925 ± 989	2,280 ± 800	4,205 ± 1,754	10,661 ± 1,415	9,939 ± 2,214	20,600 ± 2,724

Breast Cancer (BRC) Driver Mutation Identification, Selection and Design

The process for identifying, selecting, and designing driver mutations was completed for BRCA as described in Example 1 and herein. Table 6-10 shows the selected oncogenes that exhibit greater than 5% mutation frequency (percentage of samples with one or more mutations) in 4552 BRC profiled patient samples.

TABLE 6-10
Oncogenes in BRC with greater than 5% mutation frequency
		Number of samples		Percentage of samples
	Total number	with one or more	Profiled	with one or more	Is Cancer Gene
Gene	of mutations	mutations	Samples	mutations	(source: OncoKB)
PIK3CA	1825	1617	4552	35.50%	Yes
TP53	1617	1579	4552	34.70%	Yes
GATA3	518	499	4552	11.00%	Yes
CDH1	460	447	4552	9.80%	Yes
KMT2C	506	447	4552	9.80%	Yes
MAP3K1	546	382	4552	8.40%	Yes
KMT2D	261	240	4552	5.30%	Yes

Identification of Driver Mutations in Selected BRC Oncogenes

The BRC driver mutations in PIK3CA and TP53 occurring in ≥0.5% of profiled patient samples are shown in Table 6-11. There were no missense mutations occurring in ≥0.5% of profiled patient samples for the BRC oncogenes listed in Table 6-10 other than PIK3CA and TP53.

TABLE 6-11
Identified driver mutations in selected BRC oncogenes
	Driver	Number of samples	Total number of	Fre-
Gene	mutation	with mutation	samples	quency
PIK3CA	C420R	32	4552	0.7%
	E726K	43	4552	0.9%
	H1047L	76	4552	1.7%
	N345K	96	4552	2.1%
	E542K	179	4552	3.9%
	E545K	301	4552	6.6%
	H1047R	654	4552	14.4%
TP53	Y220C	28	4552	0.6%
	R273C	26	4552	0.6%
	R273H	38	4552	0.8%
	R248W	40	4552	0.9%
	R248Q	50	4552	1.1%
	R175H	73	4552	1.6%

Prioritization and Selection of Identified BRC Driver Mutations

HLA-A and HLA-B supertype-restricted 9-mer CD8 epitopes analysis was performed as described in Example 1. Based on the CD8 epitope analysis result and the frequency (%) of each mutation, a list of mutations was identified to include in the final driver mutation encoding construct(s) or for further analysis to determine the number of CD4 epitopes encoded by each driver mutation peptide as described in Example 1. The results are shown in Table 6-12.

TABLE 6-12
Prioritization and selection of identified BRC driver mutations
based on CD8 epitope analysis and frequency of each mutation
		Number of		Included as
		total CD8		vaccine
	Driver	epitopes	Frequency	target?
Gene	mutation	(SB + WB)	(%)	Yes (Y) or No (N)
PIK3CA	N345K	6	2.1	Y
	C420R	0	0.7	N
	E542K	1	3.9	Y
	E545K	0	6.6	N
	E726K	2	0.9	Y
	H1047L	8	1.7	Y
	H1047R	2	14.4	T47D
TP53	R175H	2	1.6	AU565
	Y220C	2	0.6	Y
	R248W	3	0.9	Y
	R248Q	0	1.1	N
	R273C	1	0.6	CD4 analysis
	R273H	1	0.8	CD4 analysis

Next, CD4 epitopes analysis was performed as described in Example 1 to complete the final selection of BRC driver mutations. The analysis results are shown in Table 6-13.

Among all listed mutations, PIK3CA driver mutation H1047R and TP53 driver mutation R175H were endogenously expressed by the BRC vaccine component cell lines T47D and AU565, respectively, and were excluded from the final driver mutation insert design.

Taken together, as shown in Table 6-13, seven BRC driver mutations encoded by seven peptide sequences were selected and included as driver mutation vaccine targets.

TABLE 6-13
Final selection of identified BRC driver mutations based
on CD4 epitope analysis and frequency of each mutation
		Number of		Included as
		total CD4		vaccine
	Driver	epitopes	Frequency	target?
Gene	mutation	(SB + WB)	(%)	Yes (Y) or No (N)
PIK3CA	N345K	0	2.1	Y
	E542K	0	3.9	Y
	E726K	57	0.9	Y
	H1047L	6	1.7	Yes
	H1047R	12	14.4	T47D
TP53	R175H	0	1.6	AU565
	Y220C	0	0.6	Y
	R248W	15	0.9	Y
	R273C	0	0.6	N
	R273H	0	0.8	Y

The total number of CD8 epitopes for each HLA-A and HLA-B supertype introduced by seven selected BRC driver mutations was determined as described in Example 1 encoded by seven peptide sequences. Results of the epitope prediction analysis are shown in Table 6-14.

TABLE 6-14
CD8 epitopes introduced by seven selected BRC driver
mutations encoded by seven peptide sequences
		HLA-A	HLA-B	Total number
	Driver	Supertypes	Supertypes	of CD8
Gene	Mutation	(n = 5)	(n = 7)	epitopes
PIK3CA	N345K	4	2	6
	E542K	1	0	1
	E726K	1	1	2
	H1047L	2	6	8
TP53	Y220C	0	2	2
	R248W	1	2	3
	R273H	0	1	1

The total number of CD4 epitopes for Class II locus DRB1, DRB 3/4/5, DQA1/DQB1 and DPB1 introduced by seven selected BRC driver mutations were determined as described in Example 1 encoded by seven peptide sequences and the results is shown in Table 6-15.

TABLE 6-15
CD4 epitopes introduced by seven selected BRC driver
mutations encoded by seven peptide sequences
						Total number
	Driver	DRB1	DRB3/4/5	DQA1/DQB1	DPB1	of CD4
Gene	mutation	(n = 26)	(n = 6)	(n = 8)	(n = 6)	epitopes
PIK3CA	N345K	0	0	0	0	0
	E542K	0	0	0	0	0
	E726K	39	10	0	8	57
	H1047L	0	0	0	6	6
TP53	Y220C	0	0	0	0	0
	R248W	2	4	1	9	16
	R273H	0	0	0	0	0

BRC Patient Sample Coverage by Selected Driver Mutations

As shown in Table 6-16, seven selected BRC driver mutations were assembled into a single construct insert. The final construct insert gene encodes 264 amino acids containing seven driver mutation peptide sequences (SEQ ID NO: 121, SEQ ID NO: 122) separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37).

TABLE 6-16
Seven BRC driver mutations encoded by the BRC vaccine
			Total	Total	Total CD4
	Driver	Frequency	CD8	CD4	and CD8
Gene	mutation	(%)	epitopes	epitopes	epitopes
PIK3CA	N345K	2.1	6	0	6
	E542K	3.9	1	0	1
	E726K	0.9	2	57	59
	H1047L	1.7	8	6	14
TP53	Y220C	0.6	2	0	2
	R248W	0.9	3	16	19
	R273H	0.8	1	0	1

Once the construct insert was assembled, analysis of BRC patient sample coverage was performed as described in Example 1. The results indicated that the BRC patient sample coverage by the insert was 10.6% (Table 6-17). Inclusion of driver mutations endogenously expressed by the BRC vaccine component cell lines in the population coverage analysis, the total BRC patient sample coverage was 25.8% (Table 6-18).

TABLE 6-17
Frequency of BRC patient samples targeted
by the construct encoded driver mutations
Targeted Patient
Samples Construct		Total number of	% of Patient
Insert Only	DM Target Gene	Samples with	Samples
Sample Description	PIK3CA	TP53	Driver Mutation	(n = 4423)
# of samples	354	97	451	10.2%
with one DM
# of samples	14	0	14	0.3%
with ≥2 DMs
from same antigen
# of samples			4	0.1%
with ≥2 DMs from
different antigens
		Total	469	10.60%

TABLE 6-18
Frequency of BRC patient samples targeted by construct
and cell line encoded driver mutations
Targeted Patient
Samples Construct
Insert & BRC-		Total number of	Total
Vaccine Cell Lines	DM Target Gene	Samples with	Sample
Sample Description	PIK3CA	TP53	Driver Mutation	(n = 4423)
# of samples	947	145	1092	24.7%
with one DM
# of samples	24	0	24	0.5%
with ≥2 DMs
from same antigen
# of samples			24	0.5%
with ≥2 DMs from
different antigens
		Total	1,140	25.8%

Oncogene Sequences and Insert Sequences of the BRC Driver Mutation Construct

The DNA and protein sequences of inserts encoding BRC driver mutations are included in Table 6-19. Native DNA and protein sequences of TP53 (SEQ ID NO: 41) and PIK3CA (SEQ ID NO: 47) (Table 2-10) are describe above and herein.

The BRC driver mutation construct insert gene encodes 264 amino acids containing the driver mutation peptides separated by the furin cleavage sequence RGRKRRS (SEQ ID NO: 37).

TABLE 6-19
Insert sequences for the BRC construct
BRC DM    DNA Sequence
construct 1 ATGATCAATA GCGCCCTGCG GATCAAGATC CTGTGCGCCA CCTACGTGAA AGTGAACATC
insert    61 CGGGACATCG ACAAGATCTA CGTGCGGACC GGCATCCGGG GCAGAAAGAG AAGATCCGAC
(SEQ ID   121 AAAGAGCAGC TGAAGGCCAT CAGCACCAGA GATCCTCTGA GCAAGATCAC CGAGCAAGAG
NO: 121)  181 AAGGACTTCC TGTGGTCCCA CCGGCACTAC AGAGGCCGGA AGAGAAGAAG CAAGCTGATC
          241 AACCTGACCG ACATCCTGAA GCAAGAAAAG AAGGACAAGA CCCAGAAAGT GCAGATGAAG
          301 TTCCTGGTGG AACAGATGCG GCGGAGAGGC AGAAAGCGGA GATCTGAACA AGAGGCCCTG
          361 GAATACTTTA TGAAGCAGAT GAACGACGCC CTGCACGGCG GCTGGACAAC AAAGATGGAC
          421 TGGATCTTCC ACACCATCAG AGGACGGAAG CGGCGGAGCT ACCTGGACGA CAGAAACACC
          481 TTCAGACACA GCGTGGTGGT GCCCTGCGAA CCTCCTGAAG TGGGCAGCGA TTGCACCACC
          541 ATCCACTACA ACCGGGGAAG AAAGCGCCGG TCCACAACAA TCCACTATAA CTACATGTGC
          601 AACAGCAGCT GCATGGGCGG CATGAACTGG CGGCCTATCC TGACCATCAT CACCCTGGAA
          661 GATAGCAGCG GCAACCTGCG CGGACGCAAA AGAAGAAGCG AGGACAGCTC CGGCAATCTG
          721 CTGGGCAGAA ACAGCTTCGA GGTGCACGTG TGCGCCTGTC CTGGCAGAGA CAGAAGAACC
          781 GAAGAGGAAA ACTGATAG
BRC DM    Protein Sequence*
construct 1 MINSALRIKI LCATYVKVNI RDIDKIYVRT GIRGRKRRSD KEQLKAISTR DPLSKITEQE
insert    61 KDFLWSHRHY RGRKRRSKLI NLTDILKQEK KDKTQKVQMK FLVEQMRRRG RKRRSEQEAL
(SEQ ID   121 EYFMKQMNDA LHGGWTTKMD WIFHTIRGRK RRSYLDDRNT FRHSVVVPCE PPEVGSDCTT
NO: 122)  181 IHYNRGRKRR STTIHYNYMC NSSCMGGMNW RPILTIITLE DSSGNLRGRK RRSEDSSGNL
          241 LGRNSFEVHV CACPGRDRRT EEEN
*Driver mutation is highlighted in bold.
The furin cleavage sequence is underlined.

Immune Responses to TP53 and PIK3CA Driver Mutations

BRC vaccine-A cell line AU565 modified to reduce expression of CD276, reduce secretion of TGFβ2, and express GM-CSF, membrane bound CD40L, IL-12, and modTERT was transduced with lentiviral particles expressing seven TP53 or PIK3CA driver mutations encoded by seven peptide sequences. The genes encoding each driver mutation peptide were separated by the furin cleavage sequence.

Immune responses against TP53 and PIK3CA driver mutations expressed by AU565 were characterized by IFNγ ELISpot. Specifically, 1.5×10⁶ of unmodified AU565 or BRC vaccine-A AU565 expressing TP53 and PIK3CA driver mutations were co-cultured with 1.5×10⁶ iDCs generated from six HLA diverse donors (n=4/donor). HLA-A, HLA-B, and HLA-C alleles for the six donors are described in Table 6-20. CD14-PBMCs were isolated from co-culture with DCs on day 6 and stimulated with peptide pools, 15-mers overlapping by 9 amino acids, for individual TP53 or PIK3CA driver mutations (Thermo Scientific Custom Peptide Service) for 24 hours in the ELISpot assay prior to detection of IFNγ production. Peptides were designed to span the entire sequence of the seven peptides encoding TP53 or PIK3CA driver mutations, excluding the furin cleavage sequences, but only 15-mer peptides containing TP53 or PIK3CA driver mutations were used to stimulate PBMCs in the IFNγ ELISpot assay.

TABLE 6-20
Healthy Donor MHC-I characteristics
	Donor #	HLA-A	HLA-B	HLA-C
	1	*01:01 *32:01	*35:01 *40:06	*04:01 *15:02
	2	*02:01 *03:01	*07:02 *49:01	*07:01 *07:02
	3	*02:01 *03:01	*07:02 *41:02	*07:02 *17:01
	4	*02:01 *03:01	*08:01 *51:01	*07:01 *14:02
	5	*03:01 *24:02	*07:02 *15:09	*07:02 *07:04
	6	*03:01 *24:02	*07:02 *14:02	*07:02 *08:02

FIG. 25A demonstrates IFNγ production against all three TP53 driver mutations was more robust when donor CD14-PBMCs were primed with modified AU565 compared to unmodified AU565 (Table 6-21). FIG. 25B demonstrates IFNγ production against all four PIK3CA driver mutations were more robust when priming with modified AU565 compared to unmodified AU565 (Table 6-22). The magnitude of IFNγ responses induced by modified AU565 against the Y220C (p=0.002), R248W (p=0.002) and R273H (p=0.002) TP53 driver mutations, and N345K (p=0.002), E542K (p=0.002), E726K (p=0.002), H1047R (p=0.002) PIK3CA driver mutations was significantly greater compared to unmodified AU565. Statistical analysis was completed using the Mann-Whitney U test. All six donors responded to three inserted TP53 driver mutations and four inserted PIK3CA driver mutations.

TABLE 6-21
Immune responses to TP53 driver mutations
TP53	Unmodified AU565 (SFU ± SEM)	Modified AU565 (SFU ± SEM)
mutation	Y220C	R248W	R273H	Y220C	R248W	R273H
Donor 1	0 ± 0	0 ± 0	0 ± 0	1,313 ± 450	1,170 ± 190	1,400 ± 426
Donor 2	0 ± 0	60 ± 48	110 ± 64	7,360 ± 933	8,190 ± 833	7,830 ± 546
Donor 3	0 ± 0	0 ± 0	100 ± 60	1,628 ± 738	615 ± 355	1,960 ± 770
Donor 4	0 ± 0	0 ± 0	80 ± 46	1,440 ± 949	510 ± 326	880 ± 453
Donor 5	290 ± 252	190 ± 112	150 ± 104	3,320 ± 1,859	2,600 ± 780	2,120 ± 412
Donor 6	0 ± 0	0 ± 0	0 ± 0	3,790 ± 623	2,400 ± 1,154	2,190 ± 500
Average	48 ± 48	42 ± 31	73 ± 25	3,142 ± 945	2,581 ± 1,178	2,730 ± 1,040

TABLE 6-22
Immune responses to PIK3CA driver mutations
PIK3CA	Unmodified AU565 (SFU ± SEM)	Modified AU565 (SFU ± SEM)
mutation	N345K	E542K	E726K	H1047L	N345K	E542K	E726K	H1047R
Donor 1	0 ± 0	0 ± 0	0 ± 0	0 ± 0	703 ± 346	1,450 ± 564	1,833 ± 649	1,310 ± 510
Donor 2	100 ± 53	110 ± 97	120 ± 77	70 ± 57	5,630 ± 732	7,050 ± 1,165	7,650 ± 361	7,080 ± 1,253
Donor 3	0 ± 0	0 ± 0	115 ± 74	410 ± 141	300 ± 212	830 ± 614	2,103 ± 1,036	1,770 ± 662
Donor 4	270 ± 125	0 ± 0	200 ± 71	50 ± 30	1,580 ± 1,044	1,308 ± 513	2,290 ± 1,102	1,120 ± 680
Donor 5	0 ± 0	0 ± 0	0 ± 0	0 ± 0	1,905 ± 1,332	3,280 ± 1,801	4,710 ± 1,061	3,240 ± 1,447
Donor 6	0 ± 0	0 ± 0	0 ± 0	0 ± 0	2,150 ± 1,117	3,550 ± 410	2,930 ± 779	3,580 ± 708
Average	62 ± 45	18 ± 18	73 ± 35	88 ± 65	2,045 ± 773	2,918 ± 942	3,586 ± 916	3,017 ± 912

Genetic modifications completed for BRC vaccine-A and BRC vaccine-B cell lines are described in Table 6-23 below and herein. The CD276 gene was knocked out (KO) by electroporation of zinc-finger nucleases (ZFN) (SEQ ID NO: 52) as described above. All other genetic modifications were completed by lentiviral transduction.

BRC Vaccine-A

CAMA-1 (ATCC, HTB-21) modified to reduce expression of CD276 (SEQ ID NO: 52), knockdown (KD) secretion of transforming growth factor-beta 2 (SEQ ID NO: 55) (TGFβ2), and express granulocyte macrophage-colony stimulating factor (GM-CSF) (SEQ ID NO: 7, SEQ ID NO: 8), membrane-bound CD40L (mCD40L) (SEQ ID NO: 2, SEQ ID NO: 3), interleukin 12 p70 (IL-12) (SEQ ID NO: 9, SEQ ID NO: 10) and modPSMA (SEQ ID NO: 29, SEQ ID NO: 30),

AU565 (ATCC, CRL-2351) modified to reduce expression of CD276 (SEQ ID NO: 52), reduce secretion of TGFβ2 (SEQ ID NO: 55), and express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), mCD40L (SEQ ID NO: 2, SEQ ID NO: 3), IL-12 (SEQ ID NO: 9, SEQ ID NO: 10), modTERT (SEQ ID NO: 27, SEQ ID NO: 28), and the gene encoding TP53 (SEQ ID NO: 41) driver mutations Y220C, R248W and R273H and PIK3CA (SEQ ID NO: 47) driver mutations N345K, E542K, E726K and H1047L separated by a furin cleavage sequence (SEQ ID NO: 121, SEQ ID NO: 122).

HS-578T (ATCC, HTB-126) modified to reduce expression of CD276 (SEQ ID NO: 52), reduce secretion of transforming growth factor-beta 1 (TGFβ1) (SEQ ID NO: 54) and TGFβ2 (SEQ ID NO: 55), and express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), mCD40L (SEQ ID NO: 2, SEQ ID NO: 3), IL-12 (SEQ ID NO: 9, SEQ ID NO: 10).

BRC Vaccine-B

MCF-7 (ATCC, HTB-22) modified to reduce expression of CD276 (SEQ ID NO: 52), reduce secretion of TGFβ1 (SEQ ID NO: 54) and TGFβ2 (SEQ ID NO: 54), express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), mCD40L (SEQ ID NO: 2, SEQ ID NO: 3), and IL-12 (SEQ ID NO: 9, SEQ ID NO: 10).

T47D (ATCC, HTB-133) modified to reduce expression of CD276 (SEQ ID NO: 52) and to express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), mCD40L (SEQ ID NO: 2, SEQ ID NO: 3), IL-12 (SEQ ID NO: 9, SEQ ID NO: 10) and the gene encoding modTBXT and modBORIS (SEQ ID NO: 33, SEQ ID NO: 34) separated by a furin cleavage sequence.

DMS 53 (ATCC, CRL-2062) cell line modified to reduce expression of CD276 (SEQ ID NO: 52), reduce secretion of TGFβ1 (SEQ ID NO: 54) and TGFβ2 (SEQ ID NO: 55), express GM-CSF (SEQ ID NO: 7, SEQ ID NO: 8), mCD40L (SEQ ID NO: 2, SEQ ID NO: 3) and IL-12 (SEQ ID NO: 9, SEQ ID NO: 10).

TABLE 6-23
Breast cancer vaccine cell line nomenclature and genetic modifications
								Tumor-
								Associated
	Cell	CD276	TGFβ1	TGFβ2				Antigens	Driver
Cocktail	Line	KO	KD	KD	GM-CSF	mCD40L	IL-12	(TAAs)	Mutations
A	CAMA-1	SEQ ID	—	SEQ ID	SEQ ID	SEQ ID	SEQ ID	modPSMA
		NO: 52		NO: 55	NO: 8	NO: 3	NO: 10	(SEQ ID
								NO: 30)
A	AU565	SEQ ID	—	SEQ ID	SEQ ID	SEQ ID	SEQ ID	modTERT	TP53 and PIK3CA
		NO: 52		NO: 55	NO: 8	NO: 3	NO: 10	(SEQ ID	(SEQ ID
								NO: 28)	NO: 122)
A	HS-578T	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	—	—
		NO: 52	NO: 54	NO: 55	NO: 8	NO: 3	NO: 10
B	MCF-7	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID		—
		NO: 52	NO: 54	NO: 55	NO: 8	NO: 3	NO: 10
B	T47D	SEQ ID	—	—	SEQ ID	SEQ ID	SEQ ID	modTBXT	—
		NO: 52			NO: 8	NO: 3	NO: 10	modBORIS
								(SEQ ID
								NO: 34)
B	DMS 53*	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	SEQ ID	—	—
		NO: 52	NO: 54	NO: 55	NO: 8	NO: 3	NO: 10
—, not required.
*Cell line identified as CSC-like. mCD40L, membrane bound CD40L.

Example 7: Adaptation of DMS 53 Cell Line to Growth in Xeno-Free Media

Example 7 describes adaptation of cell line DMS 53 modified to reduce expression of CD276, secretion of TGFβ1 and TGFβ2, and express GMCSF, membrane bound CD40L and IL-12 to grow in xeno-free media. Example 38 of WO/2021/113328 describes the adaptation of DMS 53—modified to reduce expression of CD276, reduce secretion of TGFβ2, and express GM-CSF and membrane bound CD40L—to grow in xeno-free media. As described in Example 4 herein, further optimization of gene editing strategies allowed inclusion of two additional adjuvant modifications to the DMS 53 cell line-reduction of TGFβ1 secretion and expression of IL-12. As further described in Example 4 and FIG. 6 herein, immune responses to eight prioritized NSCLC TAAs were significantly increased when DMS 53 was modified to reduce expression of CD276, reduce secretion of TGFβ1 and TGFβ2, express GM-CSF membrane bound CD40L and IL-12 compared to DMS 53 modified to reduce expression of CD276, reduce secretion of TGFβ2, and to express GM-CSF and membrane bound CD40L.

Cell line DMS 53 (modified to reduce expression of CD276, reduce secretion of TGFβ1 and TGFβ2, and express GMCSF, membrane bound CD40L and IL-12 as described herein) was sequentially adapted to grow in the xeno-free media as described in Example 38 of WO/2021/113328 and herein. Cell line DMS 53 (modified to reduce expression of CD276, reduce secretion of TGFβ1 and TGFβ2, and express GMCSF, membrane bound CD40L and IL-12) was sequentially adapted from growth in FBS to growth in xeno-free media using decreasing ratios of FBS to xeno-free replacement supplements. Selection of antibiotic concentration required to maintain transgene expression may depend on the protein composition of the growth media. For some cell lines, reduction of selection antibiotic concentration expedites growth in xeno-free while maintaining equivalent transgene expression levels to baseline cell lines. Adjustment of selection antibiotics used to maintain transgene did not need to be adjusted during this process (Table 7-1).

TABLE 7-1
DMS 53 selection antibiotic concentrations for inserted transgenes
before and after adaptation to grow in xeno-free media
	DMS 53	Puromycin	Blasticidin	Hygromycin
	Cell Line	(μg/mL)	(μg/mL)	(μg/mL)
	Pre-adaptation	4	8	300
	Post-adaptation	4	8	300

Two ratios of FBS to replacement supplement were used over three passages to adapt the cells to grow in serum-free xeno-free media. Following the first passage in xeno-free media the cell line was monitored for an additional four passages with an average doubling of 206 hours. The cells were then grown for six additional passages with an average doubling time of 119 hours prior to cryopreservation. Doubling time of the cell line generally decreased with subsequent passages in xeno-free media: passage 1, 148 hours; passage 2, 129 hours; passage 3, 105 hours; passage 4, 119 hours; passage 5, 109 hours and passage 6, 108 hours. Subsequent passages after cryopreservation showed the doubling time decreased further to ranging from 88 hours to 105 hours for at least two passages.

Analysis of Transgene Expression in Cell Lines Grown in Xeno-Free Media

DMS 53 cells showed stable growth in xeno-free media as described above. Expression levels of CD276, TGFβ1, TGFβ2, GMCSF, CD40L and IL-12 were compared for the cell line grown in FBS containing media to the cell line grown in xeno-free media as described in Example 4. Post-adaptation, expression of the surface protein CD40L and reduction of CD276 expression were comparable to pre-adapted cells. IL-12 and GM-CSF secretion were slightly increased in the xeno-free media (Table 7-2). TGFβ1 and TGFβ2 secretion by the cell line was not detected by ELISA before and after adaptation to xeno-free media.

TABLE 7-2
DMS 53 transgene expression pre-adaptation and
post-adaptation to grow in xeno-free media
IL-12	GMCSF	TGFB1	TGFB2
(ng/106/24 hrs)	(ng/106/24 hrs)	(pg/106/24 hrs)	(ng/106/24 hrs)
Pre-	Post-	Pre-	Post-	Pre-	Post-	Pre-	Post-
adaptation	adaptation	adaptation	adaptation	adapattion	adaptation	adaptation	adaptation
55	86	64	77	ND	ND	ND	ND
*ND, not detected/below the LLD of ELISA

In conclusion, cell line DMS 53 modified to reduce expression of CD276, secretion of TGFβ1 and TGFβ2, and express GMCSF, membrane bound CD40L and IL-12 was stably adapted to grow in xeno-free media. Expression of the surface proteins CD40L and CD276 was detected at levels similar to cells grown in FBS, and the cells retained the reduction of TGFβ1 and TGFβ2 secretion. Expression of GM-CSF and IL-12 was found to be comparable to, or increased, in the xeno-free formulation. As described in Example 4, the ability of DMS 53 to induce antigen specific IFNγ responses to eight prioritized NSCLC antigens was maintained following adaptation to xeno-free media.

Claims

1. A composition comprising a therapeutically effective amount of at least 1 modified cancer cell line, wherein the cell line or a combination of the cell lines comprises cells that express at least 5 tumor associated antigens (TAAs) associated with a cancer of a subject intended to receive said composition, and wherein said composition is capable of eliciting an immune response specific to the at least 5 TAAs, and wherein the cell line or combination of the cell lines have been modified to express at least 1 peptide comprising at least 1 oncogene driver mutation.

2.-4. (canceled)

5. The composition of claim 1, wherein the cell line or a combination of the cell lines are modified to (i) express or increase expression of at least 1 immunostimulatory factor, and (ii) inhibit or decrease expression of at least 1 immunosuppressive factor.

6. The composition of claim 1, wherein the cell line or a combination of the cell lines are modified to express or increase expression of at least 1 TAA that is either not expressed or minimally expressed by one or all of the cell lines.

7. The composition of claim 6, wherein the cell line or a combination of the cell lines are further modified to express or increase expression of at least 1 peptide comprising at least 1 tumor fitness advantage mutation selected from the group consisting of an acquired tyrosine kinase inhibitor (TKI) resistance mutation, an EGFR activating mutation, and/or a modified ALK intracellular domain (modALK-IC).

8.-11. (canceled)

12. The composition of claim 1, wherein the composition is capable of stimulating an immune response in a subject receiving the composition.

13. The composition of claim 12, wherein the cell line or a combination of the cell lines are modified to (i) express at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more peptides, wherein each peptide comprises at least 1 oncogene driver mutation, (ii) express or increase expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunostimulatory factors, (iii) inhibit or decrease expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunosuppressive factors, and/or (iv) express or increase expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 TAAs that are either not expressed or minimally expressed by one or all of the cell lines, and wherein at least one of the cell lines is a cancer stem cell line.

14.-15. (canceled)

16. The composition of claim 12, wherein the oncogene driver mutation is in one or more oncogenes selected from the group consisting of ACVR2A, AFDN, ALK, AMER1, ANKRD11, APC, AR, ARID1A, ARID1B, ARID2, ASXL1, ATM, ATR, ATRX, AXIN2, B2M, BCL9, BCL9L, BCOR, BCORL1, BRAF, BRCA2, CACNA1D, CAD, CAMTA1, CARD11, CASP8, CDH1, CDH11, CDKN1A, CDKN2A, CHD4, CIC, COL1A1, CPS1, CREBBP, CTNNB1, CUX1, DICER1, EGFR, ELF3, EP300, EP400, EPHA3, EPHA5, EPHB1, ERBB2, ERBB3, ERBB4, ERCC2, FAT1, FAT4, FBXW7, FGFR3, FLT4, FOXA1, GATA3, GNAS, GRIN2A, HGF, HRAS, IDH1, IRS1, IRS4, KAT6A, KDM2B, KDM6A, KDR, KEAP1, KMT2A, KMT2B, KMT2C, KMT2D, KRAS, LARP4B, LRP1B, LRP5, LRRK2, MAP3K1, MDC1, MEN1, MGA, MGAM, MKI67, MTOR, MYH11, MYH9, MYO18A, MYO5A, NCOA2, NCOR1, NCOR2, NF1, NFATC2, NFE2L2, NOTCH1, NOTCH2, NOTCH3, NSD1, NTRK3, NUMA1, PBRM1, PCLO, PDE4DIP, PDGFRA, PDS5B, PIK3CA, PIK3CG, PIK3R1, PLCG2, POLE, POLQ, PREX2, PRKDC, PTCH1, PTEN, PTPN13, PTPRB, PTPRC, PTPRD, PTPRK, PTPRS, PTPRT, RANBP2, RB1, RELN, RICTOR, RNF213, RNF43, ROBO1, ROS1, RPL22, RUNX1T1, SETBP1, SETD1A, SLX4, SMAD2, SMAD4, SMARCA4, SOX9, SPEN, SPOP, STAG2, STK11, TCF7L2, TET1, TGFBR2, TP53, TP53BP1, TPR, TRRAP, TSC1, UBR5, ZBTB20, ZFHX3, ZFP36L1, or ZNF521.

17.-22. (canceled)

23. The composition of claim 12, wherein (a) the at least one immunostimulatory factor is selected from the group consisting of GM-CSF, membrane-bound CD40L, GITR, IL-15, IL-23, and IL-12, and (b) wherein the at least one immunosuppressive factors are selected from the group consisting of CD276, CD47, CTLA4, HLA-E, HLA-G, IDO1, IL-10, TGFβ1, TGFβ2, and TGFβ3.

24.-58. (canceled)

59. A kit comprising 6 vials, wherein the vials each contain a composition comprising a cancer cell line, and wherein at least 2 of the 6 vials comprise a cancer cell line that is modified to (i) express or increase expression of at least 2 immunostimulatory factors, (ii) inhibit or decrease expression of at least 2 immunosuppressive factors, and (iii) express at least 1 peptide comprising at least 1 oncogene driver mutation.

60.-61. (canceled)

62. A unit dose of a medicament for treating cancer comprising at least 5 compositions of different cancer cell lines, wherein at least 2 compositions comprise a cell line that is modified to (i) express or increase expression of at least 2 immunostimulatory factors, (ii) inhibit or decrease expression of at least 2 immunosuppressive factors, and (iii) express at least 1 peptide comprising at least 1 oncogene driver mutation.

63. A unit dose of a medicament for treating cancer comprising at least 5 compositions of different cancer cell lines, wherein each cell line is modified to (i) express or increase expression of at least 2 immunostimulatory factors, (ii) inhibit or decrease expression of at least 2 immunosuppressive factors, and/or (iii) increase expression of at least 1 TAA that are either not expressed or minimally expressed by the cancer cell lines, and/or (iv) express at least 1 peptide comprising at least 1 oncogene driver mutation.

64. The unit dose of claim 62, wherein at least 2 compositions comprise a cell line that is modified to express or increase expression of at least 1 peptide comprising at least 1 tumor fitness advantage mutation selected from the group consisting of an acquired tyrosine kinase inhibitor (TKI) resistance mutation, an EGFR activating mutation, and/or a modified ALK intracellular domain.

65.-72. (canceled)

73. A method of preparing a composition comprising a modified cancer cell line, said method comprising the steps of: (a) identifying one or more mutated oncogenes with >5% mutation frequency in a cancer;(b) identifying one or more driver mutations occurring in ≥0.5% of profiled patient samples in the mutated oncogenes identified in (a);(c) determining whether a peptide sequence comprising non-mutated oncogene amino acids and the driver mutation identified in (b) comprises a CD4 epitope, a CD8 epitope, or both CD4 and CD8 epitopes;(d) inserting a nucleic acid sequence encoding the peptide sequence comprising the driver mutation of (c) into a lentiviral vector; and(e) introducing the lentiviral vector into a cancer cell line, thereby producing a composition comprising a modified cancer cell line.

74.-115. (canceled)

116. A method of stimulating an immune response in a patient comprising administering to said patient a therapeutically effective amount of a unit dose of a cancer vaccine, wherein said unit dose comprises a composition comprising a cancer stem cell line and at least 3 compositions each comprising a different modified cancer cell line; wherein the cell lines are optionally modified to (i) express at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more peptides, wherein each peptide comprises at least 1 oncogene driver mutation, and/or (ii) express or increase expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunostimulatory factors, and/or (iii) inhibit or decrease expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunosuppressive factors, and/or (iv) express or increase expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 TAAs that are either not expressed or minimally expressed by one or all of the cell lines.

117. A method of treating cancer in a patient comprising administering to said patient a therapeutically effective amount of a unit dose of a cancer vaccine, wherein said unit dose comprises a composition comprising a cancer stem cell line and at least 3 compositions each comprising a different modified cancer cell line; wherein the cell lines are optionally modified to (i) express at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more peptides, wherein each peptide comprises at least 1 oncogene driver mutation, and/or (ii) express or increase expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunostimulatory factors, and/or (iii) inhibit or decrease expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 immunosuppressive factors, and/or (iv) express or increase expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 TAAs that are either not expressed or minimally expressed by one or all of the cell lines.

118.-132. (canceled)