METHOD AND COMPOSITIONS FOR REGULATED ARMORING OF CELLS

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Month XX, 20XX, is named US sequencelisting.txt, and is X,XXX,XXX bytes in size.

BACKGROUND

Tumors employ a range of direct and indirect suppression strategies to avoid recognition and clearance by the immune system. These escape strategies can effectively shut down cell therapies. Combinatorial armoring, expression of combinations of effectors, can impact the entire cancer immunity cycle and boost the activity of cell therapies as unarmored therapies have poor efficacy in solid tumors. However, current cell and gene therapy products have no control. Uncontrolled armored therapies can have toxicity in subjects. Thus, additional methods of controlling and regulating the expression of combinations of effector molecules are required.

SUMMARY

In another aspect, provided herein are engineered expression systems comprising: (a) a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activation-conditional control polypeptide (ACP), wherein the first promoter is operably linked to the first exogenous polynucleotide; and (b) a second expression cassette comprising an ACP-responsive promoter and a second exogenous polynucleotide sequence having the formula: (L-E)_Xwherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, X=1 to 20, wherein the ACP-responsive promoter is operably linked to the second exogenous polynucleotide, wherein for the first iteration of the (L-E) unit, L is absent, and wherein the ACP is capable of inducing expression of the second expression cassette by binding to the ACP-responsive promoter. In some embodiments, the first expression cassette and the second expression cassette are encoded by separate polynucleotide sequences. In some embodiments, the first expression cassette and the second expression cassette are encoded by the same polynucleotide sequence. In some embodiments, the first expression cassette and/or the second expression cassette further comprises an additional exogenous polynucleotide sequence encoding an antigen recognizing receptor. In some embodiments, the first expression cassette further comprises an additional exogenous polynucleotide sequence encoding an antigen recognizing receptor. In some embodiments, the second expression cassette further comprises an additional exogenous polynucleotide sequence encoding an antigen recognizing receptor. In some embodiments, the engineered expression system further comprises an additional expression cassette including an additional promoter and an additional exogenous polynucleotide sequence encoding an antigen recognizing receptor, wherein the additional promoter is operably linked to the additional exogenous polynucleotide. In some embodiments, the additional exogenous polynucleotide sequence is encoded by the same polynucleotide as the first expression cassette or the second expression cassette. In some embodiments, the additional exogenous polynucleotide sequence is encoded by the same polynucleotide as the first expression cassette. In some embodiments, the additional exogenous polynucleotide sequence is encoded by the same polynucleotide as the second expression cassette. In some embodiments, a first vector comprises the first expression cassette and the additional expression cassette if present, and a second vector comprises the second expression cassette. In some embodiments, a first vector comprises the first expression cassette, and a second vector comprises the second expression cassette and the the additional expression cassette if present. In some embodiments, a first vector comprises the first expression cassette and the second expression cassette, and a second vector comprises the additional expression cassette if present. In some embodiments, the engineered expression system comprises any of the aspects, features, or embodiments of the engineered nucleic acids described herein, including, but not limited to, any of the aspects, features, or embodiments described in enumerated embodiments 1-359.

In some embodiments, when the second expression cassette comprises two or more units of (L-E)_X, each linker polynucleotide sequence is operably associated with the translation of each molecule as a separate polypeptide.

In some embodiments, the linker polynucleotide sequence encodes a 2A ribosome skipping tag.

In some embodiments, the 2A ribosome skipping tag is selected from the group consisting of: P2A, T2A, E2A, and F2A.

In some embodiments, the linker polynucleotide sequence encodes an Internal Ribosome Entry Site (IRES).

In some embodiments, the linker polynucleotide sequence encodes a cleavable polypeptide.

In some embodiments, the cleavable polypeptide comprises a furin polypeptide sequence.

In some embodiments, the second expression cassette comprising one or more units of (L-E)_Xfurther comprises a polynucleotide sequence encoding a secretion signal peptide.

In some embodiments, for each X the corresponding secretion signal peptide is operably associated with the effector molecule.

In some embodiments, each secretion signal peptide comprises a native secretion signal peptide native to the corresponding effector molecule.

In some embodiments, each secretion signal peptide comprises a non-native secretion signal peptide that is non-native to the corresponding effector molecule.

In some embodiments, the non-native secretion signal peptide is selected from the group consisting of: IL12, IL2, optimized IL2, trypsiongen-2, Gaussia luciferase, CD5, CD8, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROalpha, GM-CSFR, GM-CSF, and CXCL12.

In some embodiments, the ACP-responsive promoter comprises an ACP-binding domain and a promoter sequence.

In some embodiments, the promoter sequence is derived from a promoter selected from the group consisting of: minP, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, AP1 response element, TCF-LEF response element promoter fusion, Hypoxia responsive element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule responsive promoters, and tandem repeats thereof.

In some embodiments, the ACP-responsive promoter is a synthetic promoter.

In some embodiments, the ACP-responsive promoter comprises a minimal promoter.

In some embodiments, the ACP-binding domain comprises one or more zinc finger binding sites.

In some embodiments, the first promoter is a constitutive promoter, an inducible promoter, or a synthetic promoter.

In some embodiments, the constitutive promoter is selected from the group consisting of: CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

In some embodiments, each effector molecule is independently selected from a therapeutic class, wherein the therapeutic class is selected from the group consisting of: a cytokine, a chemokine, a homing molecule, a growth factor, a co-activation molecule, a tumor microenvironment modifier a, a receptor, a ligand, an antibody, a polynucleotide, a peptide, and an enzyme.

In some embodiments, the cytokine is selected from the group consisting of: IL1-beta, IL2, IL4, IL6, IL7, IL10, IL12, an IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, Type I interferons, Interferon-gamma, and TNF-alpha.

In some embodiments, the chemokine is selected from the group consisting of: CCL21a, CXCL10, CXCL11, CXCL13, a CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1.

In some embodiments, the homing molecule is selected from the group consisting of: anti-integrin alpha4,beta7; anti-MAdCAM; CCR9; CXCR4; SDF1; MMP-2; CXCR1; CXCR7; CCR2; CCR4; and GPR15.

In some embodiments, the growth factor is selected from the group consisting of: FLT3L and GM-CSF.

In some embodiments, the co-activation molecule is selected from the group consisting of: c-Jun, 4-1BBL and CD40L.

In some embodiments, the tumor microenvironment modifier is selected from the group consisting of: adenosine deaminase, TGFbeta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2.

In some embodiments, the TGFbeta inhibitors are selected from the group consisting of: an anti-TGFbeta peptide, an anti-TGFbeta antibody, a TGFb-TRAP, and combinations thereof.

In some embodiments, the immune checkpoint inhibitors are selected from the group consisting of: anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies, anti-TIM-3 antibodies, anti-TIGIT antibodies, anti-VISTA antibodies, anti-KIR antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-HVEM antibodies, anti-BTLA antibodies, anti-GALS antibodies, anti-A2AR antibodies, anti-phosphatidylserine antibodies, anti-CD27 antibodies, anti-TNFa antibodies, anti-TREM1 antibodies, and anti-TREM2 antibodies.

In some embodiments, the VEGF inhibitors comprise anti-VEGF antibodies, anti-VEGF peptides, or combinations thereof.

In some embodiments, each effector molecule is a human-derived effector molecule.

In some embodiments, the first exogenous polynucleotide sequence further encodes an antigen recognizing receptor.

In certain aspects, provided herein are engineered nucleic acids comprising: a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activation-conditional control polypeptide (ACP) and an antigen recognizing receptor, wherein the first promoter is operably linked to the first exogenous polynucleotide; and a second expression cassette comprising an ACP-responsive promoter and a second exogenous polynucleotide sequence having the formula: (L-E)_Xwherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, X=1 to 20, wherein the ACP-responsive promoter is operably linked to the second exogenous polynucleotide, wherein for the first iteration of the (L-E) unit, L is absent, and wherein the ACP is capable of inducing expression of the second expression cassette by binding to the ACP-responsive promoter.

In certain aspects, provided herein are engineered nucleic acids comprising: a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an antigen recognizing receptor, wherein the first promoter is operably linked to the first exogenous polynucleotide; and a second expression cassette comprising an activation-conditional control polypeptide-responsive (ACP-responsive) promoter and a second exogenous polynucleotide sequence having the formula: (L-E)_Xwherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, X=1 to 20, wherein the ACP-responsive promoter is operably linked to the second exogenous polynucleotide, wherein for the first iteration of the (L-E) unit, L is absent.

In some embodiments, the ACP is capable of inducing expression of the second expression cassette by binding to the ACP-responsive promoter.

In some embodiments, the ACP is the antigen recognizing receptor and the ACP is capable of inducing expression of the second expression cassette following binding of the ACP to a cognate antigen. In some embodiments, the ACP-responsive promoter is an inducible promoter that is capable of being induced by the ACP binding to the cognate antigen. In some embodiments, the ACP-responsive promoter is derived from a promoter region of a gene upregulated following binding of the ACP to the cognate antigen.

In some embodiments, the ACP-responsive promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, and a synthetic promoter.

In some embodiments, the ACP-responsive promoter comprises a minimal promoter.

In some embodiments, the ACP-binding domain comprises one or more zinc finger binding sites.

In some embodiments, further comprising a linker polynucleotide sequence localized between the first expression cassette and the second expression cassette.

In some embodiments, wherein the linker polynucleotide sequence is operably associated with the translation of the ACP and each effector molecule as separate polypeptides.

In some embodiments, the first exogenous polynucleotide sequence further comprises a linker polynucleotide sequence localized between the region of the first exogenous polynucleotide sequence encoding the ACP and the region of the first exogenous polynucleotide sequence encoding the antigen recognizing receptor. In some embodiments, the linker polynucleotide sequence is operably associated with the translation of the ACP and the antigen recognizing receptor as separate polypeptides.

In some embodiments, the engineered nucleic acids further comprise a linker polynucleotide sequence localized between the first expression cassette and the second expression cassette. In some embodiments, the linker polynucleotide sequence is operably associated with the translation of the antigen receptor and each effector molecule as separate polypeptides.

In some embodiments, the first promoter is operably linked to the first exogenous polynucleotide sequence encoding the ACP, linker polynucleotide sequence, and antigen recognizing receptor.

In some embodiments, the linker polynucleotide sequence encodes a 2A ribosome skipping tag. In some embodiments, the 2A ribosome skipping tag is selected from the group consisting of: P2A, T2A, E2A, and F2A. In some embodiments, the linker polynucleotide sequence encodes an Internal Ribosome Entry Site (IRES). In some embodiments, the linker polynucleotide sequence encodes a cleavable polypeptide. In some embodiments, the cleavable polypeptide comprises a furin polypeptide sequence.

In some embodiments, the antigen recognizing receptor recognizes an antigen selected from the group consisting of: 5T4, ADAMS, AFP, AXL, B7-H3, B7-H4, B7-H6, C4.4, CA6, Cadherin 3, Cadherin 6, CCR4, CD123, CD133, CD138, CD142, CD166, CD25, CD30, CD352, CD37, CD38, CD44, CD56, CD66e, CD70, CD71, CD74, CD79b, CD80, CEA, CEACAM5, Claudin18.2, cMet, CSPG4, CTLA, DLK1, DLL3, DR5, EGFR, ENPP3, EpCAM, EphA2, Ephrin A4, ETBR, FGFR2, FGFR3, FRalpha, FRb, GCC, GD2, GFRa4, gpA33, GPC3, gpNBM, GPRC5, HER2, IL-13R, IL-13Ra, IL-13Ra2, IL-8, IL-15, IL1RAP, Integrin aV, KIT, L1CAM, LAMP1, Lewis Y, LeY, LIV-1, LRRC, LY6E, MCSP, Mesothelin, MUC1, MUC16, MUC1C, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY ESO 1, Ovarin, P-cadherin, pan-Erb2, PSCA, PSMA, PTK7, ROR1, S Aures, SCT, SLAMF7, SLITRK6, SSTR2, STEAP1, Survivin, TDGF1, TIM1, TROP2, and WT1. In some embodiments, the antigen recognizing receptor recognizes GPC3. In some embodiments, the antigen recognizing receptor recognizes mesothelin (MSLN).

In some embodiments, the antigen recognizing receptor comprises an antigen-binding domain.

In some embodiments, the antigen-binding domain that binds to GPC3 comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of KNAMN (SEQ ID NO: 119), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO: 120), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY (SEQ ID NO: 121), and wherein the VL comprises: a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO: 122), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of WASSRES (SEQ ID NO: 123), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT (SEQ ID NO: 124).

In some embodiments, the VH region comprises an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of

(SEQ ID NO: 125)

EVQLVETGGGMVQPEGSLKLSCAASGFTFNKNAMNWVRQAPGKGLEWVA

RIRNKTNNYATYYADSVKARFTISRDDSQSMLYLQMNNLKIEDTAMYYC

VAGNSFAYWGQGTLVTVSA

or

(SEQ ID NO: 126)

EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVG

RIRNKTNNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYC

VAGNSFAYWGQGTLVTVSA.

In some embodiments, the VL region comprises an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of

(SEQ ID NO: 127)

DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQKNYLAWYQQKPGQS

PKLLIYWASSRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYY

NYPLTFGAGTKLELK,

or

(SEQ ID NO: 128)

DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQP

PKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYY

NYPLTFGQGTKLEIK.

In some embodiments, the antigen-binding domain that binds to MSLN comprises the three complementarity determining regions (CDRs) of a single-domain monoclonal antibody having the amino acid sequence of:

(SEQ ID NO: 129)

QVQLVESGGGTVQAGGSLKLACAASGLPRTYNVMGWFRQAPGKEREGVA

IIYTTTGATYYRDSVKGRATISQDNAKKSVSLQMNSLRPEDTAIYYCVA

RQPNSGPWEYWGQGTQVTVSS,

or

(SEQ ID NO: 130)

QVKLEESGGGSVQAGGSLRLSCTTSGYTNSYKWMGWFRQAPGQEREGVA

VIYTGNDRTYYSDSVKGRFTISRDNAKNMIYLDMTRLRPEDSAVYECAI

GHDGAWRYWGQGTQVTVSS.

In some embodiments, the antigen-binding domain comprises an antibody, an antigen-binding fragment of an antibody, a F(ab) fragment, a F(ab′) fragment, a single chain variable fragment (scFv), or a single-domain antibody (sdAb).

In some embodiments, the antigen-binding domain comprises a single chain variable fragment (scFv).

In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL).

In some embodiments, the VH and VL are separated by a peptide linker.

In some embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.

In some embodiments, the antigen recognizing receptor is a chimeric antigen receptor (CAR) or T cell receptor (TCR).

In some embodiments, the antigen recognizing receptor is a CAR.

In some embodiments, the CAR comprises one or more intracellular signaling domains, and each of the one or more intracellular signaling domains is selected from the group consisting of: a CD3zeta-chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a KIR3DS1 intracellular signaling domain, a NKp44 intracellular signaling domain, a NKp46 intracellular signaling domain, a FceRlg intracellular signaling domain, a NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling domain.

In some embodiments, the CAR comprises a transmembrane domain, and the transmembrane domain is selected from the group consisting of: a CD8 transmembrane domain, a CD28 transmembrane domain a CD3zeta-chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, a BTLA transmembrane domain, an OX40 transmembrane domain, a DAP10 transmembrane domain, a DAP12 transmembrane domain, a CD16a transmembrane domain, a DNAM-1 transmembrane domain, a KIR2DS1 transmembrane domain, a KIR3DS1 transmembrane domain, an NKp44 transmembrane domain, an NKp46 transmembrane domain, an FceRlg transmembrane domain, and an NKG2D.

In some embodiments, the CAR comprises a spacer region between the antigen-binding domain and the transmembrane domain.

In some embodiments, the ACP is a transcriptional modulator.

In some embodiments, the ACP is a transcriptional repressor.

In some embodiments, the ACP is a transcriptional activator.

In some embodiments, the ACP further comprises a repressible protease and one or more cognate cleavage sites of the repressible protease.

In some embodiments, the ACP further comprises a hormone-binding domain of estrogen receptor (ERT2 domain).

In some embodiments, the ACP is a transcription factor.

In some embodiments, the transcription factor is a zinc-finger-containing transcription factor.

In some embodiments, the ACP comprises a DNA-binding zinc finger protein domain (ZF protein domain) and a transcriptional effector domain.

In some embodiments, the ZF protein domain is modular in design and is composed of zinc finger arrays (ZFA).

In some embodiments, the ZF protein domain comprises one to ten ZFA.

In some embodiments, the effector domain is selected from the group consisting of: a Herpes Simplex Virus Protein 16 (VP16) activation domain; an activation domain consisting of four tandem copies of VP16, a VP64 activation domain; a p65 activation domain of NFκB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains, the tripartite activator is known as a VPR activation domain; a histone acetyltransferase (HAT) core domain of the human E1A-associated protein p300, known as a p300 HAT core activation domain; a Krüppel associated box (KRAB) repression domain; a truncated Krüppel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain.

In some embodiments, the one or more cognate cleavage sites of the repressible protease are localized between the ZF protein domain and the effector domain.

In some embodiments, the repressible protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3).

In some embodiments, the cognate cleavage site comprises an NS3 protease cleavage site.

In some embodiments, the NS3 protease cleavage site comprises a NS3/NS4A, a NS4A/NS4B, a NS4B/NS5A, or a NS5A/NS5B junction cleavage site.

In some embodiments, the NS3 protease can be repressed by a protease inhibitor.

In some embodiments, the protease inhibitor is selected from the group consisting of: simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir. In some embodiments, the protease inhibitor is grazoprevir. In some embodiments, the protease inhibitor is grazoprevir and and elbasvir. In some embodiments, wherein the grazoprevir and the elbasvir is co-formulated in a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a tablet. In some embodiments, the grazoprevir and the elbasvir are at a 2 to 1 weight ratio. In some embodiments, the grazoprevir is 100 mg per unit dose and the elbasvir is 50 mg per unit dose.

In some embodiments, the ACP is capable of undergoing nuclear localization upon binding of the ERT2 domain to tamoxifen or a metabolite thereof.

In some embodiments, the tamoxifen metabolite is selected from the group consisting of: 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

In some embodiments, the ACP further comprises a degron, and wherein the degron is operably linked to the ACP.

In some embodiments, the degron is selected from the group consisting of HCV NS4 degron, PEST (two copies of residues 277-307 of human IκBα), GRR (residues 352-408 of human p105), DRR (residues 210-295 of yeast Cdc34), SNS (tandem repeat of SP2 and NB (SP2-NB-SP2 of influenza A or influenza B), RPB (four copies of residues 1688-1702 of yeast RPB), SPmix (tandem repeat of SP1 and SP2 (SP2-SP1-SP2-SP1-SP2 of influenza A virus M2 protein), NS2 (three copies of residues 79-93 of influenza A virus NS protein), ODC (residues 106-142 of ornithine decarboxylase), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC including D433A and D434A point mutations), an APC/C degron, a COP1 E3 ligase binding degron motif, a CRL4-Cdt2 binding PIP degron, an actinfilin-binding degron, a KEAP1 binding degron, a KLHL2 and KLHL3 binding degron, an MDM2 binding motif, an N-degron, a hydroxyproline modification in hypoxia signaling, a phytohormone-dependent SCF-LRR-binding degron, an SCF ubiquitin ligase binding phosphodegron, a phytohormone-dependent SCF-LRR-binding degron, a DSGxxS phospho-dependent degron, an Siah binding motif, an SPOP SBC docking motif, and a PCNA binding PIP box.

In some embodiments, the CRBN polypeptide substrate domain is selected from the group consisting of: IKZF1, IKZF3, CK1a, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, or a fragment thereof that is capable of drug-inducible binding of CRBN.

In some embodiments, the CRBN polypeptide substrate domain is a chimeric fusion product of native CRBN polypeptide sequences.

In some embodiments, the CRBN polypeptide substrate domain is a IKZF3/ZFP91/IKZF3 chimeric fusion product having the amino acid sequence of

(SEQ ID NO: 131)

FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHL

CNYACQRRDAL.

In some embodiments, the IMiD is an FDA-approved drug.

In some embodiments, the IMiD is selected from the group consisting of: thalidomide, lenalidomide, and pomalidomide.

In some embodiments, the degron is localized 5′ of the repressible protease, 3′ of the repressible protease, 5′ of the ZF protein domain, 3′ of the ZF protein domain, 5′ of the effector domain, or 3′ of the effector domain.

In some embodiments, the engineered nucleic acid further comprises an insulator.

In some embodiments, the insulator is localized between the first expression cassette and the second expression cassette.

In some embodiments, the first expression cassette is localized in the same orientation relative to the second expression cassette.

In some embodiments, the first expression cassette is localized in the opposite orientation relative to the second expression cassette.

In some embodiments, the engineered nucleic acid is selected from the group consisting of: a DNA, a cDNA, an RNA, an mRNA, and a naked plasmid.

In another aspect, provided herein are expression vectors comprising the engineered nucleic acid, the expression sysem, or the first expression cassette, the second expression cassette, and/or the additional expression cassettes disclosed herein.

In another aspect, provided herein are compositions comprising the engineered nucleic acid, the expression sysem, or the first expression cassette, the second expression cassette, and/or the additional expression cassette described herein, and a pharmaceutically acceptable carrier.

In another aspect, provided herein are isolated cells comprising the engineered nucleic acid, the expression sysem, or the first expression cassette, the second expression cassette, and/or the additional expression cassette described herein or the vector as described herein.

In some embodiments, the engineered nucleic acid is recombinantly expressed.

In some embodiments, the engineered nucleic acid is expressed from a vector or a selected locus from the genome of the cell.

In some embodiments, the cell is selected from the group consisting of: a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell. In some embodiments, the cell is a Natural Killer (NK) cell.

In some embodiments, the cell is autologous.

In some embodiments, the cell is allogeneic.

In some embodiments, the cell is a tumor cell selected from the group consisting of: an adenocarcinoma cell, a bladder tumor cell, a brain tumor cell, a breast tumor cell, a cervical tumor cell, a colorectal tumor cell, an esophageal tumor cell, a glioma cell, a kidney tumor cell, a liver tumor cell, a lung tumor cell, a melanoma cell, a mesothelioma cell, an ovarian tumor cell, a pancreatic tumor cell, a gastric tumor cell, a testicular yolk sac tumor cell, a prostate tumor cell, a skin tumor cell, a thyroid tumor cell, and a uterine tumor cell.

In some embodiments, the cell is engineered via transduction with an oncolytic virus.

In some embodiments, the oncolytic virus is selected from the group consisting of: an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic sindbis virus, and any variant or derivative thereof.

In some embodiments, the oncolytic virus is a recombinant oncolytic virus comprising the first expression cassette and the second expression cassette.

In some embodiments, the cell is a bacterial cell selected from the group consisting of: Clostridium beijerinckii, Clostridium sporogenes, Clostridium novyi, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, and Salmonella choleraesuis.

In another aspect, provided herein are compositions comprising the isolated cell described herein, and a pharmaceutically acceptable carrier.

In another aspect, provided herein are methods of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of any of the isolated cells or the compositions described herein.

In another aspect, provided herein are methods of stimulating a cell-mediated immune response to a tumor cell in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of any of the isolated cells or the compositions described herein.

In another aspect, provided herein are methods of providing an anti-tumor immunity in a subject, the method comprising administering to a subject in need thereof a therapeutically effective dose of any of the isolated cells or the compositions described herein.

In another aspect, provided herein are methods of treating a subject having cancer, the method comprising administering a therapeutically effective dose of any of the isolated cells or the compositions described herein.

In another aspect, provided herein are methods of reducing tumor volume in a subject, the method comprising administering to a subject having a tumor a composition comprising any of the isolated cells or the compositions described herein.

In some embodiments, the administering comprises systemic administration.

In some embodiments, the administering comprises intratumoral administration.

In some embodiments, the isolated cell is derived from the subject.

In some embodiments, the isolated cell is allogeneic with reference to the subject.

In some embodiments, the method further comprises administering a checkpoint inhibitor.

In some embodiments, the checkpoint inhibitor is selected from the group consisting of: an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-MR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GALS antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFa antibody, an anti-TREM1 antibody, and an anti-TREM2 antibody.

In some embodiments, the method further comprises administering an anti-CD40 antibody.

In some embodiments, the tumor is selected from the group consisting of: an adenocarcinoma, a bladder tumor, a brain tumor, a breast tumor, a cervical tumor, a colorectal tumor, an esophageal tumor, a glioma, a kidney tumor, a liver tumor, a lung tumor, a melanoma, a mesothelioma, an ovarian tumor, a pancreatic tumor, a gastric tumor, a testicular yolk sac tumor, a prostate tumor, a skin tumor, a thyroid tumor, and a uterine tumor.

In another aspect, provided herein are lipid-based structures the engineered nucleic acid, the expression sysem, or the first expression cassette, the second expression cassette, and/or the additional expression cassette described herein.

In some embodiments, the lipid-based structure comprises a extracellular vesicle.

In some embodiments, the extracellular vesicle is selected from the group consisting of: a nanovesicle and an exosome.

In some embodiments, the lipid-based structure comprises a lipid nanoparticle or a micelle.

In some embodiments, the lipid-based structure comprises a liposome.

In another aspect, provided herein are compositions comprising the lipid-based structure described herein, and a pharmaceutically acceptable carrier.

In another aspect, provided herein are methods of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of any of the lipid-based structures or compositions described herein.

In another aspect, provided herein are methods of stimulating a cell-mediated immune response to a tumor cell in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of any of the lipid-based structures or compositions described herein.

In another aspect, provided herein are methods of providing an anti-tumor immunity in a subject, the method comprising administering to a subject in need thereof a therapeutically effective dose of any of the lipid-based structures or compositions described herein.

In another aspect, provided herein are methods of treating a subject having cancer, the method comprising administering a therapeutically effective dose of any of the lipid-based structures or compositions described herein.

In another aspect, provided herein are methods of reducing tumor volume in a subject, the method comprising administering to a subject having a tumor a composition comprising any of the lipid-based structures or compositions described herein.

In some embodiments, the administering comprises systemic administration.

In some embodiments, the administering comprises intratumoral administration.

In some embodiments, the lipid-based structure is capable of engineering a cell in the subject.

In some embodiments, the method further comprises administering a checkpoint inhibitor.

In some embodiments, the method further comprises administering an anti-CD40 antibody.

In another aspect, provided herein are nanoparticles the engineered nucleic acid, the expression sysem, or the first expression cassette, the second expression cassette, and/or the additional expression cassette described herein.

In some embodiments, the nanoparticle comprises an inorganic material.

In another aspect, provided herein are compositions comprising the nanoparticles described herein.

In another aspect, provided herein are methods of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of any of the nanoparticles or the compositions described herein

In another aspect, provided herein are methods of stimulating a cell-mediated immune response to a tumor cell in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of any of the nanoparticles or the compositions described herein.

In another aspect, provided herein are methods of providing an anti-tumor immunity in a subject, the method comprising administering to a subject in need thereof a therapeutically effective dose of any of the nanoparticles or the compositions described herein.

In another aspect, provided herein are methods of treating a subject having cancer, the method comprising administering a therapeutically effective dose of any of the nanoparticles or the compositions described herein.

In another aspect, provided herein are methods of reducing tumor volume in a subject, the method comprising administering to a subject having a tumor a composition comprising any of the nanoparticles or the compositions described herein.

In some embodiments, the administering comprises systemic administration.

In some embodiments, the administering comprises intratumoral administration.

In some embodiments, the nanoparticle is capable of engineering a cell in the subject.

In some embodiments, the method further comprises administering a checkpoint inhibitor.

In some embodiments, the method further comprises administering an anti-CD40 antibody.

In another aspect, provided herein are viruses engineered to comprise the engineered nucleic acid described herein.

In some embodiments, the virus is selected from the group consisting of: a lentivirus, a retrovirus, an oncolytic virus, an adenovirus, an adeno-associated virus (AAV), and a virus-like particle (VLP).

In some embodiments, the virus is an oncolytic virus.

In some embodiments, the first expression cassette and the second expression cassette are capable of being expressed in a tumor cell.

In another aspect, provided herein are compositions comprising the engineered virus or the compositions.

In another aspect, provided herein are methods of treating a subject having cancer, the method comprising administering a therapeutically effective dose of any of the engineered viruses or the compositions.

In some embodiments, the administering comprises systemic administration.

In some embodiments, the administering comprises intratumoral administration.

In some embodiments, the engineered virus infects a cell in the subject and expresses the first expression cassette and the second expression cassette.

In some embodiments, the method further comprises administering a checkpoint inhibitor.

In some embodiments, the method further comprises administering an anti-CD40 antibody.

In another aspect, provided herein are engineered cells comprising: a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activation-conditional control polypeptide (ACP), wherein the first promoter is operably linked to the first exogenous polynucleotide; and a second expression cassette comprising an ACP-responsive promoter and a second exogenous polynucleotide sequence having the formula: (L-E)_Xwherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, X=1 to 20, wherein the ACP-responsive promoter is operably linked to the second exogenous polynucleotide, wherein for the first iteration of the (L-E) unit, L is absent, and wherein the ACP is capable of inducing expression of the second expression cassette by binding to the ACP-responsive promoter.

In some embodiments, the first expression cassette and the second expression cassette are encoded by separate polynucleotide sequences.

In some embodiments, the first expression cassette and the second expression cassette are encoded by a single polynucleotide sequence.

The engineered cell of any one of claims 153-155, wherein when the second expression cassette comprises two or more units of (L₁-E)_X, each L₁linker polynucleotide sequence is operably associated with the translation of each effector molecule as a separate polypeptide.

In some embodiments, the engineered cell further comprises a second linker polynucleotide sequence, wherein the second linker polynucleotide links the first expression cassette to the second expression cassette.

In some embodiments, the second linker polynucleotide sequence is operably associated with the translation of each effector molecule and the ACP as separate polypeptides.

In some embodiments, each linker polynucleotide sequence encodes a 2A ribosome skipping tag.

In some embodiments, the 2A ribosome skipping tag is selected from the group consisting of: P2A, T2A, E2A, and F2A.

In some embodiments, each linker polynucleotide sequence encodes an Internal Ribosome Entry Site (IRES).

In some embodiments, the linker polynucleotide sequence encodes a cleavable polypeptide.

In some embodiments, the cleavable polypeptide comprises a furin polypeptide sequence.

In some embodiments, the second expression cassette comprising one or more units of (L₁-E)_Xfurther comprises a polynucleotide sequence encoding a secretion signal peptide.