BICISTRONIC INHIBITORY CHIMERIC ANTIGEN RECEPTOR (ICAR)/ACTIVATING CHIMERIC ANTIGEN RECEPTOR (ACAR) CONSTRUCTS FOR USE IN CANCER THERAPIES

SEQUENCE LISTING

A Sequence Listing is being submitted electronically via EFS in the form of a text file, created Mar. 1, 2023, and named “194110560_1.txt” (690,086 bytes), the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to the field of cancer immunotherapy by employing inhibitory chimeric antigen receptors (iCARs) paired with activating chimeric antigen receptors (aCARs) for use in cancer treatment therapies.

BACKGROUND OF THE INVENTION

The identification of targetable antigens that are exclusively expressed by tumor cells but not by healthy tissue is undoubtedly the major challenge in cancer immunotherapy today. Clinical evidence that T cells are capable of eradicating tumor cells comes from numerous studies evaluating highly diverse approaches for harnessing T cells to treat cancer (Rosenberg and Restifo, Science, 348(6230): 62-68 (2015)). These approaches employ bone marrow transplantation with donor lymphocyte infusion, adoptive transfer of tumor-infiltrating lymphocytes (TILs), treatment with T cells genetically redirected at pre-selected antigens via CARs (Gross and Eshhar, Annual Review of Pharmacology and Toxicology, 56:59-83, (2016)) or T cell receptors (TCRs), the use of immune checkpoint inhibitors, BiTEs (bispecific T-cell engager molecules) technologies; Einsele, H., et al., Cancer, 126(14):3192-3201 (2020)), or active vaccination. Of these, the use of genetically engineered T cells and different strategies for active immunization entail pre-existing information on candidate antigens which are likely to exert a durable clinical response but minimal adverse effects. Yet, as stated in the title of a review by S. Rosenberg, “Finding suitable targets is the major obstacle to cancer gene therapy” (Rosenberg, Cancer Gene Therapy, 21:45-47 (2014))).

The concept of using chimeric antigen receptors (or CARs) to genetically redirect T cells (or other killer cells of the immune system such as natural killer (NK) cells and cytokine-induced killer cells) against antigens of choice in an MHC-independent manner was first introduced by Gross and Eshhar in the late 1980s (Gross et al., PNAS, 86(24):10024-1002 (1989). They are produced synthetically from chimeric genes encoding an extracellular single-chain antibody variable fragment (scFv) fused through a flexible hinge and transmembrane domain to costimulatory domains and signaling components comprising immunoreceptor tyrosine-based activation motifs of CD3-ζ or FcRγ chains capable of T cell activation. At present, CARs are being examined in dozens of clinical trials and have shown exceptionally high efficacy in B cell malignancies (Dotti et al., 2014; Gill and June, 263(1): 68-89 (2015)); Gross and Eshhar, Annual Review of Pharmacology and Toxicology, 56:59-83, 2016). The safety of CAR-T cell therapy is determined, in large part, by its ability to discriminate between the tumor and healthy tissue. A major risk in targeting solid tumors, and the direct cause for adverse autoimmune effects that have been reported in clinical and preclinical studies, is off-tumor, on-target toxicity resulting from extra-tumor expression of the target antigen (dealt with in detail in the review (Gross and Eshhar, 2016b) and (Klebanoff, et al., Nature Medicine 22:26-36 (2016)).

While undoubtedly intriguing, these previous CAR-based approaches require tuning the affinity of CAR scFv's to selectively bind high antigen levels in tumors while minimizing recognition of lower antigen levels in healthy tissues. In addition, the magnitude of both the activating and costimulatory signals needs to be balanced to allow effective on-target, on-tumor T cell reactivity. It is worth noting that in B cell malignancies, CARs targeted antigen exclusive to B cells and did not require titration of affinity or T cell signaling. For solid tumors, whether such balance can be routinely attained in the clinical setting is questionable.

Off-tumor reactivity occurs when the tumor antigen targeted by CAR-redirected killer cells is shared with normal tissue. However, if the normal tissue expresses another surface antigen that is not present on the tumor, it can be targeted by inhibitory CARs (iCARs) that contains an inhibitory signaling moiety which when engaged prevents T-cell activation by the activating CAR (aCAR). Co-expression of aCAR and iCAR will therefore direct killer cells to target tumors while sparing normal tissue.

Instead of an activating domain (such as FcRγ or CD3-ζ), an iCAR possesses a signaling domain derived from an inhibitory receptor which can antagonize T cell activation, such as CTLA-4, PD-1, or NK inhibitory receptors.

There remains a need in the art for cancer therapies, in particular therapies that comprise iCARs in order to limit off-target effects. The present invention meets that need by providing either co-transduction of monocistronic aCAR and iCAR constructs, or bicistronic constructs comprising such iCARs and which find use in cancer treatment.

BRIEF SUMMARY OF THE INVENTION

The present invention provides bicistronic iCAR/aCAR constructs or monocistronic aCAR and iCAR constructs for co-transduction and uses thereof.

The present invention provides a bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction comprising:

- 1. an iCAR portion, wherein the iCAR portion comprises:
- 2. an iCAR single chain variable fragment (scFv) component optionally in the VH-VL or VL-VH orientation;
- 3. an iCAR hinge domain component;
- 4. an iCAR transmembrane (TM) domain component;
- 5. an iCAR inhibitory domain component; and
- 6. an aCAR portion, wherein the iCAR portion comprises:
- 7. an aCAR single chain variable fragment (scFv) component optionally in the VH-VL or VL-VH orientation;
- 8. an aCAR hinge domain component;
- 9. an aCAR co-stimulatory domain component an aCAR activation signaling domain; and
- 11. a linker that connects the iCAR portion in (i) and the aCAR portion in (ii).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the linker connecting the VH-VL or VL-VH in either orientation comprises one or more linker selected from the group consisting of (G4S)X3 linker

(SEQ ID NO:81), G4S (SEQ ID NO:153), (G4S)X3 (SEQ ID NO:154), and Whitlow linker (SEQ ID NO:82).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv component targets an HLA antigen.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the HLA antigen is selected from the group consisting of HLA-A2, HLA-A3, HLA-A, HLA-B, HLA-C, HLA-G, HLA-E, HLA-F, HLA-DPA1, HLA-DQA1, HLA-DQB1, HLA-DQB2, HLA-DRB1, and HLA-DRB5.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv component is selected from the group consisting of BB7.2, 3PF12, 3PF12/C4, 3PF12/F12, 3PF12/B11, W6/32, BBM.1, SN66E3, Ha5C2.A2, MWB1, MWB1-mod, Hz.BB7.2 VH1-69_A18VK, Hz.BB7.2 VH1-69 (27,30)_A18, Hz.BB7.2 VH1-69 (27,30,48)_A18, Hz.BB7.2 VH1-69 (27,30,67)_A18, Hz.BB7.2 VH1-69 (27,30,69)_A18, Hz.BB7.2 VH1-69 (27,30,67,69)_A18, Hz.BB7.2 VH1-3_A18, Hz.BB7.2 VH1-3(48)_A18, Hz.BB7.2 VH1-3(67)_A18, Hz.BB7.2 VH1-3(69)_A18, Hz.BB7.2 VH1-3(71)_A18, Hz.BB7.2 VH1-3(73)_A18, MWB1.2, SN66E3.2 and SN66E3.3.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv component is BB7.2.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from BB7.2 (SEQ ID NOs: 37 and 38) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 37 and 38.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from Hz.BB7.2 VH1-69_A18VK (SEQ ID NOs: 57 and 58) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 57 and 58.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, wherein the iCAR scFv comprises the Vh and Vl from Hz.BB7.2 VH1-69 (27,30) A18 (SEQ ID NOs: 59 and 60) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 59 and 60.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from Hz.BB7.2 VH1-69 (27,30,48) A18 (SEQ ID NOs: 61 and 62) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 61 and 62.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from Hz.BB7.2 VH1-69 (27,30,67) A18 (SEQ ID NOs: 63 and 64) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 63 and 64.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from Hz.BB7.2 VH1-69 (27,30,69) A18 (SEQ ID NOs: 65 and 66) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 65 and 66.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from Hz.BB7.2 VH1-69 (27,30,67,69) A18 (SEQ ID NOs: 67 and 68) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 67 and 68.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from Hz.BB7.2 VH1-3_A18 (SEQ ID NOs: 69 and 70) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 69 and 70.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from Hz.BB7.2 VH1-3(48) A18 (SEQ ID NOs: 71 and 72) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 71 and 72.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from Hz.BB7.2 VH1-3(67) A18 (SEQ ID NOs: 73 and 74) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 73 and 74.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from Hz.BB7.2 VH1-3(69) A18 (SEQ ID NOs: 75 and 76) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 75 and 76.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from Hz.BB7.2 VH1-3(71) A18 (SEQ ID NOs: 77 and 78) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 77 and 78.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from Hz.BB7.2 VH1-3(73) A18 (SEQ ID NOs: 79 and 80) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 79 and 80.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv is BB7.2 of SEQ ID NO:167.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv component is 3PF12.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from 3PF12/C4 (SEQ ID NOs: 39 and 40) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 39 and 40.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from 3PF12/F12 (SEQ ID NOs: 41 and 42) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 41 and 42.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, wherein the iCAR scFv comprises the Vh and Vl from 3PF12/B11 (SEQ ID NOs: 43 and 44) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 43 and 44.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv is 3PF12 of SEQ ID NO:168.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv component is SN66E3.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from SN66E3.1 (SEQ ID NOs: 49 and 50) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 49 and 50.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv is SN66E3.1 of SEQ ID NO:169.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from SN66E3.2 (SEQ ID NOs: 165 and 166) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 165 and 166.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv is SN66E3.2 of SEQ ID NO:285.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from SN66E3.3 (SEQ ID NOs: 283 and 284) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 283 and 284.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv is SN66E3.3 of SEQ ID NO:286.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv component is W6/32.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from W6/32 (SEQ ID NOs: 45 and 46) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 45 and 46.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv component is BBM.1.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from BBM.1 (SEQ ID NOs: 47 and 48) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 47 and 48.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv component is Ha5C2.A2.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from Ha5C2.A2 (SEQ ID NOs: 51 and 52) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 51 and 52.

T In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv component is MWB1.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from MWB1 (SEQ ID NOs: 53 and 54) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 53 and 54.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from MWB1-mod (MWB1.1) (SEQ ID NOs: 55 and 56) or vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3 from SEQ ID NOs: 55 and 56.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv comprises the Vh and Vl from MWB1.2 (SEQ ID NOs: 163 and 164).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv is MWB1.1 scFvVH_VL (SEQ ID NO:273).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR scFv is MWB1.2 scFvVH_VL (SEQ ID NO:274).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is selected from a PD-1 hinge, a CD28 hinge, and a CD8 hinge (including a CD8a hinge), a LIR1 Ig3-4 hinge, a LIR1 Ig-4 hinge, a LIR1 52 aa hinge, a LIR1 36 aa hinge, a LIR1 30 aa hinge, a LIR1 26 aa hinge, a LIR1 8 aa hinge, a CD33 hinge, a KIR2DL1 hinge, a PD-1 (47) hinge, a PD-1 (42) hinge, a PD-1 (36) hinge, a PD-1 (30) hinge, a PD-1 (26) hinge, and a PD-1 (20) hinge.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a PD-1 hinge (SEQ ID NO: 86).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a CD28 hinge (SEQ ID NO: 85).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a CD8 alpha hinge (SEQ ID NO: 84).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a LIR1 Ig3-4 hinge (SEQ ID NO: 87).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a LIR1 Ig-4 hinge (SEQ ID NO: 88).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a LIR1 52 aa hinge (SEQ ID NO: 89).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a LIR1 36 aa hinge (SEQ ID NO: 90).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a LIR1 30 aa hinge (SEQ ID NO: 91).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a LIR1 26 aa hinge (SEQ ID NO: 289).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a LIR1 8 aa hinge (SEQ ID NO:92).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a CD33 hinge (SEQ ID NO: 93).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a KIR2DL1 hinge (SEQ ID NO: 94).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a PD-1 (47) hinge (SEQ ID NO: 290).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a PD-1 (42) hinge (SEQ ID NO: 291).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a PD-1 (36) hinge (SEQ ID NO: 292).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a PD-1 (30) hinge (SEQ ID NO: 293).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a PD-1 (26) hinge (SEQ ID NO: 294).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR hinge domain component is a PD-1 (20) hinge (SEQ ID NO: 295).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR TM domain component is selected from a PD-1 TM domain, a CD28 TM domain, a CD8 TM domain (including a CD8a TM domain), a LIR1 TM domain, a CD33 TM domain, and a KIR2DL1 TM domain.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR TM domain component is a PD-1 TM domain (SEQ ID NO:97).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR TM domain component is a CD28 TM domain (SEQ ID NO:96).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR TM domain component is a CD8 alpha TM domain (SEQ ID NO:95).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR TM domain component is a LIR1 TM domain (SEQ ID NO:98).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR TM domain component is a CD33 TM domain (SEQ ID NO:99).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR TM domain component is a KIR2DL1 TM domain (SEQ ID NO:100).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is an inhibitory domain from a protein selected from the group consisting of PD-1, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR3DL1, KIR3DL2, KIR3DL3, LAIR1, CD22, CD33, SIGLEC5, SIGLEC6, SIGLEC7, SIGLEC8, SIGLEC9, SIGLEC10, SIGLEC11, SIGLEC12, PECAM1/CD31, CD200R1, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, SLAMF1, SLAMF5, BTLA, LAG3, 2B4, CD160, CEACAM1, TIM3, VISTA, TIGIT, SIRPalpha, FcγRIIB, CD5, CD300a, CD300f, LIR1, LIR2, LIR3, LIR5, LIRE, Ly9, 2xPD1(G4S), 2xPD1(PD1), PVRIg, and AA2AR.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a PD-1 inhibitory domain (SEQ ID NO:101).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR component is a KIR2DL1 inhibitory domain (SEQ ID NO:102).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR component is a KIR2DL2 inhibitory domain (SEQ ID NO:103).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR component is a KIR2DL3 inhibitory domain (SEQ ID NO:104).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a KIR2DL4 inhibitory domain (SEQ ID NO:105).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a KIR2DL5A inhibitory domain (SEQ ID NO:106).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a KIR3DL1 inhibitory domain (SEQ ID NO:107).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a KIR3DL2 inhibitory domain (SEQ ID NO:108).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a KIR3DL3 inhibitory domain (SEQ ID NO:109).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a LAIR1 inhibitory domain (SEQ ID NO:110).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a CD22 inhibitory domain (SEQ ID NO:111).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a CD33 inhibitory domain (SEQ ID NO:112).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a SIGLEC5 inhibitory domain (SEQ ID NO:113).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a SIGLEC6 inhibitory domain (SEQ ID NO:114).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a SIGLEC7 inhibitory domain (SEQ ID NO:115).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a SIGLEC8 inhibitory domain (SEQ ID NO:116).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a SIGLEC9 inhibitory domain (SEQ ID NO:117).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a SIGLEC10inhibitory domain (SEQ ID NO:118).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a SIGLEC11inhibitory domain (SEQ ID NO:119).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a SIGLEC12inhibitory domain (SEQ ID NO:120).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a PECAM1/CD31 inhibitory domain (SEQ ID NO:121).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a CD200R1inhibitory domain (SEQ ID NO:122).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a FCRL1inhibitory domain (SEQ ID NO:123).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a FCRL2inhibitory domain (SEQ ID NO:124).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a FCRL3inhibitory domain (SEQ ID NO:125).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a FCRL4 inhibitory domain (SEQ ID NO:126).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a FCRL5 inhibitory domain (SEQ ID NO:127).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a SLAMF1 inhibitory domain (SEQ ID NO:128).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a SLAMF5 inhibitory domain (SEQ ID NO:129).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a BTLA inhibitory domain (SEQ ID NO:130).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a LAG3 inhibitory domain (SEQ ID NO:131).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a 2B4 inhibitory domain (SEQ ID NO:132).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a CD160 inhibitory domain (SEQ ID NO:133).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a CEACAM1 inhibitory domain (SEQ ID NO:134).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a TIM3 inhibitory domain (SEQ ID NO:135).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a VISTA inhibitory domain (SEQ ID NO:136).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a TIGIT inhibitory domain (SEQ ID NO:137).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a SIRPalpha inhibitory domain (SEQ ID NO:138).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a FcγRIIB inhibitory domain (SEQ ID NO:139).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a CD5 inhibitory domain (SEQ ID NO:140).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a CD300a inhibitory domain (SEQ ID NO:141).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a CD300f inhibitory domain (SEQ ID NO:142).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a LIR1 inhibitory domain (SEQ ID NO:143).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a LIR2 inhibitory domain (SEQ ID NO:144).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a LIR3 inhibitory domain (SEQ ID NO:145).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a LIR5 inhibitory domain (SEQ ID NO:146).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a LIR8 inhibitory domain (SEQ ID NO:147).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a Ly9 inhibitory domain (SEQ ID NO:148).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a PVRIg inhibitory domain (SEQ ID NO:151).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the iCAR inhibitory domain component is a AA2AR inhibitory domain (SEQ ID NO:152).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR single chain variable fragment (scFv) component targets Her2.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from trastuzumab (SEQ ID NOs:170 and 171, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv is SEQ ID NO:172.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from pertuzumab (SEQ ID NOs:173 and 174, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv is SEQ ID NO:175.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from FRP5 (SEQ ID NOs:176 and 177, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from A21 (SEQ ID NOs:178 and 179, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from XMT1517 (SEQ ID NOs:180 and 181, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from XMT1518 (SEQ ID NOs:182 and 183, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from XMT1519 (SEQ ID NOs:184 and 185, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from FWP51 (SEQ ID NOs:186 and 187, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises SEQ ID NOs: 188.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR single chain variable fragment (scFv) component targets EGFR.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from cetuximab (SEQ ID NOs:189 and 190, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv is SEQ ID NO:191.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from panitumumab (SEQ ID NOs:192 and 193, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv is SEQ ID NO:194.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from Imgatuzumab (SEQ ID NOs:195 and 196, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from Nimotuzumab (SEQ ID NOs:197 and 198, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from Necitumumab (SEQ ID NOs:199 and 200, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from ICR62 (SEQ ID NOs:201 and 202, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from Matuzumab (SEQ ID NOs:204 and 205, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from C10 (SEQ ID NOs:206 and 207, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from Zalutumumab (SEQ ID NOs:208 and 209, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from P1X (SEQ ID NOs:210 and 211, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from P2X (SEQ ID NOs:212 and 213, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the Vh and Vl from P3X (SEQ ID NOs:214 and 215, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the VH from EGFR-1a1-VHH (SEQ ID NO:216).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprises the VH from EGFR-VHH (SEQ ID NO:312).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR single chain variable fragment (scFv) component targets Mesothelin.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprise the Vh and Vl from Amatuximab (SEQ ID NOs:217 and 218, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprise the Vh and Vl from P4 (SEQ ID NOs:219 and 220, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprise the Vh and Vl from SS1 (SEQ ID NOs:222 and 223, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprise the VHH from SD1 (SEQ ID NO:225).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprise the VHH from SD2 (SEQ ID NO:226).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprise the Vh and Vl from 1H7 (SEQ ID NOs:227 and 228, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the aCAR scFv comprise the Vh and Vl from 3C02 (SEQ ID NOs:230 and 231, respectively).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the hinge TM domain component is selected from the group consisting of a CD28 hinge and a CD8 hinge (including a CD8a hinge domain).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the hinge TM domain component is a CD28 hinge domain (SEQ ID NO: 85).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the hinge TM domain component is a CD8 alpha hinge domain (SEQ ID NO: 84).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the co-stimulatory domain component is selected from the group consisting of a CD137 (4-1BB) co-stimulatory domain, a CD28 co-stimulatory domain, a 28BB co-stimulatory domain, and a CD3z co-stimulatory domain.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the co-stimulatory domain component is a CD137 (4-1BB) co-stimulatory domain (SEQ ID NO:233).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the co-stimulatory domain component is a CD28 co-stimulatory domain (SEQ ID NO:234).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the co-stimulatory domain component a CD3z activation signaling domain (SEQ ID NO:235).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the ITAM is a CD3 zeta domain.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the ITAM is a CD3 zeta domain (SEQ ID NO:236).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the ITAM is a CD3 zeta 3F domain (SEQ ID NO:237).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the ITAM is a CD3 zeta 4F domain (SEQ ID NO:238).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the ITAM is a CD3 zeta 4OF domain (SEQ ID NO:239).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the linker connecting the iCAR portion and the aCAR portion comprises one or more linker selected from the group consisting of T2A (SEQ ID NO:155), F2A (SEQ ID NO:156), P2A (SEQ ID NO:157), E2A (SEQ ID NO:158), and an IRES sequence (SEQ ID NO:159 or 160).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the bicistronic iCAR/aCAR construct comprises an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the bicistronic iCAR/aCAR construct comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:322, SEQ ID NO:324, and SEQ ID NO:326.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, the bicistronic iCAR/aCAR construct further comprises a short hairpin RNA (shRNA).

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction as described herein comprise an iCAR that comprises a synthetic PD-1 or LIR1 sequence as shown in Table 8, including one selected from the group consisting of SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, and SEQ ID NO:304.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction as described herein comprise an iCAR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:305, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:327, SEQ ID NO:328, SEQ ID NO:329, SEQ ID NO:330, SEQ ID NO:331, SEQ ID NO:332, SEQ ID NO:333, and SEQ ID NO:334.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction as described herein comprise a construct as described in Table 1, Table 11 and/or Table 12.

In some embodiments of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction as described herein comprise a construct or portion thereof as described in any one of Tables 1 to 22.

The present invention also provides for a nucleic acid composition comprising a nucleic acid that encodes a bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction according to any one of the preceding claims.

The present invention also provides for a vector comprising a nucleic acid sequence encoding for a bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction according to any one of the preceding claims.

The present invention also provides for a vector composition comprising the vector according to paragraphs [00192].

In some embodiments, the iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction comprises a signal peptide upstream of the iCAR and/or aCAR portions. In some embodiments, the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

The present invention also provides for a safe effector cell comprising a nucleic acid or nucleic acid sequence composition as described herein.

The present invention also provides for a safe effector cell comprising a vector or vector composition o as described herein.

A safe effector immune cell expressing a bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction as described herein.

A method for treating cancer in a patient having a tumor characterized by LOH, comprising administering to the patient a safe effector immune cell as described herein.

A method for treating cancer in a patient having a tumor characterized by a genetic mutation resulting in a complete loss of expression of a target gene or target extracellular polymorphic epitope gene, comprising administering to the patient a safe effector immune cell as described herein.

A method for treating cancer in a patient having a tumor characterized by loss of heterozygosity (LOH), or other genetic loss or allelic imbalance phenotypes including, without limitation, loss of function or expression, resulting from mutations affecting one or more nucleotides, comprising administering to the patient a safe effector immune cell as described herein.

In some embodiments, the cancer is selected from the group consisting of Acute Myeloid Leukemia [LAML], Adrenocortical carcinoma [ACC], Bladder Urothelial Carcinoma [BLCA], Brain Lower Grade Glioma [LGG], Breast invasive carcinoma [BRCA], Cervical squamous cell carcinoma and endocervical adenocarcinoma [CESC], Cholangiocarcinoma [CHOL], Colon adenocarcinoma [COAD], Esophageal carcinoma [ESCA], Glioblastoma multiforme [GBM], Head and Neck squamous cell carcinoma [HNSC], Kidney Chromophobe [KICH], Kidney renal clear cell carcinoma [KIRC], Kidney renal papillary cell carcinoma [KIRP], Liver hepatocellular carcinoma [LIHC], Lung adenocarcinoma [LUAD], Lung squamous cell carcinoma [LUSC], Lymphoid Neoplasm Diffuse Large B-cell Lymphoma [DLBC], Mesothelioma [MESO], Ovarian serous cystadenocarcinoma [OV], Pancreatic adenocarcinoma [PAAD], Pheochromocytoma and Paraganglioma [PCPG], Prostate adenocarcinoma [PRAD], Rectum adenocarcinoma [READ], Sarcoma [SARC], Skin Cutaneous Melanoma [SKCM], Stomach adenocarcinoma [STAD], Testicular Germ Cell Tumors [TGCT], Thymoma [THYM], Thyroid carcinoma [THCA], Uterine Carcinosarcoma [UCS], Uterine Corpus Endometrial Carcinoma [UCEC], Uveal Melanoma [UVM], Non-small cell lung carcinoma [NSCLC], and Small cell lung cancer [SCLC].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows bicistronic construct design overview and component table.

FIGS. 2A-2H show bicistronic survey—constructs MC0280-MC0300, MC0428, MC0447, MC0449, HLA-A2 shRNA.

FIG. 3 shows BTLA & KIR2DL2 as new iCAR leads.

FIG. 4 shows identification of fully human scFv constructs with higher HLA-A binding avidity.

FIG. 5 shows 3PF12 & SN66E3 PD-1 iCAR exhibit are more stably expressed.

FIG. 6 shows a schematic for luciferase-based cytotoxicity assays.

FIGS. 7A-7B. A) Expression of HER2 Bicistronics Day 9—Donor 466. B) Expression of HER2 Bicistronics Day 9-Donor 149.

FIG. 8 shows luciferase killing results for LIR1 & KIR2DL1 dual CAR. LIR1 inhibits efficiently the aCAR, enabling high protection for MCF7. KIR2DL1 inhibits the aCAR, enabling moderate protection for MCF7.

FIG. 9 shows IFNg ELISA assays showing LIR1 and KIR2DL1 inhibition. LIR1 and KIR2DL1 very efficiently inhibit IFNg secretion against MCF7.

FIG. 10 shows KIR2DL1/2 and LIR1 confirmed as hits in Jurkat assay.

FIGS. 11A-11B shows low dual CAR lentiviral transduction efficiency and variable expression.

FIGS. 12A-12B shows experimental set-up and data regarding target cell killing and CAR-T activation correlate with E/T ratio.

FIG. 13 shows target cell killing and CAR-T activation correlate with E/T ratio.

FIG. 14 shows a quantum bead assay to determine CAR cell surface level.

FIG. 15 shows exceptional differential PD-1 iCAR expression relative to HER2 aCAR.

FIG. 16 shows target antigen quantifications in screen cell-line panel.

FIG. 17 shows PD-1 iCAR directs HLA-A2 specific EGFR a CAR killing (E/T=2).

FIG. 18 shows HLA-A2 POS cancer cells specifically inhibit dual CAR T-cells

FIG. 19 shows iCAR inhibits T-cell degranulation across a wide range of HLA-A2 level.

FIG. 20 shows a PD-1 iCAR directs HLA-A2 specific HER2 aCAR killing.

FIG. 21 shows dual CAR lentiviral expression is highly variable (HER2 aCAR).

FIG. 22 shows cetuximab scFv lentiviral expression is relatively low.

FIG. 23 shows bicistronic constructs express well on Day 8.

FIG. 24 shows bicistronic expression is lower on Day 12

FIG. 25 shows anti-HLA-A2 iCAR screen—construct design

FIGS. 26A-26B shows alternative scFvs with higher HLA binding than BB7.2 identified.

FIG. 27 shows iCAR single chain options.

FIG. 28 shows BB7.2 (two versions), 3PF12, and SN66E3 PD-1 iCAR exhibit are more stably expressed.

FIG. 29 shows KIR2DL1 iCAR identified as hit in FaDu/U87-LUC immune cell killing assay.

FIGS. 30A-30B shows a schematic for IMPT001: A dual CART system designed to kill based on tumor specific loss-of-HLA-A2 gene expression.

FIGS. 31A-31G. shows donor 149 Expression: HER2 Bicistronics Day 12.

FIGS. 32A-32G shows donor 466 Expression: HER2 Bicistronics Day 12.

FIG. 33 shows D149 Luciferase Kill Assay Results Day 12. LIR1 inhibits efficiently the aCAR, enabling high protection for H1703, H1650 and MDA-MB231. KIR2DL1 and CD33 inhibit the aCAR, enabling moderate protection for H1703, H1650 and MDA-MB231.

FIG. 34 shows D466 Luciferase Kill Assay Results Day 12. LIR1 and CD33 inhibits efficiently the aCAR, enabling protection for H1703, H1650. LIR1 and CD33 inhibit very efficiently IFNγ secretion against H1703, H1650 and MCF7.

FIG. 35 shows HER2 Bicistronic Expression Day 8 from an exemplary experiment.

FIG. 36 shows VR51 (LIR1 iDomain) protect HLA-A2POS targets. LIR1 inhibits efficiently the aCAR, allowing high protection for H1650 and moderate protection for MDA-MB-231 cells from an exemplary experiment.

FIG. 37 provides CAR expression on the cell surface. Note: VR52 had very low aCAR expression (excluded from analysis). VR55,56 had no iCAR expression (data not shown) (excluded from analysis). The MFI is of the positive CAR fraction only. To clarify, the aCAR+ fraction of the untransduced cells (3%) has an MFI of 766.

FIGS. 38A-38C showcell staining of transduced PBMCs (raw data).

FIG. 39 show bicistronic iCAR/aCAR constructs show efficacy against A2NEG cell lines.

FIG. 40 show iCAR RNA expression is transient.

FIGS. 41A-41F show in vitro analysis of bicistronic iCAR-aCAR constructs described herein. VR354 was identified as a superior LIR bicistronic construct for protection against HER2 aCAR killing.

FIG. 42 show screen of HLA-A2 scFv as aCAR. All humanized BB7.2 versions expressed well and showed both binding and efficacy against an A2 POS target. The top hit seemed to be VR375 due to even lower EC50 compared to VR370.

FIG. 43 show HLA-A2 enrichment. Anti-PE beads and Miltenyi LS columns were used to achieve successful enrichment of VR51 bicistronic construct in the bound fraction.

FIGS. 44A-44K show screen of synthetic PD1 constructs. Enriched synthetic PD1 constructs screened using the luciferase assay on H1703 isogenic cell lines showed that synthetic constructs containing 1-5 PD1 ITSM repeats showed superior protection compared to 1-5 PD1 ITIM repeats.

FIG. 45 showscreen of 1×vs 2×PD1 constructs. Enriched PD1 constructs screened using luciferase assay and isogenic H1703 cell lines showed that 2×PD1 construct showed better protection than the naturally occurring 1×PD1 construct, with the G4S linker (VR68) providing superior protection over the PD1 linker (VR69).

FIG. 46 show iCAR Engagement Regulates CAR-T Activation. Singular aCAR engagement by iTarget NEG cells induces T-cell activation. Dual aCAR+iCAR engagement inhibits CAR-T activation with iTarget POS cells.

FIG. 47 show iCAR target POS cancer cells inhibit dual CAR T cells.

FIG. 48 show iCAR targeted killing of cancer cell lines.

FIGS. 49A-49B show screen of SN66E3 iCAR scFv constructs. Enriched bicistronic constructs screened using the luciferase assay on H1703 isogenic cell lines showed that constructs containing SN66E3 iCAR scFv showed superior protection.

FIG. 50 show functional Luc results-Screen of Camel VHH EGFR scFv cotransduced with mBB7.2 scFv with LIR1 or PD1x2 iDomains.

FIG. 51 show scheme of the in-vivo study design.

FIG. 52 show scheme of the in-vivo process.

FIGS. 53A-53D show tumor growth kinetics of a representative in-vivo study with main constructs. Both protection and efficacy are observed for the VR354 and VR51.

constructs.

FIGS. 54A-54F show series F in-vivo screen Tumor growth kinetics. The model is the H1703 WT where protection are observed. Top hit for both CAR-T doses is VR428.

FIGS. 55A-55F show series F in-vivo screen Tumor growth kinetics. The model is the H1703 KO where efficacy are observed. Top hit for both CAR-T doses is VR428.

FIG. 56 show in vitro screen for synthetic iDomains comprising variations in the LIR1 ITIM and PD-1 ITSM motifs of the iCAR.

FIG. 57 show series G in vitro screen for humanized and fully human iCAR scFv specific against the HLA-A2 target.

FIG. 58 show in vitro screen for synthetic LIR1 iDomain comprising variations in the ITIM and ITSM motifs of the iCAR.

FIG. 59 show series F in vitro screen for humanized iCAR scFv specific against the HLA-A2 target. Validation of HzBB7.2 iCAR scFv in-vitro.

DETAILED DESCRIPTION OF THE INVENTION
I. Introduction

The present invention provides bicistronic and co-administered monocistronic constructs specifically targeting tumor cells while keeping the normal cells protected. The constructs provided herein provide iCAR/aCAR constructs that target single allelic variants of polymorphic cell surface epitopes, which are lost from tumor cells due to loss of heterozygosity (LOH) of the chromosomal region they reside in, while remaining expressed on normal tissue. Because of the polymorphic variation, the iCAR/aCAR pair is able to distinguish the two alleles and target only the tumor cells missing the target allele due to LOH.

II. Select Definitions

The term “nucleic acid molecule” as used herein refers to a DNA or RNA molecule.

The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

The term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

“Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

The term “genomic variant” as used herein refers to a change of at least one nucleotide at the genomic level in a sequenced sample compared to the reference or consensus sequence at the same genomic position.

The term “corresponding reference allele” as used herein with reference to a variant means the reference or consensus sequence or nucleotide at the same genomic position as the variant.

The term “extracellular domain” as used herein with reference to a protein means a region of the protein which is outside of the cell membrane.

The term “loss of heterozygosity” or “LOH” as used herein means the loss of chromosomal materials such as a complete chromosome or a part thereof, in one copy of the two chromosomes in a somatic cell.

The term “sequence region” as used herein with reference to a variant or a reference allele means a sequence starting upstream and ending downstream from the position of the variant, which can be translated into an “epitope peptide” that can be recognized by an antibody.

The term “CAR”, as that term is used herein, refers to a chimeric polypeptide that shares structural and functional properties with a cell immune-function receptor or adaptor molecule, from e.g., a T cell or a NK cell. CARs include TCARs and NKR-CARs. Upon binding to cognate antigen, a CAR can activate or inactivate the cytotoxic cell in which it is disposed, or modulate the cell's antitumor activity or otherwise modulate the cells immune response.

The term “specific binding” as used herein in the context of an extracellular domain, such as an scFv, that specifically binds to a single allelic variant of a polymorphic cell surface epitope, refers to the relative binding of the scFv to one allelic variant and its failure to bind to the corresponding different allelic variant of the same polymorphic cell surface epitope. Since this depends on the avidity (number of CAR copies on the T cell, number of antigen molecules on the surface of target cells (or cells to be protected) and the affinity of the specific CARs used, a functional definition would be that the specific scFv would provide a significant signal in an ELISA against the single allelic variant of a polymorphic cell surface epitope to which it is specific or cells transfected with a CAR displaying the scFv would be clearly labeled with the single allelic variant of a polymorphic cell surface epitope in a FACS assay, while the same assays using the corresponding different allelic variant of the same polymorphic cell surface epitope would not give any detectable signal.

The term “treating” as used herein refers to means of obtaining a desired physiological effect. The effect may be therapeutic in terms of partially or completely curing a disease and/or symptoms attributed to the disease. The term refers to inhibiting the disease, e.g., arresting its development; or ameliorating the disease, e.g., causing regression of the disease.

As used herein, the terms “subject” or “individual” or “animal” or “patient” or “mammal,” refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired, for example, a human.

The phrase “safe effector immune cell” or “safe effector cell” includes those cells described by the invention that express at least one bicistronic iCAR/aCAR construct, or portion thereof, as described herein, or exhibit co-expression of monocistronic aCAR and iCAR constructs. In some embodiments, the “safe effector immune cell” or “safe effector cell” is capable of administration to a subject. In some embodiments, the “safe effector immune cell” or “safe effector cell” further expresses at least one bicistronic iCAR/aCAR construct, or portion thereof, or exhibit co-expression of monocistronic aCAR and iCAR constructs, as described herein.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

The phrase “effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result.

The term “peripheral blood mononuclear cell (PBMC)” as used herein refers to any blood cell having a round nucleus, such as a lymphocyte, or a monocyte. Methods for isolating PBMCs from blood are readily apparent to those skilled in the art. A non-limiting example is the extraction of these cells from whole blood using ficoll, a hydrophilic polysaccharide that separates layers of blood, with monocytes and lymphocytes forming a buffy coat under a layer of plasma or by leukapheresis, the preparation of leukocyte concentrates with the return of red cells and leukocyte-poor plasma to the donor.

The term “cancer” as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, glioma, and the like.

III. CAR-T SYSTEM: iCARs and aCARs

LOH, being a genomic event, results in a total loss of a specific variant from the tumor with a very rare probability of gaining back the lost allele. If the LOH event occurs very early in the development of tumors, it ensures a uniform target signature in all tumor cells derived from the initial pre-malignant tissue including metastatic tumors. Additionally, LOH occurs in almost all types of cancer and this concept can therefore be relied upon as a universal tool for developing markers relevant to all these cancer types. Since the LOH events are to some extent random, the present invention further provides for selection of personalized tumor markers for each individual cancer patient, based on the specific LOH events which took place in that patient. The tools relied upon to execute this concept, the aCARs and the iCARs, are well-known and can be easily prepared using methods well-known in the art as taught for example, in WO 2015/142314 and in U.S. Pat. No. 9,745,368, both incorporated by reference as if fully disclosed herein.

According to one strategy, the two CARs in every given pair specifically recognize the product of a different allelic variant of the same target gene for which the patient is heterozygous. The basic principle is as follows: the aCAR targets an allelic variant of a selected cell surface protein that is expressed by the given tumor cells and is not affected by LOH while the iCAR targets the product encoded by the allelic variant of the same gene that has been lost from these tumor cells due to LOH. In other normal tissues of that individual patient that express the said gene, both alleles are present and are known to be equally functional, that is, expression is biallelic in all tissues (in contrast to other genes which may exhibit random monoallelic expression (Chess, 2012; Savova et al., 2016). In one scenario, the two CARs target two related epitopes residing at the same location on the protein product, which differ by one, or only few amino acids. In another scenario, the aCAR targets a non-polymorphic epitope on the same protein while the iCAR is allele-specific. In these embodiments, the density of the aCAR epitope on normal cells would generally be two-fold higher than that of the iCAR one. In some embodiments, a single nucleic acid vector encodes both the aCAR and iCAR, as exemplified with the bicistronic constructs described herein. In some embodiments, the aCAR and iCAR are encoded by separate nucleic acid vectors and co-expressed.

Care must be taken to ensure that the inhibitory signal transmitted by the iCAR is dominant over the aCAR signal and that cross-recognition between the iCAR and the aCAR is limited and/or negligible. Dominance of the iCAR guarantees that activation of the killer cell upon encounter with normal cells expressing both alleles would be prevented. This default brake would not operate upon engagement with tumor cells: in the absence of its target antigen the iCAR would not deliver inhibitory signals, thus unleashing the anticipated aCAR-mediated cellular activation and subsequent tumor cell lysis. Dominance of the iCARs over their aCARs counterparts is a significant portion of how the system functions. The present invention provides novel bicistronic iCAR/aCAR constructs that function in this manner, as well as methods for co-transduction of monocistronic aCAR and iCAR constructs.

The bicistronic constructs of the present invention comprise the following components: an iCAR and aCAR connected via a linker domain. In some embodiments, the iCAR (protective) portion comprises an iCAR scFv, a hinge transmembrane (TM) domain, and inhibitory domain. In some embodiments, the aCAR (efficacy) portion comprises an aCAR scFv, a hinge transmembrane (TM) domain, a co-stimulatory domain, and a CD3 zeta domain.

I. Bicistronic Sequences

In some embodiments, the bicistronic iCAR/aCAR comprises an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325, as provided in Table 1 below. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:1. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:3. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:5. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:7. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:9. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:11. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:13. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:15. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:17. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:19. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:21. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:23. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:25. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:27. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:29. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:31. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:33. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:35. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:275. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:277. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:279. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:281. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:321. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:323. In some embodiments, the bicistronic iCAR/aCAR comprise an amino acid sequence encoded by a nucleic acid sequence comprising SEQ ID NO:325.

In some embodiments, the bicistronic iCAR/aCAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:322, SEQ ID NO:324, and SEQ ID NO:326 as provided in Table 1.

below. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:2. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:4. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:6. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:8. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:10. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:12. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:14. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:16. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:18. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:20. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:22. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:24. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:26. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:28. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:30. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:32, SEQ ID NO:34. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:36. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:276. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:278. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:280. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:282. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:322. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:324. In some embodiments, the bicistronic iCAR/aCAR comprises SEQ ID NO:326.

TABLE 1

Bicistonic iCAR/aCARs: nucleic acid and amino acid sequences

Sequence
SEQ ID

name
NO:
Polynucleotide or polypeptide sequences

MC0280-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

BB7.2_28_
NO: 1
GCTGCTGCACGCAGCCAGACCCCAGGTGCAGCTGCAGCAGT

PD1_HER2

CTGGACCTGAGCTGGTGAAGCCAGGAGCCTCCGTGAAGATG

Nucleotide

TCTTGCAAGGCCAGCGGCTACACCTTCACATCTTATCACATC

sequence

CAGTGGGTGAAGCAGCGGCCCGGACAGGGCCTGGAGTGGA

(VR280)

TCGGATGGATCTACCCAGGCGACGGCTCCACACAGTATAAC

GAGAAGTTCAAGGGCAAGACCACACTGACCGCCGATAAGA

GCAGCAGCACCGCCTACATGCTGCTGAGCAGCCTGACCAGC

GAGGACAGCGCCATCTACTTTTGCGCCAGGGAGGGCACATA

CTATGCTATGGACTATTGGGGCCAGGGCACCAGCGTGACAG

TGTCTAGCGGAGGAGGAGGCTCCGGAGGAGGAGGCTCTGG

CGGCGGCGGCAGCGACGTGCTGATGACCCAGACACCACTG

AGCCTGCCCGTGAGCCTGGGCGATCAGGTGAGCATCTCCTG

TAGATCCTCTCAGAGCATCGTGCACTCCAACGGCAATACCT

ACCTGGAGTGGTATCTGCAGAAGCCAGGCCAGTCCCCCAAG

CTGCTGATCTATAAGGTGTCTAATCGGTTCAGCGGCGTGCCT

GACAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCT

GAAGATCAGCCGGGTGGAGGCAGAGGATCTGGGCGTGTAC

TATTGTTTCCAGGGCTCCCACGTGCCACGCACCTTTGGCGGC

GGTACCAAGCTGGAGATCAAGATTGAAGTTATGTATCCTCC

TCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCC

ATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCC

GGGCCTTCGAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGG

AGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTAGCGTTTAT

TATTTTCTGGGTGTGCAGCAGGGCCGCCCGCGGCACCATCG

GCGCCAGGCGCACAGGCCAGCCTCTGAAGGAGGACCCTTCC

GCCGTGCCAGTGTTCTCTGTGGACTACGGCGAGCTGGATTTT

CAGTGGCGGGAGAAAACCCCAGAGCCACCTGTGCCCTGCGT

GCCTGAGCAGACCGAGTATGCCACAATCGTGTTTCCATCCG

GAATGGGCACAAGCTCCCCTGCAAGGAGAGGCAGCGCCGA

CGGACCACGGTCCGCCCAGCCACTGCGGCCCGAGGATGGCC

ACTGTTCTTGGCCCCTGCGGAGAAAGCGTGGATCCGGGGAA

GGCCGAGGCTCCCTTCTAACATGTGGAGATGTCGAGGAAAA

CCCTGGCCCTATGGCGCTGCCAGTCACTGCATTGTTATTGCC

TCTGGCCCTGCTTCTCCATGCGGCGCGCCCAGAAGTGCAGC

TGGTCGAGAGCGGAGGCGGACTGGTTCAACCCGGAGGCAG

CTTGAGACTGTCCTGCGCGGCCAGCGGCTTCAACATCAAGG

ATACCTATATCCACTGGGTGAGGCAGGCTCCAGGAAAGGGC

CTGGAGTGGGTGGCAAGGATTTACCCTACTAATGGATATAC

ACGCTACGCTGATTCCGTGAAGGGACGCTTTACAATCTCAG

CAGATACATCCAAAAACACGGCCTATTTACAGATGAATAGT

TTGCGGGCCGAAGACACGGCTGTATACTATTGTTCTCGGTG

GGGGGGCGATGGATTTTATGCGATGGATTACTGGGGCCAGG

GCACCCTGGTAACCGTGTCAAGCGGCTCAACATCCGGGTCC

GGTAAGCCGGGCTCCGGCGAGGGGTCTACAAAGGGAGATA

TACAGATGACACAGTCCCCCAGTTCCCTGTCCGCCTCAGTG

GGAGACCGAGTGACGATTACCTGTCGTGCCAGCCAGGACGT

CAATACCGCCGTCGCTTGGTATCAGCAAAAACCAGGCAAGG

CCCCGAAACTATTGATCTACAGTGCCTCTTTTCTGTACTCCG

GGGTGCCGAGCAGATTTAGTGGCTCCAGGAGCGGAACCGAT

TTCACCCTAACCATTTCCAGTTTGCAGCCAGAGGATTTCGCG

ACCTATTACTGCCAGCAACACTACACCACACCGCCAACTTT

CGGACAAGGAACCAAGGTTGAAATCAAAACTACGACCCCA

GCACCTAGACCTCCCACCCCAGCTCCAACTATAGCTTCCCA

GCCATTGTCTCTCCGGCCAGAGGCGTGTCGACCAGCCGCTG

GAGGGGCCGTTCATACAAGAGGACTCGATTTCGCTTGCGAT

ATCTACATATGGGCCCCTCTTGCCGGGACATGCGGTGTCCT

GCTTCTAAGCTTGGTTATTACCCTCTATTGCAAACGCGGCCG

CAAGAAACTGCTCTACATCTTTAAACAGCCGTTCATGAGGC

CTGTGCAGACAACGCAGGAAGAGGATGGCTGTAGTTGTCGG

TTTCCGGAAGAGGAAGAGGGGGGCTGCGAGTTGCGTGTCA

AATTTTCTCGGTCTGCCGACGCCCCCGCGTACCAGCAAGGG

CAGAACCAGCTTTATAATGAGCTGAATCTTGGACGACGGGA

GGAATATGACGTGCTTGACAAGAGGCGAGGTAGGGACCCT

GAGATGGGGGGAAAACCTCGGAGGAAAAACCCACAGGAAG

GCCTGTATAACGAACTGCAGAAGGACAAGATGGCTGAAGC

CTACTCTGAGATTGGAATGAAAGGGGAACGCAGACGCGGC

AAGGGCCATGATGGCCTCTACCAAGGTCTAAGCACTGCCAC

CAAGGACACCTATGACGCACTCCACATGCAAGCTCTACCTC

CCCGTTGATAA

MC0280-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKMS

BB7.2_28_
NO: 2
CKASGYTFTSYHIQWVKQRPGQGLEWIGWIYPGDGSTQYNEK

PD1_HER2

FKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCAREGTYYAMD

Protein

YWGQGTSVTVSSGGGGSGGGGSGGGGSDVLMTQTPLSLPVSL

sequence

GDQVSISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNR

(VR280)

FSGVPDRESGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTF

GGGTKLEIKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPG

PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVCSRAARGTIGAR

RTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQT

EYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLR

RKRGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAA

RPEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP

GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQM

NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGSTS

GSGKPGSGEGSTKGDIQMTQSPSSLSASVGDRVTITCRASQDV

NTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFT

LTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKTTTPAPRPPT

PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAG

TCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGC

SCRFPEEEEGGCELRVKESRSADAPAYQQGQNQLYNELNLGR

REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE

AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP

PR

MC0281-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

BB7.2_28_
NO: 3
GCTGCTGCACGCAGCCAGACCCCAGGTGCAGCTGCAGCAGT

PD1_EGFR

CTGGACCTGAGCTGGTGAAGCCAGGAGCCTCCGTGAAGATG

nucleotide

TCTTGCAAGGCCAGCGGCTACACCTTCACATCTTATCACATC

Sequence

CAGTGGGTGAAGCAGCGGCCCGGACAGGGCCTGGAGTGGA

(VR281)

TCGGATGGATCTACCCAGGCGACGGCTCCACACAGTATAAC

GAGAAGTTCAAGGGCAAGACCACACTGACCGCCGATAAGA

GCAGCAGCACCGCCTACATGCTGCTGAGCAGCCTGACCAGC

GAGGACAGCGCCATCTACTTTTGCGCCAGGGAGGGCACATA

CTATGCTATGGACTATTGGGGCCAGGGCACCAGCGTGACAG

TGTCTAGCGGAGGAGGAGGCTCCGGAGGAGGAGGCTCTGG

CGGCGGCGGCAGCGACGTGCTGATGACCCAGACACCACTG

AGCCTGCCCGTGAGCCTGGGCGATCAGGTGAGCATCTCCTG

TAGATCCTCTCAGAGCATCGTGCACTCCAACGGCAATACCT

ACCTGGAGTGGTATCTGCAGAAGCCAGGCCAGTCCCCCAAG

CTGCTGATCTATAAGGTGTCTAATCGGTTCAGCGGCGTGCCT

GACAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCT

GAAGATCAGCCGGGTGGAGGCAGAGGATCTGGGCGTGTAC

TATTGTTTCCAGGGCTCCCACGTGCCACGCACCTTTGGCGGC

GGTACCAAGCTGGAGATCAAGATTGAAGTTATGTATCCTCC

TCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCC

ATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCC

GGGCCTTCGAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGG

AGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTAGCGTTTAT

TATTTTCTGGGTGTGCAGCAGGGCCGCCCGCGGCACCATCG

GCGCCAGGCGCACAGGCCAGCCTCTGAAGGAGGACCCTTCC

GCCGTGCCAGTGTTCTCTGTGGACTACGGCGAGCTGGATTTT

CAGTGGCGGGAGAAAACCCCAGAGCCACCTGTGCCCTGCGT

GCCTGAGCAGACCGAGTATGCCACAATCGTGTTTCCATCCG

GAATGGGCACAAGCTCCCCTGCAAGGAGAGGCAGCGCCGA

CGGACCACGGTCCGCCCAGCCACTGCGGCCCGAGGATGGCC

ACTGTTCTTGGCCCCTGCGGAGAAAGCGTGGATCCGGGGAA

GGCCGAGGCTCCCTTCTAACATGTGGAGATGTCGAGGAAAA

CCCTGGCCCTATGGCGCTGCCAGTCACTGCATTGTTATTGCC

TCTGGCCCTGCTTCTCCATGCGGCGCGCCCACAAGTGCAGC

TGAAACAGAGCGGACCAGGACTGGTTCAACCCAGCCAGAG

CTTGAGCATCACGTGCACGGTTAGCGGCTTCAGTCTGACCA

ATTATGGTGTGCACTGGGTGAGGCAGTCTCCAGGAAAGGGC

CTGGAGTGGCTTGGAGTCATTTGGAGCGGTGGGAATACAGA

TTACAATACACCTTTTACGTCACGTCTCTCCATTAACAAGGA

CAACTCCAAATCCCAAGTATTTTTCAAAATGAATAGCCTGC

AGAGTAATGATACCGCCATCTATTACTGTGCACGAGCTTTG

ACATATTACGACTATGAATTTGCCTATTGGGGTCAAGGCAC

GCTGGTGACCGTATCAGGCTCAACATCCGGGTCCGGTAAGC

CGGGCTCCGGCGAGGGGTCTACAAAGGGAGACATCCTTCTG

ACACAGAGCCCCGTGATCCTGTCCGTGTCCCCCGGCGAGAG

AGTATCATTTTCCTGTAGGGCTTCTCAGAGCATCGGAACAA

ATATCCACTGGTATCAGCAACGGACTAACGGATCACCTCGC

CTGCTCATAAAGTACGCCAGTGAATCTATTAGTGGCATACC

GAGCCGCTTCAGCGGGAGTGGCTCCGGCACAGACTTTACTC

TGAGTATAAATTCCGTGGAATCTGAGGACATCGCGGACTAT

TACTGCCAGCAAAACAATAACTGGCCCACCACGTTCGGCGC

GGGAACTAAACTAGAACTAAAGACTACGACCCCAGCACCT

AGACCTCCCACCCCAGCTCCAACTATAGCTTCCCAGCCATT

GTCTCTCCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGGG

CCGTTCATACAAGAGGACTCGATTTCGCTTGCGATATCTAC

ATATGGGCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCTA

AGCTTGGTTATTACCCTCTATTGCAAACGCGGCCGCAAGAA

ACTGCTCTACATCTTTAAACAGCCGTTCATGAGGCCTGTGCA

GACAACGCAGGAAGAGGATGGCTGTAGTTGTCGGTTTCCGG

AAGAGGAAGAGGGGGGCTGCGAGTTGCGTGTCAAATTTTCT

CGGTCTGCCGACGCCCCCGCGTACCAGCAAGGGCAGAACCA

GCTTTATAATGAGCTGAATCTTGGACGACGGGAGGAATATG

ACGTGCTTGACAAGAGGCGAGGTAGGGACCCTGAGATGGG

GGGAAAACCTCGGAGGAAAAACCCACAGGAAGGCCTGTAT

AACGAACTGCAGAAGGACAAGATGGCTGAAGCCTACTCTG

AGATTGGAATGAAAGGGGAACGCAGACGCGGCAAGGGCCA

TGATGGCCTCTACCAAGGTCTAAGCACTGCCACCAAGGACA

CCTATGACGCACTCCACATGCAAGCTCTACCTCCCCGTTGAT

AA

MC0281-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKMS

BB7.2_28_
NO: 4
CKASGYTFTSYHIQWVKQRPGQGLEWIGWIYPGDGSTQYNEK

PD1_EGFR

FKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCAREGTYYAMD

Protein

YWGQGTSVTVSSGGGGSGGGGSGGGGSDVLMTQTPLSLPVSL

Sequence

GDQVSISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNR

(VR281)

FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTF

GGGTKLEIKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPG

PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVCSRAARGTIGAR

RTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQT

EYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLR

RKRGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAA

RPQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSP

GKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMN

SLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSGSTSGSG

KPGSGEGSTKGDILLTQSPVILSVSPGERVSFSCRASQSIGTNIH

WYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVES

EDIADYYCQQNNNWPTTFGAGTKLELKTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL

LSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE

EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL

DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM

KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

MC0282-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

3PF12_28_
NO: 5
GCTGCTGCACGCAGCCAGACCCCAAGTGCAACTAGTCCAAT

PD1_HER2

CAGGTGGAGGCGTCGTGCAACCTGGAGGGTCCCTCCGCGTT

Nucleotide

AGCTGCGCCGCATCAGGCGTTACCTTGTCAGACTACGGCAT

Sequence

GCATTGGGTTAGGCAAGCCCCCGGCAAGGGGCTCGAATGG

(VR282)

ATGGCTTTCATTCGGAATGACGGGAGCGATAAATATTACGC

GGATTCAGTTAAAGGGCGGTTCACCATCAGCCGCGACAATA

GCAAAAAGACGGTCTCCTTACAGATGTCCAGCTTGCGGGCC

GAAGACACGGCTGTATACTATTGTGCTAAAAATGGCGAGAG

CGGCCCCCTGGATTACTGGTACTTTGACCTGTGGGGCAGAG

GCACCCTGGTCACGGTGTCCTCTGGAGGAGGAGGCTCCGGA

GGAGGAGGCTCTGGCGGCGGCGGCAGCGACATTGTAATGA

CCCAGTCACCCTCCTTCCTTAGTGCCTCAGTCGGAGACCGCG

TGACTATCACTTGTCGTGCCTCACACGGAATTAATAACTACC

TCGCTTGGTATCAGCAAAAACCAGGCAAGGCCCCGAAACTA

TTGATCTACGCCGCATCTACTCTGCAGAGCGGAGTACCGAG

CAGATTTAGTGGTTCCGGCAGCGGAACCGAGTTCACCCTAA

CCATTTCCAGTTTGCAGCCAGAGGATTTCGCGACCTATTACT

GCCAGCAATACGATTCATACCCGCCAACTTTCGGAAGAGGT

ACCAAGGTTGAAATCAAGATTGAAGTTATGTATCCTCCTCC

TTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATG

TGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGG

CCTTCGAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTC

CTGGCTTGCTATAGCTTGCTAGTAACAGTAGCGTTTATTATT

TTCTGGGTGTGCAGCAGGGCCGCCCGCGGCACCATCGGCGC

CAGGCGCACAGGCCAGCCTCTGAAGGAGGACCCTTCCGCCG

TGCCAGTGTTCTCTGTGGACTACGGCGAGCTGGATTTTCAGT

GGCGGGAGAAAACCCCAGAGCCACCTGTGCCCTGCGTGCCT

GAGCAGACCGAGTATGCCACAATCGTGTTTCCATCCGGAAT

GGGCACAAGCTCCCCTGCAAGGAGAGGCAGCGCCGACGGA

CCACGGTCCGCCCAGCCACTGCGGCCCGAGGATGGCCACTG

TTCTTGGCCCCTGCGGAGAAAGCGTGGATCCGGGGAAGGCC

GAGGCTCCCTTCTAACATGTGGAGATGTCGAGGAAAACCCT

GGCCCTATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCTG

GCCCTGCTTCTCCATGCGGCGCGCCCAGAAGTGCAGCTGGT

CGAGAGCGGAGGCGGACTGGTTCAACCCGGAGGCAGCTTG

AGACTGTCCTGCGCGGCCAGCGGCTTCAACATCAAGGATAC

CTATATCCACTGGGTGAGGCAGGCTCCAGGAAAGGGCCTGG

AGTGGGTGGCAAGGATTTACCCTACTAATGGATATACACGC

TACGCTGATTCCGTGAAGGGACGCTTTACAATCTCAGCAGA

TACATCCAAAAACACGGCCTATTTACAGATGAATAGTTTGC

GGGCCGAAGACACGGCTGTATACTATTGTTCTCGGTGGGGG

GGCGATGGATTTTATGCGATGGATTACTGGGGCCAGGGCAC

CCTGGTAACCGTGTCAAGCGGCTCAACATCCGGGTCCGGTA

AGCCGGGCTCCGGCGAGGGGTCTACAAAGGGAGATATACA

GATGACACAGTCCCCCAGTTCCCTGTCCGCCTCAGTGGGAG

ACCGAGTGACGATTACCTGTCGTGCCAGCCAGGACGTCAAT

ACCGCCGTCGCTTGGTATCAGCAAAAACCAGGCAAGGCCCC

GAAACTATTGATCTACAGTGCCTCTTTTCTGTACTCCGGGGT

GCCGAGCAGATTTAGTGGCTCCAGGAGCGGAACCGATTTCA

CCCTAACCATTTCCAGTTTGCAGCCAGAGGATTTCGCGACCT

ATTACTGCCAGCAACACTACACCACACCGCCAACTTTCGGA

CAAGGAACCAAGGTTGAAATCAAAACTACGACCCCAGCAC

CTAGACCTCCCACCCCAGCTCCAACTATAGCTTCCCAGCCAT

TGTCTCTCCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGG

GCCGTTCATACAAGAGGACTCGATTTCGCTTGCGATATCTA

CATATGGGCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCT

AAGCTTGGTTATTACCCTCTATTGCAAACGCGGCCGCAAGA

AACTGCTCTACATCTTTAAACAGCCGTTCATGAGGCCTGTGC

AGACAACGCAGGAAGAGGATGGCTGTAGTTGTCGGTTTCCG

GAAGAGGAAGAGGGGGGCTGCGAGTTGCGTGTCAAATTTTC

TCGGTCTGCCGACGCCCCCGCGTACCAGCAAGGGCAGAACC

AGCTTTATAATGAGCTGAATCTTGGACGACGGGAGGAATAT

GACGTGCTTGACAAGAGGCGAGGTAGGGACCCTGAGATGG

GGGGAAAACCTCGGAGGAAAAACCCACAGGAAGGCCTGTA

TAACGAACTGCAGAAGGACAAGATGGCTGAAGCCTACTCTG

AGATTGGAATGAAAGGGGAACGCAGACGCGGCAAGGGCCA

TGATGGCCTCTACCAAGGTCTAAGCACTGCCACCAAGGACA

CCTATGACGCACTCCACATGCAAGCTCTACCTCCCCGTTGAT

AA

MC0282-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLVQSGGGVVQPGGSLRVS

3PF12_28_
NO: 6
CAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYA

PD1_HER2

DSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGP

Protein

LDYWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSDIVMTQS

Sequence

PSFLSASVGDRVTITCRASHGINNYLAWYQQKPGKAPKLLIYA

(VR282)

ASTLQSGVPSRESGSGSGTEFTLTISSLQPEDFATYYCQQYDSY

PPTFGRGTKVEIKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPL

FPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVCSRAARGTI

GARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP

EQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSW

PLRRKRGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLL

HAARPEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVR

QAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY

LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

GSTSGSGKPGSGEGSTKGDIQMTQSPSSLSASVGDRVTITCRAS

QDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGT

DFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKTTTPAP

RPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA

PLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEE

DGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELN

LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK

MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ

ALPPR

MC0283-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

3PF12_28_
NO: 7
GCTGCTGCACGCAGCCAGACCCCAAGTGCAACTAGTCCAAT

PD1_EGFR

CAGGTGGAGGCGTCGTGCAACCTGGAGGGTCCCTCCGCGTT

Nucleotide

AGCTGCGCCGCATCAGGCGTTACCTTGTCAGACTACGGCAT

Sequence

GCATTGGGTTAGGCAAGCCCCCGGCAAGGGGCTCGAATGG

(VR283)

ATGGCTTTCATTCGGAATGACGGGAGCGATAAATATTACGC

GGATTCAGTTAAAGGGCGGTTCACCATCAGCCGCGACAATA

GCAAAAAGACGGTCTCCTTACAGATGTCCAGCTTGCGGGCC

GAAGACACGGCTGTATACTATTGTGCTAAAAATGGCGAGAG

CGGCCCCCTGGATTACTGGTACTTTGACCTGTGGGGCAGAG

GCACCCTGGTCACGGTGTCCTCTGGAGGAGGAGGCTCCGGA

GGAGGAGGCTCTGGCGGCGGCGGCAGCGACATTGTAATGA

CCCAGTCACCCTCCTTCCTTAGTGCCTCAGTCGGAGACCGCG

TGACTATCACTTGTCGTGCCTCACACGGAATTAATAACTACC

TCGCTTGGTATCAGCAAAAACCAGGCAAGGCCCCGAAACTA

TTGATCTACGCCGCATCTACTCTGCAGAGCGGAGTACCGAG

CAGATTTAGTGGTTCCGGCAGCGGAACCGAGTTCACCCTAA

CCATTTCCAGTTTGCAGCCAGAGGATTTCGCGACCTATTACT

GCCAGCAATACGATTCATACCCGCCAACTTTCGGAAGAGGT

ACCAAGGTTGAAATCAAGATTGAAGTTATGTATCCTCCTCC

TTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATG

TGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGG

CCTTCGAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTC

CTGGCTTGCTATAGCTTGCTAGTAACAGTAGCGTTTATTATT

TTCTGGGTGTGCAGCAGGGCCGCCCGCGGCACCATCGGCGC

CAGGCGCACAGGCCAGCCTCTGAAGGAGGACCCTTCCGCCG

TGCCAGTGTTCTCTGTGGACTACGGCGAGCTGGATTTTCAGT

GGCGGGAGAAAACCCCAGAGCCACCTGTGCCCTGCGTGCCT

GAGCAGACCGAGTATGCCACAATCGTGTTTCCATCCGGAAT

GGGCACAAGCTCCCCTGCAAGGAGAGGCAGCGCCGACGGA

CCACGGTCCGCCCAGCCACTGCGGCCCGAGGATGGCCACTG

TTCTTGGCCCCTGCGGAGAAAGCGTGGATCCGGGGAAGGCC

GAGGCTCCCTTCTAACATGTGGAGATGTCGAGGAAAACCCT

GGCCCTATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCTG

GCCCTGCTTCTCCATGCGGCGCGCCCACAAGTGCAGCTGAA

ACAGAGCGGACCAGGACTGGTTCAACCCAGCCAGAGCTTG

AGCATCACGTGCACGGTTAGCGGCTTCAGTCTGACCAATTA

TGGTGTGCACTGGGTGAGGCAGTCTCCAGGAAAGGGCCTGG

AGTGGCTTGGAGTCATTTGGAGCGGTGGGAATACAGATTAC

AATACACCTTTTACGTCACGTCTCTCCATTAACAAGGACAA

CTCCAAATCCCAAGTATTTTTCAAAATGAATAGCCTGCAGA

GTAATGATACCGCCATCTATTACTGTGCACGAGCTTTGACAT

ATTACGACTATGAATTTGCCTATTGGGGTCAAGGCACGCTG

GTGACCGTATCAGGCTCAACATCCGGGTCCGGTAAGCCGGG

CTCCGGCGAGGGGTCTACAAAGGGAGACATCCTTCTGACAC

AGAGCCCCGTGATCCTGTCCGTGTCCCCCGGCGAGAGAGTA

TCATTTTCCTGTAGGGCTTCTCAGAGCATCGGAACAAATATC

CACTGGTATCAGCAACGGACTAACGGATCACCTCGCCTGCT

CATAAAGTACGCCAGTGAATCTATTAGTGGCATACCGAGCC

GCTTCAGCGGGAGTGGCTCCGGCACAGACTTTACTCTGAGT

ATAAATTCCGTGGAATCTGAGGACATCGCGGACTATTACTG

CCAGCAAAACAATAACTGGCCCACCACGTTCGGCGCGGGA

ACTAAACTAGAACTAAAGACTACGACCCCAGCACCTAGACC

TCCCACCCCAGCTCCAACTATAGCTTCCCAGCCATTGTCTCT

CCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGGGCCGTTC

ATACAAGAGGACTCGATTTCGCTTGCGATATCTACATATGG

GCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCTAAGCTTG

GTTATTACCCTCTATTGCAAACGCGGCCGCAAGAAACTGCT

CTACATCTTTAAACAGCCGTTCATGAGGCCTGTGCAGACAA

CGCAGGAAGAGGATGGCTGTAGTTGTCGGTTTCCGGAAGAG

GAAGAGGGGGGCTGCGAGTTGCGTGTCAAATTTTCTCGGTC

TGCCGACGCCCCCGCGTACCAGCAAGGGCAGAACCAGCTTT

ATAATGAGCTGAATCTTGGACGACGGGAGGAATATGACGTG

CTTGACAAGAGGCGAGGTAGGGACCCTGAGATGGGGGGAA

AACCTCGGAGGAAAAACCCACAGGAAGGCCTGTATAACGA

ACTGCAGAAGGACAAGATGGCTGAAGCCTACTCTGAGATTG

GAATGAAAGGGGAACGCAGACGCGGCAAGGGCCATGATGG

CCTCTACCAAGGTCTAAGCACTGCCACCAAGGACACCTATG

ACGCACTCCACATGCAAGCTCTACCTCCCCGTTGATAA

MC0283-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLVQSGGGVVQPGGSLRVS

3PF12_28_
NO: 8
CAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYA

PD1_EGFR

DSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGP

Protein

LDYWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSDIVMTQS

Sequence

PSFLSASVGDRVTITCRASHGINNYLAWYQQKPGKAPKLLIYA

(VR283)

ASTLQSGVPSRESGSGSGTEFTLTISSLQPEDFATYYCQQYDSY

PPTFGRGTKVEIKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPL

FPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVCSRAARGTI

GARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVP

EQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSW

PLRRKRGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLL

HAARPQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVR

QSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFK

MNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSGSTSG

SGKPGSGEGSTKGDILLTQSPVILSVSPGERVSFSCRASQSIGTNI

HWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSV

ESEDIADYYCQQNNNWPTTFGAGTKLELKTTTPAPRPPTPAPTI

ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGV

LLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP

EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD

VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI

GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

MC0284-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

BB7.2_8_
NO: 9
GCTGCTGCACGCAGCCAGACCCCAGGTGCAGCTGCAGCAGT

PD1_HER2

CTGGACCTGAGCTGGTGAAGCCAGGAGCCTCCGTGAAGATG

Nucleotide

TCTTGCAAGGCCAGCGGCTACACCTTCACATCTTATCACATC

Sequence

CAGTGGGTGAAGCAGCGGCCCGGACAGGGCCTGGAGTGGA

(VR284)

TCGGATGGATCTACCCAGGCGACGGCTCCACACAGTATAAC

GAGAAGTTCAAGGGCAAGACCACACTGACCGCCGATAAGA

GCAGCAGCACCGCCTACATGCTGCTGAGCAGCCTGACCAGC

GAGGACAGCGCCATCTACTTTTGCGCCAGGGAGGGCACATA

CTATGCTATGGACTATTGGGGCCAGGGCACCAGCGTGACAG

TGTCTAGCGGAGGAGGAGGCTCCGGAGGAGGAGGCTCTGG

CGGCGGCGGCAGCGACGTGCTGATGACCCAGACACCACTG

AGCCTGCCCGTGAGCCTGGGCGATCAGGTGAGCATCTCCTG

TAGATCCTCTCAGAGCATCGTGCACTCCAACGGCAATACCT

ACCTGGAGTGGTATCTGCAGAAGCCAGGCCAGTCCCCCAAG

CTGCTGATCTATAAGGTGTCTAATCGGTTCAGCGGCGTGCCT

GACAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCT

GAAGATCAGCCGGGTGGAGGCAGAGGATCTGGGCGTGTAC

TATTGTTTCCAGGGCTCCCACGTGCCACGCACCTTTGGCGGC

GGTACCAAGCTGGAGATCAAGACTACGACCCCAGCACCTAG

ACCTCCCACCCCAGCTCCAACTATAGCTTCCCAGCCATTGTC

TCTCCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGGGCCG

TTCATACAAGAGGACTCGATTTCGCTTGCGATATCTACATAT

GGGCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCTAAGCT

TGGTTATTACCCTCTATTGCTGCAGCAGGGCCGCCCGCGGC

ACCATCGGCGCCAGGCGCACAGGCCAGCCTCTGAAGGAGG

ACCCTTCCGCCGTGCCAGTGTTCTCTGTGGACTACGGCGAG

CTGGATTTTCAGTGGCGGGAGAAAACCCCAGAGCCACCTGT

GCCCTGCGTGCCTGAGCAGACCGAGTATGCCACAATCGTGT

TTCCATCCGGAATGGGCACAAGCTCCCCTGCAAGGAGAGGC

AGCGCCGACGGACCACGGTCCGCCCAGCCACTGCGGCCCGA

GGATGGCCACTGTTCTTGGCCCCTGCGGAGAAAGCGTGGAT

CCGGGGAAGGCCGAGGCTCCCTTCTAACATGTGGAGATGTC

GAGGAAAACCCTGGCCCTATGGCGCTGCCAGTCACTGCATT

GTTATTGCCTCTGGCCCTGCTTCTCCATGCGGCGCGCCCAGA

AGTGCAGCTGGTCGAGAGCGGAGGCGGACTGGTTCAACCC

GGAGGCAGCTTGAGACTGTCCTGCGCGGCCAGCGGCTTCAA

CATCAAGGATACCTATATCCACTGGGTGAGGCAGGCTCCAG

GAAAGGGCCTGGAGTGGGTGGCAAGGATTTACCCTACTAAT

GGATATACACGCTACGCTGATTCCGTGAAGGGACGCTTTAC

AATCTCAGCAGATACATCCAAAAACACGGCCTATTTACAGA

TGAATAGTTTGCGGGCCGAAGACACGGCTGTATACTATTGT

TCTCGGTGGGGGGGCGATGGATTTTATGCGATGGATTACTG

GGGCCAGGGCACCCTGGTAACCGTGTCAAGCGGCTCAACAT

CCGGGTCCGGTAAGCCGGGCTCCGGCGAGGGGTCTACAAA

GGGAGATATACAGATGACACAGTCCCCCAGTTCCCTGTCCG

CCTCAGTGGGAGACCGAGTGACGATTACCTGTCGTGCCAGC

CAGGACGTCAATACCGCCGTCGCTTGGTATCAGCAAAAACC

AGGCAAGGCCCCGAAACTATTGATCTACAGTGCCTCTTTTCT

GTACTCCGGGGTGCCGAGCAGATTTAGTGGCTCCAGGAGCG

GAACCGATTTCACCCTAACCATTTCCAGTTTGCAGCCAGAG

GATTTCGCGACCTATTACTGCCAGCAACACTACACCACACC

GCCAACTTTCGGACAAGGAACCAAGGTTGAAATCAAAATTG

AAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGC

AATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCC

AAGTCCCCTATTTCCCGGGCCTTCGAAGCCCTTTTGGGTGCT

GGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGT

AACAGTAGCGTTTATTATTTTCTGGGTGAAACGCGGCCGCA

AGAAACTGCTCTACATCTTTAAACAGCCGTTCATGAGGCCT

GTGCAGACAACGCAGGAAGAGGATGGCTGTAGTTGTCGGTT

TCCGGAAGAGGAAGAGGGGGGCTGCGAGTTGCGTGTCAAA

TTTTCTCGGTCTGCCGACGCCCCCGCGTACCAGCAAGGGCA

GAACCAGCTTTATAATGAGCTGAATCTTGGACGACGGGAGG

AATATGACGTGCTTGACAAGAGGCGAGGTAGGGACCCTGA

GATGGGGGGAAAACCTCGGAGGAAAAACCCACAGGAAGGC

CTGTATAACGAACTGCAGAAGGACAAGATGGCTGAAGCCT

ACTCTGAGATTGGAATGAAAGGGGAACGCAGACGCGGCAA

GGGCCATGATGGCCTCTACCAAGGTCTAAGCACTGCCACCA

AGGACACCTATGACGCACTCCACATGCAAGCTCTACCTCCC

CGTTGATAA

MC0284-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKMS

BB7.2_8_
NO: 10
CKASGYTFTSYHIQWVKQRPGQGLEWIGWIYPGDGSTQYNEK

PD1_HER2

FKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCAREGTYYAMD

Protein

YWGQGTSVTVSSGGGGSGGGGSGGGGSDVLMTQTPLSLPVSL

Sequence

GDQVSISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNR

(VR284)

FSGVPDRESGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTF

GGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV

HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCSRAARGTIG

ARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPE

QTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWP

LRRKRGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLH

AARPEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQ

APGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYL

QMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSG

STSGSGKPGSGEGSTKGDIQMTQSPSSLSASVGDRVTITCRASQ

DVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRESGSRSGTD

FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKIEVMYPPP

YLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLA

CYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGC

SCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGR

REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE

AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP

PR

MC0285-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

3PF12_8_
NO: 11
GCTGCTGCACGCAGCCAGACCCCAAGTGCAACTAGTCCAAT

PD1_HER2

CAGGTGGAGGCGTCGTGCAACCTGGAGGGTCCCTCCGCGTT

nucleotide

AGCTGCGCCGCATCAGGCGTTACCTTGTCAGACTACGGCAT

Sequence

GCATTGGGTTAGGCAAGCCCCCGGCAAGGGGCTCGAATGG

(VR285)

ATGGCTTTCATTCGGAATGACGGGAGCGATAAATATTACGC

GGATTCAGTTAAAGGGCGGTTCACCATCAGCCGCGACAATA

GCAAAAAGACGGTCTCCTTACAGATGTCCAGCTTGCGGGCC

GAAGACACGGCTGTATACTATTGTGCTAAAAATGGCGAGAG

CGGCCCCCTGGATTACTGGTACTTTGACCTGTGGGGCAGAG

GCACCCTGGTCACGGTGTCCTCTGGAGGAGGAGGCTCCGGA

GGAGGAGGCTCTGGCGGCGGCGGCAGCGACATTGTAATGA

CCCAGTCACCCTCCTTCCTTAGTGCCTCAGTCGGAGACCGCG

TGACTATCACTTGTCGTGCCTCACACGGAATTAATAACTACC

TCGCTTGGTATCAGCAAAAACCAGGCAAGGCCCCGAAACTA

TTGATCTACGCCGCATCTACTCTGCAGAGCGGAGTACCGAG

CAGATTTAGTGGTTCCGGCAGCGGAACCGAGTTCACCCTAA

CCATTTCCAGTTTGCAGCCAGAGGATTTCGCGACCTATTACT

GCCAGCAATACGATTCATACCCGCCAACTTTCGGAAGAGGT

ACCAAGGTTGAAATCAAGACTACGACCCCAGCACCTAGACC

TCCCACCCCAGCTCCAACTATAGCTTCCCAGCCATTGTCTCT

CCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGGGCCGTTC

ATACAAGAGGACTCGATTTCGCTTGCGATATCTACATATGG

GCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCTAAGCTTG

GTTATTACCCTCTATTGCTGCAGCAGGGCCGCCCGCGGCAC

CATCGGCGCCAGGCGCACAGGCCAGCCTCTGAAGGAGGAC

CCTTCCGCCGTGCCAGTGTTCTCTGTGGACTACGGCGAGCTG

GATTTTCAGTGGCGGGAGAAAACCCCAGAGCCACCTGTGCC

CTGCGTGCCTGAGCAGACCGAGTATGCCACAATCGTGTTTC

CATCCGGAATGGGCACAAGCTCCCCTGCAAGGAGAGGCAG

CGCCGACGGACCACGGTCCGCCCAGCCACTGCGGCCCGAGG

ATGGCCACTGTTCTTGGCCCCTGCGGAGAAAGCGTGGATCC

GGGGAAGGCCGAGGCTCCCTTCTAACATGTGGAGATGTCGA

GGAAAACCCTGGCCCTATGGCGCTGCCAGTCACTGCATTGT

TATTGCCTCTGGCCCTGCTTCTCCATGCGGCGCGCCCAGAAG

TGCAGCTGGTCGAGAGCGGAGGCGGACTGGTTCAACCCGG

AGGCAGCTTGAGACTGTCCTGCGCGGCCAGCGGCTTCAACA

TCAAGGATACCTATATCCACTGGGTGAGGCAGGCTCCAGGA

AAGGGCCTGGAGTGGGTGGCAAGGATTTACCCTACTAATGG

ATATACACGCTACGCTGATTCCGTGAAGGGACGCTTTACAA

TCTCAGCAGATACATCCAAAAACACGGCCTATTTACAGATG

AATAGTTTGCGGGCCGAAGACACGGCTGTATACTATTGTTC

TCGGTGGGGGGGCGATGGATTTTATGCGATGGATTACTGGG

GCCAGGGCACCCTGGTAACCGTGTCAAGCGGCTCAACATCC

GGGTCCGGTAAGCCGGGCTCCGGCGAGGGGTCTACAAAGG

GAGATATACAGATGACACAGTCCCCCAGTTCCCTGTCCGCC

TCAGTGGGAGACCGAGTGACGATTACCTGTCGTGCCAGCCA

GGACGTCAATACCGCCGTCGCTTGGTATCAGCAAAAACCAG

GCAAGGCCCCGAAACTATTGATCTACAGTGCCTCTTTTCTGT

ACTCCGGGGTGCCGAGCAGATTTAGTGGCTCCAGGAGCGGA

ACCGATTTCACCCTAACCATTTCCAGTTTGCAGCCAGAGGA

TTTCGCGACCTATTACTGCCAGCAACACTACACCACACCGC

CAACTTTCGGACAAGGAACCAAGGTTGAAATCAAAATTGAA

GTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAA

TGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAA

GTCCCCTATTTCCCGGGCCTTCGAAGCCCTTTTGGGTGCTGG

TGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAA

CAGTAGCGTTTATTATTTTCTGGGTGAAACGCGGCCGCAAG

AAACTGCTCTACATCTTTAAACAGCCGTTCATGAGGCCTGT

GCAGACAACGCAGGAAGAGGATGGCTGTAGTTGTCGGTTTC

CGGAAGAGGAAGAGGGGGGCTGCGAGTTGCGTGTCAAATT

TTCTCGGTCTGCCGACGCCCCCGCGTACCAGCAAGGGCAGA

ACCAGCTTTATAATGAGCTGAATCTTGGACGACGGGAGGAA

TATGACGTGCTTGACAAGAGGCGAGGTAGGGACCCTGAGAT

GGGGGGAAAACCTCGGAGGAAAAACCCACAGGAAGGCCTG

TATAACGAACTGCAGAAGGACAAGATGGCTGAAGCCTACTC

TGAGATTGGAATGAAAGGGGAACGCAGACGCGGCAAGGGC

CATGATGGCCTCTACCAAGGTCTAAGCACTGCCACCAAGGA

CACCTATGACGCACTCCACATGCAAGCTCTACCTCCCCGTTG

ATAAMC

MC0285-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLVQSGGGVVQPGGSLRVS

3PF12_8_
NO: 12
CAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYA

PD1_HER2

DSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGP

Protein

LDYWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSDIVMTQS

Sequence

PSFLSASVGDRVTITCRASHGINNYLAWYQQKPGKAPKLLIYA

(VR285)

ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYDSY

PPTFGRGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG

GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCSRAAR

GTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP

CVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGH

CSWPLRRKRGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLA

LLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH

WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKN

TAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVT

VSSGSTSGSGKPGSGEGSTKGDIQMTQSPSSLSASVGDRVTITC

RASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSR

SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKIEV

MYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVV

GGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQ

EEDGCSCRFPEEEEGGCELRVKESRSADAPAYQQGQNQLYNEL

NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD

KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPR

MC0286-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

BB7.2_8_
NO: 13
GCTGCTGCACGCAGCCAGACCCCAGGTGCAGCTGCAGCAGT

PD1_EGFR

CTGGACCTGAGCTGGTGAAGCCAGGAGCCTCCGTGAAGATG

nucleotide

TCTTGCAAGGCCAGCGGCTACACCTTCACATCTTATCACATC

Sequence

CAGTGGGTGAAGCAGCGGCCCGGACAGGGCCTGGAGTGGA

(VR286)

TCGGATGGATCTACCCAGGCGACGGCTCCACACAGTATAAC

GAGAAGTTCAAGGGCAAGACCACACTGACCGCCGATAAGA

GCAGCAGCACCGCCTACATGCTGCTGAGCAGCCTGACCAGC

GAGGACAGCGCCATCTACTTTTGCGCCAGGGAGGGCACATA

CTATGCTATGGACTATTGGGGCCAGGGCACCAGCGTGACAG

TGTCTAGCGGAGGAGGAGGCTCCGGAGGAGGAGGCTCTGG

CGGCGGCGGCAGCGACGTGCTGATGACCCAGACACCACTG

AGCCTGCCCGTGAGCCTGGGCGATCAGGTGAGCATCTCCTG

TAGATCCTCTCAGAGCATCGTGCACTCCAACGGCAATACCT

ACCTGGAGTGGTATCTGCAGAAGCCAGGCCAGTCCCCCAAG

CTGCTGATCTATAAGGTGTCTAATCGGTTCAGCGGCGTGCCT

GACAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCT

GAAGATCAGCCGGGTGGAGGCAGAGGATCTGGGCGTGTAC

TATTGTTTCCAGGGCTCCCACGTGCCACGCACCTTTGGCGGC

GGTACCAAGCTGGAGATCAAGACTACGACCCCAGCACCTAG

ACCTCCCACCCCAGCTCCAACTATAGCTTCCCAGCCATTGTC

TCTCCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGGGCCG

TTCATACAAGAGGACTCGATTTCGCTTGCGATATCTACATAT

GGGCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCTAAGCT

TGGTTATTACCCTCTATTGCTGCAGCAGGGCCGCCCGCGGC

ACCATCGGCGCCAGGCGCACAGGCCAGCCTCTGAAGGAGG

ACCCTTCCGCCGTGCCAGTGTTCTCTGTGGACTACGGCGAG

CTGGATTTTCAGTGGCGGGAGAAAACCCCAGAGCCACCTGT

GCCCTGCGTGCCTGAGCAGACCGAGTATGCCACAATCGTGT

TTCCATCCGGAATGGGCACAAGCTCCCCTGCAAGGAGAGGC

AGCGCCGACGGACCACGGTCCGCCCAGCCACTGCGGCCCGA

GGATGGCCACTGTTCTTGGCCCCTGCGGAGAAAGCGTGGAT

CCGGGGAAGGCCGAGGCTCCCTTCTAACATGTGGAGATGTC

GAGGAAAACCCTGGCCCTATGGCGCTGCCAGTCACTGCATT

GTTATTGCCTCTGGCCCTGCTTCTCCATGCGGCGCGCCCACA

AGTGCAGCTGAAACAGAGCGGACCAGGACTGGTTCAACCC

AGCCAGAGCTTGAGCATCACGTGCACGGTTAGCGGCTTCAG

TCTGACCAATTATGGTGTGCACTGGGTGAGGCAGTCTCCAG

GAAAGGGCCTGGAGTGGCTTGGAGTCATTTGGAGCGGTGGG

AATACAGATTACAATACACCTTTTACGTCACGTCTCTCCATT

AACAAGGACAACTCCAAATCCCAAGTATTTTTCAAAATGAA

TAGCCTGCAGAGTAATGATACCGCCATCTATTACTGTGCAC

GAGCTTTGACATATTACGACTATGAATTTGCCTATTGGGGTC

AAGGCACGCTGGTGACCGTATCAGGCTCAACATCCGGGTCC

GGTAAGCCGGGCTCCGGCGAGGGGTCTACAAAGGGAGACA

TCCTTCTGACACAGAGCCCCGTGATCCTGTCCGTGTCCCCCG

GCGAGAGAGTATCATTTTCCTGTAGGGCTTCTCAGAGCATC

GGAACAAATATCCACTGGTATCAGCAACGGACTAACGGATC

ACCTCGCCTGCTCATAAAGTACGCCAGTGAATCTATTAGTG

GCATACCGAGCCGCTTCAGCGGGAGTGGCTCCGGCACAGAC

TTTACTCTGAGTATAAATTCCGTGGAATCTGAGGACATCGC

GGACTATTACTGCCAGCAAAACAATAACTGGCCCACCACGT

TCGGCGCGGGAACTAAACTAGAACTAAAGATTGAAGTTATG

TATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAAC

CATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCC

TATTTCCCGGGCCTTCGAAGCCCTTTTGGGTGCTGGTGGTGG

TTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTA

GCGTTTATTATTTTCTGGGTGAAACGCGGCCGCAAGAAACT

GCTCTACATCTTTAAACAGCCGTTCATGAGGCCTGTGCAGA

CAACGCAGGAAGAGGATGGCTGTAGTTGTCGGTTTCCGGAA

GAGGAAGAGGGGGGCTGCGAGTTGCGTGTCAAATTTTCTCG

GTCTGCCGACGCCCCCGCGTACCAGCAAGGGCAGAACCAGC

TTTATAATGAGCTGAATCTTGGACGACGGGAGGAATATGAC

GTGCTTGACAAGAGGCGAGGTAGGGACCCTGAGATGGGGG

GAAAACCTCGGAGGAAAAACCCACAGGAAGGCCTGTATAA

CGAACTGCAGAAGGACAAGATGGCTGAAGCCTACTCTGAG

ATTGGAATGAAAGGGGAACGCAGACGCGGCAAGGGCCATG

ATGGCCTCTACCAAGGTCTAAGCACTGCCACCAAGGACACC

TATGACGCACTCCACATGCAAGCTCTACCTCCCCGTTGATA

A

MC0286-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKMS

BB7.2_8_
NO: 14
CKASGYTFTSYHIQWVKQRPGQGLEWIGWIYPGDGSTQYNEK

PD1_EGFR

FKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCAREGTYYAMD

Protein

YWGQGTSVTVSSGGGGSGGGGSGGGGSDVLMTQTPLSLPVSL

Sequence

GDQVSISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNR

(VR286)

FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTF

GGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV

HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCSRAARGTIG

ARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPE

QTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWP

LRRKRGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLH

AARPQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQ

SPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKM

NSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSGSTSGS

GKPGSGEGSTKGDILLTQSPVILSVSPGERVSFSCRASQSIGTNI

HWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSV

ESEDIADYYCQQNNNWPTTFGAGTKLELKIEVMYPPPYLDNE

KSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLL

VTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE

EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV

LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG

MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

MC0287-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

3PF12_8_P
NO: 15
GCTGCTGCACGCAGCCAGACCCCAAGTGCAACTAGTCCAAT

D1_EGFR

CAGGTGGAGGCGTCGTGCAACCTGGAGGGTCCCTCCGCGTT

Nucleotide

AGCTGCGCCGCATCAGGCGTTACCTTGTCAGACTACGGCAT

Sequence

GCATTGGGTTAGGCAAGCCCCCGGCAAGGGGCTCGAATGG

(VR287)

ATGGCTTTCATTCGGAATGACGGGAGCGATAAATATTACGC

GGATTCAGTTAAAGGGCGGTTCACCATCAGCCGCGACAATA

GCAAAAAGACGGTCTCCTTACAGATGTCCAGCTTGCGGGCC

GAAGACACGGCTGTATACTATTGTGCTAAAAATGGCGAGAG

CGGCCCCCTGGATTACTGGTACTTTGACCTGTGGGGCAGAG

GCACCCTGGTCACGGTGTCCTCTGGAGGAGGAGGCTCCGGA

GGAGGAGGCTCTGGCGGCGGCGGCAGCGACATTGTAATGA

CCCAGTCACCCTCCTTCCTTAGTGCCTCAGTCGGAGACCGCG

TGACTATCACTTGTCGTGCCTCACACGGAATTAATAACTACC

TCGCTTGGTATCAGCAAAAACCAGGCAAGGCCCCGAAACTA

TTGATCTACGCCGCATCTACTCTGCAGAGCGGAGTACCGAG

CAGATTTAGTGGTTCCGGCAGCGGAACCGAGTTCACCCTAA

CCATTTCCAGTTTGCAGCCAGAGGATTTCGCGACCTATTACT

GCCAGCAATACGATTCATACCCGCCAACTTTCGGAAGAGGT

ACCAAGGTTGAAATCAAGACTACGACCCCAGCACCTAGACC

TCCCACCCCAGCTCCAACTATAGCTTCCCAGCCATTGTCTCT

CCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGGGCCGTTC

ATACAAGAGGACTCGATTTCGCTTGCGATATCTACATATGG

GCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCTAAGCTTG

GTTATTACCCTCTATTGCTGCAGCAGGGCCGCCCGCGGCAC

CATCGGCGCCAGGCGCACAGGCCAGCCTCTGAAGGAGGAC

CCTTCCGCCGTGCCAGTGTTCTCTGTGGACTACGGCGAGCTG

GATTTTCAGTGGCGGGAGAAAACCCCAGAGCCACCTGTGCC

CTGCGTGCCTGAGCAGACCGAGTATGCCACAATCGTGTTTC

CATCCGGAATGGGCACAAGCTCCCCTGCAAGGAGAGGCAG

CGCCGACGGACCACGGTCCGCCCAGCCACTGCGGCCCGAGG

ATGGCCACTGTTCTTGGCCCCTGCGGAGAAAGCGTGGATCC

GGGGAAGGCCGAGGCTCCCTTCTAACATGTGGAGATGTCGA

GGAAAACCCTGGCCCTATGGCGCTGCCAGTCACTGCATTGT

TATTGCCTCTGGCCCTGCTTCTCCATGCGGCGCGCCCACAAG

TGCAGCTGAAACAGAGCGGACCAGGACTGGTTCAACCCAG

CCAGAGCTTGAGCATCACGTGCACGGTTAGCGGCTTCAGTC

TGACCAATTATGGTGTGCACTGGGTGAGGCAGTCTCCAGGA

AAGGGCCTGGAGTGGCTTGGAGTCATTTGGAGCGGTGGGAA

TACAGATTACAATACACCTTTTACGTCACGTCTCTCCATTAA

CAAGGACAACTCCAAATCCCAAGTATTTTTCAAAATGAATA

GCCTGCAGAGTAATGATACCGCCATCTATTACTGTGCACGA

GCTTTGACATATTACGACTATGAATTTGCCTATTGGGGTCAA

GGCACGCTGGTGACCGTATCAGGCTCAACATCCGGGTCCGG

TAAGCCGGGCTCCGGCGAGGGGTCTACAAAGGGAGACATC

CTTCTGACACAGAGCCCCGTGATCCTGTCCGTGTCCCCCGGC

GAGAGAGTATCATTTTCCTGTAGGGCTTCTCAGAGCATCGG

AACAAATATCCACTGGTATCAGCAACGGACTAACGGATCAC

CTCGCCTGCTCATAAAGTACGCCAGTGAATCTATTAGTGGC

ATACCGAGCCGCTTCAGCGGGAGTGGCTCCGGCACAGACTT

TACTCTGAGTATAAATTCCGTGGAATCTGAGGACATCGCGG

ACTATTACTGCCAGCAAAACAATAACTGGCCCACCACGTTC

GGCGCGGGAACTAAACTAGAACTAAAGATTGAAGTTATGTA

TCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCA

TTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTA

TTTCCCGGGCCTTCGAAGCCCTTTTGGGTGCTGGTGGTGGTT

GGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTAGC

GTTTATTATTTTCTGGGTGAAACGCGGCCGCAAGAAACTGC

TCTACATCTTTAAACAGCCGTTCATGAGGCCTGTGCAGACA

ACGCAGGAAGAGGATGGCTGTAGTTGTCGGTTTCCGGAAGA

GGAAGAGGGGGGCTGCGAGTTGCGTGTCAAATTTTCTCGGT

CTGCCGACGCCCCCGCGTACCAGCAAGGGCAGAACCAGCTT

TATAATGAGCTGAATCTTGGACGACGGGAGGAATATGACGT

GCTTGACAAGAGGCGAGGTAGGGACCCTGAGATGGGGGGA

AAACCTCGGAGGAAAAACCCACAGGAAGGCCTGTATAACG

AACTGCAGAAGGACAAGATGGCTGAAGCCTACTCTGAGATT

GGAATGAAAGGGGAACGCAGACGCGGCAAGGGCCATGATG

GCCTCTACCAAGGTCTAAGCACTGCCACCAAGGACACCTAT

GACGCACTCCACATGCAAGCTCTACCTCCCCGTTGATAA

MC0287-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLVQSGGGVVQPGGSLRVS

3PF12_8_
NO: 16
CAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYA

PD1_EGFR

DSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGP

Protein

LDYWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSDIVMTQS

Sequence

PSFLSASVGDRVTITCRASHGINNYLAWYQQKPGKAPKLLIYA

(VR287)

ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYDSY

PPTFGRGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG

GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCSRAAR

GTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP

CVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGH

CSWPLRRKRGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLA

LLLHAARPQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVH

WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQ

VFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSG

STSGSGKPGSGEGSTKGDILLTQSPVILSVSPGERVSFSCRASQSI

GTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSI

NSVESEDIADYYCQQNNNWPTTFGAGTKLELKIEVMYPPPYLD

NEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS

LLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF

PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEY

DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

MC0288-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

BB7.2_28_
NO: 17
GCTGCTGCACGCAGCCAGACCCCAGGTGCAGCTGCAGCAGT

Pdel_HER2

CTGGACCTGAGCTGGTGAAGCCAGGAGCCTCCGTGAAGATG

Nucleotide

TCTTGCAAGGCCAGCGGCTACACCTTCACATCTTATCACATC

Sequence

CAGTGGGTGAAGCAGCGGCCCGGACAGGGCCTGGAGTGGA

(VR288)

TCGGATGGATCTACCCAGGCGACGGCTCCACACAGTATAAC

GAGAAGTTCAAGGGCAAGACCACACTGACCGCCGATAAGA

GCAGCAGCACCGCCTACATGCTGCTGAGCAGCCTGACCAGC

GAGGACAGCGCCATCTACTTTTGCGCCAGGGAGGGCACATA

CTATGCTATGGACTATTGGGGCCAGGGCACCAGCGTGACAG

TGTCTAGCGGAGGAGGAGGCTCCGGAGGAGGAGGCTCTGG

CGGCGGCGGCAGCGACGTGCTGATGACCCAGACACCACTG

AGCCTGCCCGTGAGCCTGGGCGATCAGGTGAGCATCTCCTG

TAGATCCTCTCAGAGCATCGTGCACTCCAACGGCAATACCT

ACCTGGAGTGGTATCTGCAGAAGCCAGGCCAGTCCCCCAAG

CTGCTGATCTATAAGGTGTCTAATCGGTTCAGCGGCGTGCCT

GACAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCT

GAAGATCAGCCGGGTGGAGGCAGAGGATCTGGGCGTGTAC

TATTGTTTCCAGGGCTCCCACGTGCCACGCACCTTTGGCGGC

GGTACCAAGCTGGAGATCAAGATTGAAGTTATGTATCCTCC

TCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCC

ATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCC

GGGCCTTCGAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGG

AGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTAGCGTTTAT

TATTTTCTGGGTGCGGAGAAAGCGTGGATCCGGGGAAGGCC

GAGGCTCCCTTCTAACATGTGGAGATGTCGAGGAAAACCCT

GGCCCTATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCTG

GCCCTGCTTCTCCATGCGGCGCGCCCAGAAGTGCAGCTGGT

CGAGAGCGGAGGCGGACTGGTTCAACCCGGAGGCAGCTTG

AGACTGTCCTGCGCGGCCAGCGGCTTCAACATCAAGGATAC

CTATATCCACTGGGTGAGGCAGGCTCCAGGAAAGGGCCTGG

AGTGGGTGGCAAGGATTTACCCTACTAATGGATATACACGC

TACGCTGATTCCGTGAAGGGACGCTTTACAATCTCAGCAGA

TACATCCAAAAACACGGCCTATTTACAGATGAATAGTTTGC

GGGCCGAAGACACGGCTGTATACTATTGTTCTCGGTGGGGG

GGCGATGGATTTTATGCGATGGATTACTGGGGCCAGGGCAC

CCTGGTAACCGTGTCAAGCGGCTCAACATCCGGGTCCGGTA

AGCCGGGCTCCGGCGAGGGGTCTACAAAGGGAGATATACA

GATGACACAGTCCCCCAGTTCCCTGTCCGCCTCAGTGGGAG

ACCGAGTGACGATTACCTGTCGTGCCAGCCAGGACGTCAAT

ACCGCCGTCGCTTGGTATCAGCAAAAACCAGGCAAGGCCCC

GAAACTATTGATCTACAGTGCCTCTTTTCTGTACTCCGGGGT

GCCGAGCAGATTTAGTGGCTCCAGGAGCGGAACCGATTTCA

CCCTAACCATTTCCAGTTTGCAGCCAGAGGATTTCGCGACCT

ATTACTGCCAGCAACACTACACCACACCGCCAACTTTCGGA

CAAGGAACCAAGGTTGAAATCAAAACTACGACCCCAGCAC

CTAGACCTCCCACCCCAGCTCCAACTATAGCTTCCCAGCCAT

TGTCTCTCCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGG

GCCGTTCATACAAGAGGACTCGATTTCGCTTGCGATATCTA

CATATGGGCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCT

AAGCTTGGTTATTACCCTCTATTGCAAACGCGGCCGCAAGA

AACTGCTCTACATCTTTAAACAGCCGTTCATGAGGCCTGTGC

AGACAACGCAGGAAGAGGATGGCTGTAGTTGTCGGTTTCCG

GAAGAGGAAGAGGGGGGCTGCGAGTTGCGTGTCAAATTTTC

TCGGTCTGCCGACGCCCCCGCGTACCAGCAAGGGCAGAACC

AGCTTTATAATGAGCTGAATCTTGGACGACGGGAGGAATAT

GACGTGCTTGACAAGAGGCGAGGTAGGGACCCTGAGATGG

GGGGAAAACCTCGGAGGAAAAACCCACAGGAAGGCCTGTA

TAACGAACTGCAGAAGGACAAGATGGCTGAAGCCTACTCTG

AGATTGGAATGAAAGGGGAACGCAGACGCGGCAAGGGCCA

TGATGGCCTCTACCAAGGTCTAAGCACTGCCACCAAGGACA

CCTATGACGCACTCCACATGCAAGCTCTACCTCCCCGTTGAT

AA

MC0288-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKMS

BB7.2_28_
NO: 18
CKASGYTFTSYHIQWVKQRPGQGLEWIGWIYPGDGSTQYNEK

Pdel_HER2

FKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCAREGTYYAMD

Protein

YWGQGTSVTVSSGGGGSGGGGSGGGGSDVLMTQTPLSLPVSL

Sequence

GDQVSISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNR

(VR288)

FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTF

GGGTKLEIKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPG

PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRRKRGSGEGRGS

LLTCGDVEENPGPMALPVTALLLPLALLLHAARPEVQLVESGG

GLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARI

YPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV

YYCSRWGGDGFYAMDYWGQGTLVTVSSGSTSGSGKPGSGEG

STKGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ

KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDF

ATYYCQQHYTTPPTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLS

LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLV

ITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG

CELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR

GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER

RRGKGHDGLYQGLSTATKDTYDALHMQALPPR

MC0289-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

3PF12_28
NO: 19
GCTGCTGCACGCAGCCAGACCCCAAGTGCAACTAGTCCAAT

Pdel_HER2

CAGGTGGAGGCGTCGTGCAACCTGGAGGGTCCCTCCGCGTT

Nucleotide

AGCTGCGCCGCATCAGGCGTTACCTTGTCAGACTACGGCAT

Sequence

GCATTGGGTTAGGCAAGCCCCCGGCAAGGGGCTCGAATGG

(VR289)

ATGGCTTTCATTCGGAATGACGGGAGCGATAAATATTACGC

GGATTCAGTTAAAGGGCGGTTCACCATCAGCCGCGACAATA

GCAAAAAGACGGTCTCCTTACAGATGTCCAGCTTGCGGGCC

GAAGACACGGCTGTATACTATTGTGCTAAAAATGGCGAGAG

CGGCCCCCTGGATTACTGGTACTTTGACCTGTGGGGCAGAG

GCACCCTGGTCACGGTGTCCTCTGGAGGAGGAGGCTCCGGA

GGAGGAGGCTCTGGCGGCGGCGGCAGCGACATTGTAATGA

CCCAGTCACCCTCCTTCCTTAGTGCCTCAGTCGGAGACCGCG

TGACTATCACTTGTCGTGCCTCACACGGAATTAATAACTACC

TCGCTTGGTATCAGCAAAAACCAGGCAAGGCCCCGAAACTA

TTGATCTACGCCGCATCTACTCTGCAGAGCGGAGTACCGAG

CAGATTTAGTGGTTCCGGCAGCGGAACCGAGTTCACCCTAA

CCATTTCCAGTTTGCAGCCAGAGGATTTCGCGACCTATTACT

GCCAGCAATACGATTCATACCCGCCAACTTTCGGAAGAGGT

ACCAAGGTTGAAATCAAGATTGAAGTTATGTATCCTCCTCC

TTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATG

TGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGG

CCTTCGAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTC

CTGGCTTGCTATAGCTTGCTAGTAACAGTAGCGTTTATTATT

TTCTGGGTGCGGAGAAAGCGTGGATCCGGGGAAGGCCGAG

GCTCCCTTCTAACATGTGGAGATGTCGAGGAAAACCCTGGC

CCTATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCTGGCC

CTGCTTCTCCATGCGGCGCGCCCAGAAGTGCAGCTGGTCGA

GAGCGGAGGCGGACTGGTTCAACCCGGAGGCAGCTTGAGA

CTGTCCTGCGCGGCCAGCGGCTTCAACATCAAGGATACCTA

TATCCACTGGGTGAGGCAGGCTCCAGGAAAGGGCCTGGAGT

GGGTGGCAAGGATTTACCCTACTAATGGATATACACGCTAC

GCTGATTCCGTGAAGGGACGCTTTACAATCTCAGCAGATAC

ATCCAAAAACACGGCCTATTTACAGATGAATAGTTTGCGGG

CCGAAGACACGGCTGTATACTATTGTTCTCGGTGGGGGGGC

GATGGATTTTATGCGATGGATTACTGGGGCCAGGGCACCCT

GGTAACCGTGTCAAGCGGCTCAACATCCGGGTCCGGTAAGC

CGGGCTCCGGCGAGGGGTCTACAAAGGGAGATATACAGAT

GACACAGTCCCCCAGTTCCCTGTCCGCCTCAGTGGGAGACC

GAGTGACGATTACCTGTCGTGCCAGCCAGGACGTCAATACC

GCCGTCGCTTGGTATCAGCAAAAACCAGGCAAGGCCCCGAA

ACTATTGATCTACAGTGCCTCTTTTCTGTACTCCGGGGTGCC

GAGCAGATTTAGTGGCTCCAGGAGCGGAACCGATTTCACCC

TAACCATTTCCAGTTTGCAGCCAGAGGATTTCGCGACCTATT

ACTGCCAGCAACACTACACCACACCGCCAACTTTCGGACAA

GGAACCAAGGTTGAAATCAAAACTACGACCCCAGCACCTA

GACCTCCCACCCCAGCTCCAACTATAGCTTCCCAGCCATTGT

CTCTCCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGGGCC

GTTCATACAAGAGGACTCGATTTCGCTTGCGATATCTACAT

ATGGGCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCTAA

GCTTGGTTATTACCCTCTATTGCAAACGCGGCCGCAAGAAA

CTGCTCTACATCTTTAAACAGCCGTTCATGAGGCCTGTGCAG

ACAACGCAGGAAGAGGATGGCTGTAGTTGTCGGTTTCCGGA

AGAGGAAGAGGGGGGCTGCGAGTTGCGTGTCAAATTTTCTC

GGTCTGCCGACGCCCCCGCGTACCAGCAAGGGCAGAACCA

GCTTTATAATGAGCTGAATCTTGGACGACGGGAGGAATATG

ACGTGCTTGACAAGAGGCGAGGTAGGGACCCTGAGATGGG

GGGAAAACCTCGGAGGAAAAACCCACAGGAAGGCCTGTAT

AACGAACTGCAGAAGGACAAGATGGCTGAAGCCTACTCTG

AGATTGGAATGAAAGGGGAACGCAGACGCGGCAAGGGCCA

TGATGGCCTCTACCAAGGTCTAAGCACTGCCACCAAGGACA

CCTATGACGCACTCCACATGCAAGCTCTACCTCCCCGTTGAT

AA

MC0289-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLVQSGGGVVQPGGSLRVS

3PF12_28
NO: 20
CAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYA

Pdel_HER2

DSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGP

Protein

LDYWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSDIVMTQS

Sequence

PSFLSASVGDRVTITCRASHGINNYLAWYQQKPGKAPKLLIYA

(VR289)

ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYDSY

PPTFGRGTKVEIKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPL

FPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRRKRGSGEG

RGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPEVQLVE

SGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV

ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDT

AVYYCSRWGGDGFYAMDYWGQGTLVTVSSGSTSGSGKPGSG

EGSTKGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWY

QQKPGKAPKLLIYSASFLYSGVPSRESGSRSGTDFTLTISSLQPE

DFATYYCQQHYTTPPTFGQGTKVEIKTTTPAPRPPTPAPTIASQ

PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLL

SLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE

EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD

KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK

GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

MC0290-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

3PF12_28
NO: 21
GCTGCTGCACGCAGCCAGACCCCAAGTGCAACTAGTCCAAT

LIR1_HER2

CAGGTGGAGGCGTCGTGCAACCTGGAGGGTCCCTCCGCGTT

Nucleic

AGCTGCGCCGCATCAGGCGTTACCTTGTCAGACTACGGCAT

acid

GCATTGGGTTAGGCAAGCCCCCGGCAAGGGGCTCGAATGG

sequence

ATGGCTTTCATTCGGAATGACGGGAGCGATAAATATTACGC

(VR290)

GGATTCAGTTAAAGGGCGGTTCACCATCAGCCGCGACAATA

GCAAAAAGACGGTCTCCTTACAGATGTCCAGCTTGCGGGCC

GAAGACACGGCTGTATACTATTGTGCTAAAAATGGCGAGAG

CGGCCCCCTGGATTACTGGTACTTTGACCTGTGGGGCAGAG

GCACCCTGGTCACGGTGTCCTCTGGAGGAGGAGGCTCCGGA

GGAGGAGGCTCTGGCGGCGGCGGCAGCGACATTGTAATGA

CCCAGTCACCCTCCTTCCTTAGTGCCTCAGTCGGAGACCGCG

TGACTATCACTTGTCGTGCCTCACACGGAATTAATAACTACC

TCGCTTGGTATCAGCAAAAACCAGGCAAGGCCCCGAAACTA

TTGATCTACGCCGCATCTACTCTGCAGAGCGGAGTACCGAG

CAGATTTAGTGGTTCCGGCAGCGGAACCGAGTTCACCCTAA

CCATTTCCAGTTTGCAGCCAGAGGATTTCGCGACCTATTACT

GCCAGCAATACGATTCATACCCGCCAACTTTCGGAAGAGGT

ACCAAGGTTGAAATCAAGATTGAAGTTATGTATCCTCCTCC

TTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATG

TGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGG

CCTTCGAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTC

CTGGCTTGCTATAGCTTGCTAGTAACAGTAGCGTTTATTATT

TTCTGGGTGCTGCGCCACAGGAGACAGGGCAAGCACTGGAC

CAGCACCCAGCGGAAGGCCGACTTTCAGCACCCTGCCGGCG

CCGTGGGCCCTGAGCCTACCGACAGGGGCCTGCAGTGGAGG

AGCTCCCCAGCCGCCGATGCCCAGGAGGAGAATCTGTACGC

CGCCGTGAAGCACACCCAGCCAGAGGACGGCGTGGAGATG

GACACCCGCTCCCCACACGACGAGGATCCACAGGCCGTGAC

CTACGCCGAGGTGAAGCACAGCCGCCCCAGACGCGAGATG

GCCAGCCCACCCAGCCCCCTGTCCGGCGAGTTCCTGGACAC

CAAGGACAGGCAGGCCGAGGAGGACCGGCAGATGGACACC

GAGGCCGCCGCCTCCGAGGCCCCCCAGGACGTGACCTACGC

CCAGCTGCACTCCCTGACCCTGCGGAGAGAGGCCACCGAGC

CCCCACCCAGCCAGGAGGGCCCCTCCCCCGCCGTGCCTAGC

ATCTACGCCACCCTGGCCATCCACCGGAGAAAGCGTGGATC

CGGGGAAGGCCGAGGCTCCCTTCTAACATGTGGAGATGTCG

AGGAAAACCCTGGCCCTATGGCGCTGCCAGTCACTGCATTG

TTATTGCCTCTGGCCCTGCTTCTCCATGCGGCGCGCCCAGAA

GTGCAGCTGGTCGAGAGCGGAGGCGGACTGGTTCAACCCG

GAGGCAGCTTGAGACTGTCCTGCGCGGCCAGCGGCTTCAAC

ATCAAGGATACCTATATCCACTGGGTGAGGCAGGCTCCAGG

AAAGGGCCTGGAGTGGGTGGCAAGGATTTACCCTACTAATG

GATATACACGCTACGCTGATTCCGTGAAGGGACGCTTTACA

ATCTCAGCAGATACATCCAAAAACACGGCCTATTTACAGAT

GAATAGTTTGCGGGCCGAAGACACGGCTGTATACTATTGTT

CTCGGTGGGGGGGCGATGGATTTTATGCGATGGATTACTGG

GGCCAGGGCACCCTGGTAACCGTGTCAAGCGGCTCAACATC

CGGGTCCGGTAAGCCGGGCTCCGGCGAGGGGTCTACAAAG

GGAGATATACAGATGACACAGTCCCCCAGTTCCCTGTCCGC

CTCAGTGGGAGACCGAGTGACGATTACCTGTCGTGCCAGCC

AGGACGTCAATACCGCCGTCGCTTGGTATCAGCAAAAACCA

GGCAAGGCCCCGAAACTATTGATCTACAGTGCCTCTTTTCTG

TACTCCGGGGTGCCGAGCAGATTTAGTGGCTCCAGGAGCGG

AACCGATTTCACCCTAACCATTTCCAGTTTGCAGCCAGAGG

ATTTCGCGACCTATTACTGCCAGCAACACTACACCACACCG

CCAACTTTCGGACAAGGAACCAAGGTTGAAATCAAAACTAC

GACCCCAGCACCTAGACCTCCCACCCCAGCTCCAACTATAG

CTTCCCAGCCATTGTCTCTCCGGCCAGAGGCGTGTCGACCA

GCCGCTGGAGGGGCCGTTCATACAAGAGGACTCGATTTCGC

TTGCGATATCTACATATGGGCCCCTCTTGCCGGGACATGCG

GTGTCCTGCTTCTAAGCTTGGTTATTACCCTCTATTGCAAAC

GCGGCCGCAAGAAACTGCTCTACATCTTTAAACAGCCGTTC

ATGAGGCCTGTGCAGACAACGCAGGAAGAGGATGGCTGTA

GTTGTCGGTTTCCGGAAGAGGAAGAGGGGGGCTGCGAGTTG

CGTGTCAAATTTTCTCGGTCTGCCGACGCCCCCGCGTACCAG

CAAGGGCAGAACCAGCTTTATAATGAGCTGAATCTTGGACG

ACGGGAGGAATATGACGTGCTTGACAAGAGGCGAGGTAGG

GACCCTGAGATGGGGGGAAAACCTCGGAGGAAAAACCCAC

AGGAAGGCCTGTATAACGAACTGCAGAAGGACAAGATGGC

TGAAGCCTACTCTGAGATTGGAATGAAAGGGGAACGCAGA

CGCGGCAAGGGCCATGATGGCCTCTACCAAGGTCTAAGCAC

TGCCACCAAGGACACCTATGACGCACTCCACATGCAAGCTC

TACCTCCCCGTTGATAA

MC0290-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLVQSGGGVVQPGGSLRVS

3PF12_28
NO: 22
CAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYA

LIR1_HER2

DSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGP

Protein

LDYWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSDIVMTQS

sequence

PSFLSASVGDRVTITCRASHGINNYLAWYQQKPGKAPKLLIYA

(VR290)

ASTLQSGVPSRESGSGSGTEFTLTISSLQPEDFATYYCQQYDSY

PPTFGRGTKVEIKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPL

FPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVLRHRRQGKH

WTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENL

YAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRRE

MASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYA

QLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIHRRKRGSGEGR

GSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPEVQLVES

GGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA

RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA

VYYCSRWGGDGFYAMDYWGQGTLVTVSSGSTSGSGKPGSGE

GSTKGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQ

QKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPED

FATYYCQQHYTTPPTFGQGTKVEIKTTTPAPRPPTPAPTIASQPL

SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL

VITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG

GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR

RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE

RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

MC0291-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

3PF12_28_
NO: 23
GCTGCTGCACGCAGCCAGACCCCAAGTGCAACTAGTCCAAT

KIR2DL1_

CAGGTGGAGGCGTCGTGCAACCTGGAGGGTCCCTCCGCGTT

HER2

AGCTGCGCCGCATCAGGCGTTACCTTGTCAGACTACGGCAT

nucleotide

GCATTGGGTTAGGCAAGCCCCCGGCAAGGGGCTCGAATGG

Sequence

ATGGCTTTCATTCGGAATGACGGGAGCGATAAATATTACGC

(VR291)

GGATTCAGTTAAAGGGCGGTTCACCATCAGCCGCGACAATA

GCAAAAAGACGGTCTCCTTACAGATGTCCAGCTTGCGGGCC

GAAGACACGGCTGTATACTATTGTGCTAAAAATGGCGAGAG

CGGCCCCCTGGATTACTGGTACTTTGACCTGTGGGGCAGAG

GCACCCTGGTCACGGTGTCCTCTGGAGGAGGAGGCTCCGGA

GGAGGAGGCTCTGGCGGCGGCGGCAGCGACATTGTAATGA

CCCAGTCACCCTCCTTCCTTAGTGCCTCAGTCGGAGACCGCG

TGACTATCACTTGTCGTGCCTCACACGGAATTAATAACTACC

TCGCTTGGTATCAGCAAAAACCAGGCAAGGCCCCGAAACTA

TTGATCTACGCCGCATCTACTCTGCAGAGCGGAGTACCGAG

CAGATTTAGTGGTTCCGGCAGCGGAACCGAGTTCACCCTAA

CCATTTCCAGTTTGCAGCCAGAGGATTTCGCGACCTATTACT

GCCAGCAATACGATTCATACCCGCCAACTTTCGGAAGAGGT

ACCAAGGTTGAAATCAAGATTGAAGTTATGTATCCTCCTCC

TTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATG

TGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGG

CCTTCGAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTC

CTGGCTTGCTATAGCTTGCTAGTAACAGTAGCGTTTATTATT

TTCTGGGTGCATAGGTGGTGCTCAAACAAAAAGAATGCTGC

CGTCATGGACCAGGAGAGCGCGGGCAATCGGACCGCAAAC

TCAGAGGACTCAGATGAACAAGATCCACAGGAAGTGACCT

ACACTCAGCTGAACCATTGTGTGTTTACACAGCGCAAGATT

ACTCGTCCAAGCCAGCGTCCTAAGACCCCCCCGACCGATAT

CATTGTGTATACCGAGCTTCCTAATGCCGAATCCCGCAGCA

AGGTGGTCTCCTGCCCGCGGAGAAAGCGTGGATCCGGGGA

AGGCCGAGGCTCCCTTCTAACATGTGGAGATGTCGAGGAAA

ACCCTGGCCCTATGGCGCTGCCAGTCACTGCATTGTTATTGC

CTCTGGCCCTGCTTCTCCATGCGGCGCGCCCAGAAGTGCAG

CTGGTCGAGAGCGGAGGCGGACTGGTTCAACCCGGAGGCA

GCTTGAGACTGTCCTGCGCGGCCAGCGGCTTCAACATCAAG

GATACCTATATCCACTGGGTGAGGCAGGCTCCAGGAAAGGG

CCTGGAGTGGGTGGCAAGGATTTACCCTACTAATGGATATA

CACGCTACGCTGATTCCGTGAAGGGACGCTTTACAATCTCA

GCAGATACATCCAAAAACACGGCCTATTTACAGATGAATAG

TTTGCGGGCCGAAGACACGGCTGTATACTATTGTTCTCGGT

GGGGGGGCGATGGATTTTATGCGATGGATTACTGGGGCCAG

GGCACCCTGGTAACCGTGTCAAGCGGCTCAACATCCGGGTC

CGGTAAGCCGGGCTCCGGCGAGGGGTCTACAAAGGGAGAT

ATACAGATGACACAGTCCCCCAGTTCCCTGTCCGCCTCAGT

GGGAGACCGAGTGACGATTACCTGTCGTGCCAGCCAGGACG

TCAATACCGCCGTCGCTTGGTATCAGCAAAAACCAGGCAAG

GCCCCGAAACTATTGATCTACAGTGCCTCTTTTCTGTACTCC

GGGGTGCCGAGCAGATTTAGTGGCTCCAGGAGCGGAACCG

ATTTCACCCTAACCATTTCCAGTTTGCAGCCAGAGGATTTCG

CGACCTATTACTGCCAGCAACACTACACCACACCGCCAACT

TTCGGACAAGGAACCAAGGTTGAAATCAAAACTACGACCCC

AGCACCTAGACCTCCCACCCCAGCTCCAACTATAGCTTCCC

AGCCATTGTCTCTCCGGCCAGAGGCGTGTCGACCAGCCGCT

GGAGGGGCCGTTCATACAAGAGGACTCGATTTCGCTTGCGA

TATCTACATATGGGCCCCTCTTGCCGGGACATGCGGTGTCCT

GCTTCTAAGCTTGGTTATTACCCTCTATTGCAAACGCGGCCG

CAAGAAACTGCTCTACATCTTTAAACAGCCGTTCATGAGGC

CTGTGCAGACAACGCAGGAAGAGGATGGCTGTAGTTGTCGG

TTTCCGGAAGAGGAAGAGGGGGGCTGCGAGTTGCGTGTCA

AATTTTCTCGGTCTGCCGACGCCCCCGCGTACCAGCAAGGG

CAGAACCAGCTTTATAATGAGCTGAATCTTGGACGACGGGA

GGAATATGACGTGCTTGACAAGAGGCGAGGTAGGGACCCT

GAGATGGGGGGAAAACCTCGGAGGAAAAACCCACAGGAAG

GCCTGTATAACGAACTGCAGAAGGACAAGATGGCTGAAGC

CTACTCTGAGATTGGAATGAAAGGGGAACGCAGACGCGGC

AAGGGCCATGATGGCCTCTACCAAGGTCTAAGCACTGCCAC

CAAGGACACCTATGACGCACTCCACATGCAAGCTCTACCTC

CCCGTTGATAA

MC0291-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLVQSGGGVVQPGGSLRVS

3PF12_28
NO: 24
CAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYA

KIR2DL1_

DSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGP

HER2

LDYWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSDIVMTQS

Protein

PSFLSASVGDRVTITCRASHGINNYLAWYQQKPGKAPKLLIYA

Sequence

ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYDSY

(VR291)

PPTFGRGTKVEIKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPL

FPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVHRWCSNKK

NAAVMDQESAGNRTANSEDSDEQDPQEVTYTQLNHCVFTQR

KITRPSQRPKTPPTDIIVYTELPNAESRSKVVSCPRRKRGSGEGR

GSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPEVQLVES

GGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA

RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA

VYYCSRWGGDGFYAMDYWGQGTLVTVSSGSTSGSGKPGSGE

GSTKGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQ

QKPGKAPKLLIYSASFLYSGVPSRESGSRSGTDFTLTISSLQPED

FATYYCQQHYTTPPTFGQGTKVEIKTTTPAPRPPTPAPTIASQPL

SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL

VITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG

GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR

RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE

RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

MC0292-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

BB7.2_28_
NO: 25
GCTGCTGCACGCAGCCAGACCCCAGGTGCAGCTGCAGCAGT

LIR1_HER2

CTGGACCTGAGCTGGTGAAGCCAGGAGCCTCCGTGAAGATG

nucleotide

TCTTGCAAGGCCAGCGGCTACACCTTCACATCTTATCACATC

Sequence

CAGTGGGTGAAGCAGCGGCCCGGACAGGGCCTGGAGTGGA

(VR292)

TCGGATGGATCTACCCAGGCGACGGCTCCACACAGTATAAC

GAGAAGTTCAAGGGCAAGACCACACTGACCGCCGATAAGA

GCAGCAGCACCGCCTACATGCTGCTGAGCAGCCTGACCAGC

GAGGACAGCGCCATCTACTTTTGCGCCAGGGAGGGCACATA

CTATGCTATGGACTATTGGGGCCAGGGCACCAGCGTGACAG

TGTCTAGCGGAGGAGGAGGCTCCGGAGGAGGAGGCTCTGG

CGGCGGCGGCAGCGACGTGCTGATGACCCAGACACCACTG

AGCCTGCCCGTGAGCCTGGGCGATCAGGTGAGCATCTCCTG

TAGATCCTCTCAGAGCATCGTGCACTCCAACGGCAATACCT

ACCTGGAGTGGTATCTGCAGAAGCCAGGCCAGTCCCCCAAG

CTGCTGATCTATAAGGTGTCTAATCGGTTCAGCGGCGTGCCT

GACAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCT

GAAGATCAGCCGGGTGGAGGCAGAGGATCTGGGCGTGTAC

TATTGTTTCCAGGGCTCCCACGTGCCACGCACCTTTGGCGGC

GGTACCAAGCTGGAGATCAAGATTGAAGTTATGTATCCTCC

TCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCC

ATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCC

GGGCCTTCGAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGG

AGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTAGCGTTTAT

TATTTTCTGGGTGCTGCGCCACAGGAGACAGGGCAAGCACT

GGACCAGCACCCAGCGGAAGGCCGACTTTCAGCACCCTGCC

GGCGCCGTGGGCCCTGAGCCTACCGACAGGGGCCTGCAGTG

GAGGAGCTCCCCAGCCGCCGATGCCCAGGAGGAGAATCTGT

ACGCCGCCGTGAAGCACACCCAGCCAGAGGACGGCGTGGA

GATGGACACCCGCTCCCCACACGACGAGGATCCACAGGCCG

TGACCTACGCCGAGGTGAAGCACAGCCGCCCCAGACGCGA

GATGGCCAGCCCACCCAGCCCCCTGTCCGGCGAGTTCCTGG

ACACCAAGGACAGGCAGGCCGAGGAGGACCGGCAGATGGA

CACCGAGGCCGCCGCCTCCGAGGCCCCCCAGGACGTGACCT

ACGCCCAGCTGCACTCCCTGACCCTGCGGAGAGAGGCCACC

GAGCCCCCACCCAGCCAGGAGGGCCCCTCCCCCGCCGTGCC

TAGCATCTACGCCACCCTGGCCATCCACCGGAGAAAGCGTG

GATCCGGGGAAGGCCGAGGCTCCCTTCTAACATGTGGAGAT

GTCGAGGAAAACCCTGGCCCTATGGCGCTGCCAGTCACTGC

ATTGTTATTGCCTCTGGCCCTGCTTCTCCATGCGGCGCGCCC

AGAAGTGCAGCTGGTCGAGAGCGGAGGCGGACTGGTTCAA

CCCGGAGGCAGCTTGAGACTGTCCTGCGCGGCCAGCGGCTT

CAACATCAAGGATACCTATATCCACTGGGTGAGGCAGGCTC

CAGGAAAGGGCCTGGAGTGGGTGGCAAGGATTTACCCTACT

AATGGATATACACGCTACGCTGATTCCGTGAAGGGACGCTT

TACAATCTCAGCAGATACATCCAAAAACACGGCCTATTTAC

AGATGAATAGTTTGCGGGCCGAAGACACGGCTGTATACTAT

TGTTCTCGGTGGGGGGGCGATGGATTTTATGCGATGGATTA

CTGGGGCCAGGGCACCCTGGTAACCGTGTCAAGCGGCTCAA

CATCCGGGTCCGGTAAGCCGGGCTCCGGCGAGGGGTCTACA

AAGGGAGATATACAGATGACACAGTCCCCCAGTTCCCTGTC

CGCCTCAGTGGGAGACCGAGTGACGATTACCTGTCGTGCCA

GCCAGGACGTCAATACCGCCGTCGCTTGGTATCAGCAAAAA

CCAGGCAAGGCCCCGAAACTATTGATCTACAGTGCCTCTTT

TCTGTACTCCGGGGTGCCGAGCAGATTTAGTGGCTCCAGGA

GCGGAACCGATTTCACCCTAACCATTTCCAGTTTGCAGCCA

GAGGATTTCGCGACCTATTACTGCCAGCAACACTACACCAC

ACCGCCAACTTTCGGACAAGGAACCAAGGTTGAAATCAAA

ACTACGACCCCAGCACCTAGACCTCCCACCCCAGCTCCAAC

TATAGCTTCCCAGCCATTGTCTCTCCGGCCAGAGGCGTGTCG

ACCAGCCGCTGGAGGGGCCGTTCATACAAGAGGACTCGATT

TCGCTTGCGATATCTACATATGGGCCCCTCTTGCCGGGACAT

GCGGTGTCCTGCTTCTAAGCTTGGTTATTACCCTCTATTGCA

AACGCGGCCGCAAGAAACTGCTCTACATCTTTAAACAGCCG

TTCATGAGGCCTGTGCAGACAACGCAGGAAGAGGATGGCT

GTAGTTGTCGGTTTCCGGAAGAGGAAGAGGGGGGCTGCGA

GTTGCGTGTCAAATTTTCTCGGTCTGCCGACGCCCCCGCGTA

CCAGCAAGGGCAGAACCAGCTTTATAATGAGCTGAATCTTG

GACGACGGGAGGAATATGACGTGCTTGACAAGAGGCGAGG

TAGGGACCCTGAGATGGGGGGAAAACCTCGGAGGAAAAAC

CCACAGGAAGGCCTGTATAACGAACTGCAGAAGGACAAGA

TGGCTGAAGCCTACTCTGAGATTGGAATGAAAGGGGAACGC

AGACGCGGCAAGGGCCATGATGGCCTCTACCAAGGTCTAAG

CACTGCCACCAAGGACACCTATGACGCACTCCACATGCAAG

CTCTACCTCCCCGTTGATAA

MC0292-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKMS

BB7.2_28_
NO: 26
CKASGYTFTSYHIQWVKQRPGQGLEWIGWIYPGDGSTQYNEK

LIR1_HER2

FKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCAREGTYYAMD

Protein

YWGQGTSVTVSSGGGGSGGGGSGGGGSDVLMTQTPLSLPVSL

Sequence

GDQVSISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNR

(VR292)

FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTF

GGGTKLEIKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPG

PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVLRHRRQGKHWT

STQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAA

VKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASP

PSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHS

LTLRREATEPPPSQEGPSPAVPSIYATLAIHRRKRGSGEGRGSLL

TCGDVEENPGPMALPVTALLLPLALLLHAARPEVQLVESGGGL

VQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPT

NGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC

SRWGGDGFYAMDYWGQGTLVTVSSGSTSGSGKPGSGEGSTK

GDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPG

KAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATY

YCQQHYTTPPTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRP

EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL

YCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE

LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR

RGKGHDGLYQGLSTATKDTYDALHMQALPPR

MC0293-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

BB7.2_28_
NO: 27
GCTGCTGCACGCAGCCAGACCCCAGGTGCAGCTGCAGCAGT

KIR2DL1

CTGGACCTGAGCTGGTGAAGCCAGGAGCCTCCGTGAAGATG

HER2

TCTTGCAAGGCCAGCGGCTACACCTTCACATCTTATCACATC

nucleotide

CAGTGGGTGAAGCAGCGGCCCGGACAGGGCCTGGAGTGGA

sequence

TCGGATGGATCTACCCAGGCGACGGCTCCACACAGTATAAC

(VR293)

GAGAAGTTCAAGGGCAAGACCACACTGACCGCCGATAAGA

GCAGCAGCACCGCCTACATGCTGCTGAGCAGCCTGACCAGC

GAGGACAGCGCCATCTACTTTTGCGCCAGGGAGGGCACATA

CTATGCTATGGACTATTGGGGCCAGGGCACCAGCGTGACAG

TGTCTAGCGGAGGAGGAGGCTCCGGAGGAGGAGGCTCTGG

CGGCGGCGGCAGCGACGTGCTGATGACCCAGACACCACTG

AGCCTGCCCGTGAGCCTGGGCGATCAGGTGAGCATCTCCTG

TAGATCCTCTCAGAGCATCGTGCACTCCAACGGCAATACCT

ACCTGGAGTGGTATCTGCAGAAGCCAGGCCAGTCCCCCAAG

CTGCTGATCTATAAGGTGTCTAATCGGTTCAGCGGCGTGCCT

GACAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCT

GAAGATCAGCCGGGTGGAGGCAGAGGATCTGGGCGTGTAC

TATTGTTTCCAGGGCTCCCACGTGCCACGCACCTTTGGCGGC

GGTACCAAGCTGGAGATCAAGATTGAAGTTATGTATCCTCC

TCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCC

ATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCC

GGGCCTTCGAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGG

AGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTAGCGTTTAT

TATTTTCTGGGTGCATAGGTGGTGCTCAAACAAAAAGAATG

CTGCCGTCATGGACCAGGAGAGCGCGGGCAATCGGACCGC

AAACTCAGAGGACTCAGATGAACAAGATCCACAGGAAGTG

ACCTACACTCAGCTGAACCATTGTGTGTTTACACAGCGCAA

GATTACTCGTCCAAGCCAGCGTCCTAAGACCCCCCCGACCG

ATATCATTGTGTATACCGAGCTTCCTAATGCCGAATCCCGCA

GCAAGGTGGTCTCCTGCCCGCGGAGAAAGCGTGGATCCGGG

GAAGGCCGAGGCTCCCTTCTAACATGTGGAGATGTCGAGGA

AAACCCTGGCCCTATGGCGCTGCCAGTCACTGCATTGTTATT

GCCTCTGGCCCTGCTTCTCCATGCGGCGCGCCCAGAAGTGC

AGCTGGTCGAGAGCGGAGGCGGACTGGTTCAACCCGGAGG

CAGCTTGAGACTGTCCTGCGCGGCCAGCGGCTTCAACATCA

AGGATACCTATATCCACTGGGTGAGGCAGGCTCCAGGAAAG

GGCCTGGAGTGGGTGGCAAGGATTTACCCTACTAATGGATA

TACACGCTACGCTGATTCCGTGAAGGGACGCTTTACAATCT

CAGCAGATACATCCAAAAACACGGCCTATTTACAGATGAAT

AGTTTGCGGGCCGAAGACACGGCTGTATACTATTGTTCTCG

GTGGGGGGGCGATGGATTTTATGCGATGGATTACTGGGGCC

AGGGCACCCTGGTAACCGTGTCAAGCGGCTCAACATCCGGG

TCCGGTAAGCCGGGCTCCGGCGAGGGGTCTACAAAGGGAG

ATATACAGATGACACAGTCCCCCAGTTCCCTGTCCGCCTCA

GTGGGAGACCGAGTGACGATTACCTGTCGTGCCAGCCAGGA

CGTCAATACCGCCGTCGCTTGGTATCAGCAAAAACCAGGCA

AGGCCCCGAAACTATTGATCTACAGTGCCTCTTTTCTGTACT

CCGGGGTGCCGAGCAGATTTAGTGGCTCCAGGAGCGGAACC

GATTTCACCCTAACCATTTCCAGTTTGCAGCCAGAGGATTTC

GCGACCTATTACTGCCAGCAACACTACACCACACCGCCAAC

TTTCGGACAAGGAACCAAGGTTGAAATCAAAACTACGACCC

CAGCACCTAGACCTCCCACCCCAGCTCCAACTATAGCTTCC

CAGCCATTGTCTCTCCGGCCAGAGGCGTGTCGACCAGCCGC

TGGAGGGGCCGTTCATACAAGAGGACTCGATTTCGCTTGCG

ATATCTACATATGGGCCCCTCTTGCCGGGACATGCGGTGTC

CTGCTTCTAAGCTTGGTTATTACCCTCTATTGCAAACGCGGC

CGCAAGAAACTGCTCTACATCTTTAAACAGCCGTTCATGAG

GCCTGTGCAGACAACGCAGGAAGAGGATGGCTGTAGTTGTC

GGTTTCCGGAAGAGGAAGAGGGGGGCTGCGAGTTGCGTGT

CAAATTTTCTCGGTCTGCCGACGCCCCCGCGTACCAGCAAG

GGCAGAACCAGCTTTATAATGAGCTGAATCTTGGACGACGG

GAGGAATATGACGTGCTTGACAAGAGGCGAGGTAGGGACC

CTGAGATGGGGGGAAAACCTCGGAGGAAAAACCCACAGGA

AGGCCTGTATAACGAACTGCAGAAGGACAAGATGGCTGAA

GCCTACTCTGAGATTGGAATGAAAGGGGAACGCAGACGCG

GCAAGGGCCATGATGGCCTCTACCAAGGTCTAAGCACTGCC

ACCAAGGACACCTATGACGCACTCCACATGCAAGCTCTACC

TCCCCGTTGATAA

MC0293-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKMS

BB7.2_28_
NO: 28
CKASGYTFTSYHIQWVKQRPGQGLEWIGWIYPGDGSTQYNEK

KIR2DL1_

FKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCAREGTYYAMD

HER2

YWGQGTSVTVSSGGGGSGGGGSGGGGSDVLMTQTPLSLPVSL

Protein

GDQVSISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNR

Sequence

FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTF

(VR293)

GGGTKLEIKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPG

PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVHRWCSNKKNAA

VMDQESAGNRTANSEDSDEQDPQEVTYTQLNHCVFTQRKITR

PSQRPKTPPTDIIVYTELPNAESRSKVVSCPRRKRGSGEGRGSLL

TCGDVEENPGPMALPVTALLLPLALLLHAARPEVQLVESGGGL

VQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPT

NGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC

SRWGGDGFYAMDYWGQGTLVTVSSGSTSGSGKPGSGEGSTK

GDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPG

KAPKLLIYSASFLYSGVPSRESGSRSGTDFTLTISSLQPEDFATY

YCQQHYTTPPTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRP

EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL

YCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE

LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR

RGKGHDGLYQGLSTATKDTYDALHMQALPPR

MC0294-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

3PF12_CD8_
NO: 29
GCTGCTGCACGCAGCCAGACCCCAAGTGCAACTAGTCCAAT

LIR1_HER2

CAGGTGGAGGCGTCGTGCAACCTGGAGGGTCCCTCCGCGTT

Nucleotide

AGCTGCGCCGCATCAGGCGTTACCTTGTCAGACTACGGCAT

sequence

GCATTGGGTTAGGCAAGCCCCCGGCAAGGGGCTCGAATGG

(VR294)

ATGGCTTTCATTCGGAATGACGGGAGCGATAAATATTACGC

GGATTCAGTTAAAGGGCGGTTCACCATCAGCCGCGACAATA

GCAAAAAGACGGTCTCCTTACAGATGTCCAGCTTGCGGGCC

GAAGACACGGCTGTATACTATTGTGCTAAAAATGGCGAGAG

CGGCCCCCTGGATTACTGGTACTTTGACCTGTGGGGCAGAG

GCACCCTGGTCACGGTGTCCTCTGGAGGAGGAGGCTCCGGA

GGAGGAGGCTCTGGCGGCGGCGGCAGCGACATTGTAATGA

CCCAGTCACCCTCCTTCCTTAGTGCCTCAGTCGGAGACCGCG

TGACTATCACTTGTCGTGCCTCACACGGAATTAATAACTACC

TCGCTTGGTATCAGCAAAAACCAGGCAAGGCCCCGAAACTA

TTGATCTACGCCGCATCTACTCTGCAGAGCGGAGTACCGAG

CAGATTTAGTGGTTCCGGCAGCGGAACCGAGTTCACCCTAA

CCATTTCCAGTTTGCAGCCAGAGGATTTCGCGACCTATTACT

GCCAGCAATACGATTCATACCCGCCAACTTTCGGAAGAGGT

ACCAAGGTTGAAATCAAGACTACGACCCCAGCACCTAGACC

TCCCACCCCAGCTCCAACTATAGCTTCCCAGCCATTGTCTCT

CCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGGGCCGTTC

ATACAAGAGGACTCGATTTCGCTTGCGATATCTACATATGG

GCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCTAAGCTTG

GTTATTACCCTCTATTGCCTGCGCCACAGGAGACAGGGCAA

GCACTGGACCAGCACCCAGCGGAAGGCCGACTTTCAGCACC

CTGCCGGCGCCGTGGGCCCTGAGCCTACCGACAGGGGCCTG

CAGTGGAGGAGCTCCCCAGCCGCCGATGCCCAGGAGGAGA

ATCTGTACGCCGCCGTGAAGCACACCCAGCCAGAGGACGGC

GTGGAGATGGACACCCGCTCCCCACACGACGAGGATCCACA

GGCCGTGACCTACGCCGAGGTGAAGCACAGCCGCCCCAGA

CGCGAGATGGCCAGCCCACCCAGCCCCCTGTCCGGCGAGTT

CCTGGACACCAAGGACAGGCAGGCCGAGGAGGACCGGCAG

ATGGACACCGAGGCCGCCGCCTCCGAGGCCCCCCAGGACGT

GACCTACGCCCAGCTGCACTCCCTGACCCTGCGGAGAGAGG

CCACCGAGCCCCCACCCAGCCAGGAGGGCCCCTCCCCCGCC

GTGCCTAGCATCTACGCCACCCTGGCCATCCACCGGAGAAA

GCGTGGATCCGGGGAAGGCCGAGGCTCCCTTCTAACATGTG

GAGATGTCGAGGAAAACCCTGGCCCTATGGCGCTGCCAGTC

ACTGCATTGTTATTGCCTCTGGCCCTGCTTCTCCATGCGGCG

CGCCCAGAAGTGCAGCTGGTCGAGAGCGGAGGCGGACTGG

TTCAACCCGGAGGCAGCTTGAGACTGTCCTGCGCGGCCAGC

GGCTTCAACATCAAGGATACCTATATCCACTGGGTGAGGCA

GGCTCCAGGAAAGGGCCTGGAGTGGGTGGCAAGGATTTAC

CCTACTAATGGATATACACGCTACGCTGATTCCGTGAAGGG

ACGCTTTACAATCTCAGCAGATACATCCAAAAACACGGCCT

ATTTACAGATGAATAGTTTGCGGGCCGAAGACACGGCTGTA

TACTATTGTTCTCGGTGGGGGGGCGATGGATTTTATGCGAT

GGATTACTGGGGCCAGGGCACCCTGGTAACCGTGTCAAGCG

GCTCAACATCCGGGTCCGGTAAGCCGGGCTCCGGCGAGGGG

TCTACAAAGGGAGATATACAGATGACACAGTCCCCCAGTTC

CCTGTCCGCCTCAGTGGGAGACCGAGTGACGATTACCTGTC

GTGCCAGCCAGGACGTCAATACCGCCGTCGCTTGGTATCAG

CAAAAACCAGGCAAGGCCCCGAAACTATTGATCTACAGTGC

CTCTTTTCTGTACTCCGGGGTGCCGAGCAGATTTAGTGGCTC

CAGGAGCGGAACCGATTTCACCCTAACCATTTCCAGTTTGC

AGCCAGAGGATTTCGCGACCTATTACTGCCAGCAACACTAC

ACCACACCGCCAACTTTCGGACAAGGAACCAAGGTTGAAAT

CAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGA

GAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACAC

CTTTGTCCAAGTCCCCTATTTCCCGGGCCTTCGAAGCCCTTT

TGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAG

CTTGCTAGTAACAGTAGCGTTTATTATTTTCTGGGTGAAACG

CGGCCGCAAGAAACTGCTCTACATCTTTAAACAGCCGTTCA

TGAGGCCTGTGCAGACAACGCAGGAAGAGGATGGCTGTAG

TTGTCGGTTTCCGGAAGAGGAAGAGGGGGGCTGCGAGTTGC

GTGTCAAATTTTCTCGGTCTGCCGACGCCCCCGCGTACCAGC

AAGGGCAGAACCAGCTTTATAATGAGCTGAATCTTGGACGA

CGGGAGGAATATGACGTGCTTGACAAGAGGCGAGGTAGGG

ACCCTGAGATGGGGGGAAAACCTCGGAGGAAAAACCCACA

GGAAGGCCTGTATAACGAACTGCAGAAGGACAAGATGGCT

GAAGCCTACTCTGAGATTGGAATGAAAGGGGAACGCAGAC

GCGGCAAGGGCCATGATGGCCTCTACCAAGGTCTAAGCACT

GCCACCAAGGACACCTATGACGCACTCCACATGCAAGCTCT

ACCTCCCCGTTGATAA

MC0294-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLVQSGGGVVQPGGSLRVS

3PF12_CD8_
NO: 30
CAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYA

LIR1_HER2

DSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGP

Protein

LDYWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSDIVMTQS

Sequence

PSFLSASVGDRVTITCRASHGINNYLAWYQQKPGKAPKLLIYA

(VR294)

ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYDSY

PPTFGRGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG

GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCLRHRRQ

GKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQE

ENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPR

REMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVT

YAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIHRRKRGSG

EGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPEVQL

VESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE

WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRA

EDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGSTSGSGKP

GSGEGSTKGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVA

WYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSL

QPEDFATYYCQQHYTTPPTFGQGTKVEIKIEVMYPPPYLDNEK

SNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLV

TVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE

EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL

DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM

KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

MC0295-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

3PF12_CD8_
NO: 31
GCTGCTGCACGCAGCCAGACCCCAAGTGCAACTAGTCCAAT

KIR2DL1_

CAGGTGGAGGCGTCGTGCAACCTGGAGGGTCCCTCCGCGTT

HER2

AGCTGCGCCGCATCAGGCGTTACCTTGTCAGACTACGGCAT

nucleotide

GCATTGGGTTAGGCAAGCCCCCGGCAAGGGGCTCGAATGG

Sequence

ATGGCTTTCATTCGGAATGACGGGAGCGATAAATATTACGC

(VR295)

GGATTCAGTTAAAGGGCGGTTCACCATCAGCCGCGACAATA

GCAAAAAGACGGTCTCCTTACAGATGTCCAGCTTGCGGGCC

GAAGACACGGCTGTATACTATTGTGCTAAAAATGGCGAGAG

CGGCCCCCTGGATTACTGGTACTTTGACCTGTGGGGCAGAG

GCACCCTGGTCACGGTGTCCTCTGGAGGAGGAGGCTCCGGA

GGAGGAGGCTCTGGCGGCGGCGGCAGCGACATTGTAATGA

CCCAGTCACCCTCCTTCCTTAGTGCCTCAGTCGGAGACCGCG

TGACTATCACTTGTCGTGCCTCACACGGAATTAATAACTACC

TCGCTTGGTATCAGCAAAAACCAGGCAAGGCCCCGAAACTA

TTGATCTACGCCGCATCTACTCTGCAGAGCGGAGTACCGAG

CAGATTTAGTGGTTCCGGCAGCGGAACCGAGTTCACCCTAA

CCATTTCCAGTTTGCAGCCAGAGGATTTCGCGACCTATTACT

GCCAGCAATACGATTCATACCCGCCAACTTTCGGAAGAGGT

ACCAAGGTTGAAATCAAGACTACGACCCCAGCACCTAGACC

TCCCACCCCAGCTCCAACTATAGCTTCCCAGCCATTGTCTCT

CCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGGGCCGTTC

ATACAAGAGGACTCGATTTCGCTTGCGATATCTACATATGG

GCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCTAAGCTTG

GTTATTACCCTCTATTGCCATAGGTGGTGCTCAAACAAAAA

GAATGCTGCCGTCATGGACCAGGAGAGCGCGGGCAATCGG

ACCGCAAACTCAGAGGACTCAGATGAACAAGATCCACAGG

AAGTGACCTACACTCAGCTGAACCATTGTGTGTTTACACAG

CGCAAGATTACTCGTCCAAGCCAGCGTCCTAAGACCCCCCC

GACCGATATCATTGTGTATACCGAGCTTCCTAATGCCGAAT

CCCGCAGCAAGGTGGTCTCCTGCCCGCGGAGAAAGCGTGGA

TCCGGGGAAGGCCGAGGCTCCCTTCTAACATGTGGAGATGT

CGAGGAAAACCCTGGCCCTATGGCGCTGCCAGTCACTGCAT

TGTTATTGCCTCTGGCCCTGCTTCTCCATGCGGCGCGCCCAG

AAGTGCAGCTGGTCGAGAGCGGAGGCGGACTGGTTCAACC

CGGAGGCAGCTTGAGACTGTCCTGCGCGGCCAGCGGCTTCA

ACATCAAGGATACCTATATCCACTGGGTGAGGCAGGCTCCA

GGAAAGGGCCTGGAGTGGGTGGCAAGGATTTACCCTACTAA

TGGATATACACGCTACGCTGATTCCGTGAAGGGACGCTTTA

CAATCTCAGCAGATACATCCAAAAACACGGCCTATTTACAG

ATGAATAGTTTGCGGGCCGAAGACACGGCTGTATACTATTG

TTCTCGGTGGGGGGGCGATGGATTTTATGCGATGGATTACT

GGGGCCAGGGCACCCTGGTAACCGTGTCAAGCGGCTCAACA

TCCGGGTCCGGTAAGCCGGGCTCCGGCGAGGGGTCTACAAA

GGGAGATATACAGATGACACAGTCCCCCAGTTCCCTGTCCG

CCTCAGTGGGAGACCGAGTGACGATTACCTGTCGTGCCAGC

CAGGACGTCAATACCGCCGTCGCTTGGTATCAGCAAAAACC

AGGCAAGGCCCCGAAACTATTGATCTACAGTGCCTCTTTTCT

GTACTCCGGGGTGCCGAGCAGATTTAGTGGCTCCAGGAGCG

GAACCGATTTCACCCTAACCATTTCCAGTTTGCAGCCAGAG

GATTTCGCGACCTATTACTGCCAGCAACACTACACCACACC

GCCAACTTTCGGACAAGGAACCAAGGTTGAAATCAAAATTG

AAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGC

AATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCC

AAGTCCCCTATTTCCCGGGCCTTCGAAGCCCTTTTGGGTGCT

GGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGT

AACAGTAGCGTTTATTATTTTCTGGGTGAAACGCGGCCGCA

AGAAACTGCTCTACATCTTTAAACAGCCGTTCATGAGGCCT

GTGCAGACAACGCAGGAAGAGGATGGCTGTAGTTGTCGGTT

TCCGGAAGAGGAAGAGGGGGGCTGCGAGTTGCGTGTCAAA

TTTTCTCGGTCTGCCGACGCCCCCGCGTACCAGCAAGGGCA

GAACCAGCTTTATAATGAGCTGAATCTTGGACGACGGGAGG

AATATGACGTGCTTGACAAGAGGCGAGGTAGGGACCCTGA

GATGGGGGGAAAACCTCGGAGGAAAAACCCACAGGAAGGC

CTGTATAACGAACTGCAGAAGGACAAGATGGCTGAAGCCT

ACTCTGAGATTGGAATGAAAGGGGAACGCAGACGCGGCAA

GGGCCATGATGGCCTCTACCAAGGTCTAAGCACTGCCACCA

AGGACACCTATGACGCACTCCACATGCAAGCTCTACCTCCC

CGTTGATAA

MC0295-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLVQSGGGVVQPGGSLRVS

3PF12_CD8
NO: 32
CAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYA

KIR2DL1_

DSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGP

HER2

LDYWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSDIVMTQS

Protein

PSFLSASVGDRVTITCRASHGINNYLAWYQQKPGKAPKLLIYA

Sequence

ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYDSY

(VR295)

PPTFGRGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG

GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHRWCSN

KKNAAVMDQESAGNRTANSEDSDEQDPQEVTYTQLNHCVFT

QRKITRPSQRPKTPPTDIIVYTELPNAESRSKVVSCPRRKRGSGE

GRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPEVQLV

ESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW

VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED

TAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGSTSGSGKPGS

GEGSTKGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW

YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQP

EDFATYYCQQHYTTPPTFGQGTKVEIKIEVMYPPPYLDNEKSN

GTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTV

AFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE

GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD

KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK

GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

GCTGCTGCACGCAGCCAGACCCCAGGTGCAGCTGCAGCAGT

CTGGACCTGAGCTGGTGAAGCCAGGAGCCTCCGTGAAGATG

TCTTGCAAGGCCAGCGGCTACACCTTCACATCTTATCACATC

CAGTGGGTGAAGCAGCGGCCCGGACAGGGCCTGGAGTGGA

TCGGATGGATCTACCCAGGCGACGGCTCCACACAGTATAAC

GAGAAGTTCAAGGGCAAGACCACACTGACCGCCGATAAGA

GCAGCAGCACCGCCTACATGCTGCTGAGCAGCCTGACCAGC

GAGGACAGCGCCATCTACTTTTGCGCCAGGGAGGGCACATA

CTATGCTATGGACTATTGGGGCCAGGGCACCAGCGTGACAG

MC00296-
SEQ ID
TGTCTAGCGGAGGAGGAGGCTCCGGAGGAGGAGGCTCTGG

BB7.2_CD8_
NO: 33
CGGCGGCGGCAGCGACGTGCTGATGACCCAGACACCACTG

LIR1_HER2

AGCCTGCCCGTGAGCCTGGGCGATCAGGTGAGCATCTCCTG

(VR296)

TAGATCCTCTCAGAGCATCGTGCACTCCAACGGCAATACCT

ACCTGGAGTGGTATCTGCAGAAGCCAGGCCAGTCCCCCAAG

CTGCTGATCTATAAGGTGTCTAATCGGTTCAGCGGCGTGCCT

GACAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCT

GAAGATCAGCCGGGTGGAGGCAGAGGATCTGGGCGTGTAC

TATTGTTTCCAGGGCTCCCACGTGCCACGCACCTTTGGCGGC

GGTACCAAGCTGGAGATCAAGACTACGACCCCAGCACCTAG

ACCTCCCACCCCAGCTCCAACTATAGCTTCCCAGCCATTGTC

TCTCCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGGGCCG

TTCATACAAGAGGACTCGATTTCGCTTGCGATATCTACATAT

GGGCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCTAAGCT

TGGTTATTACCCTCTATTGCCTGCGCCACAGGAGACAGGGC

AAGCACTGGACCAGCACCCAGCGGAAGGCCGACTTTCAGC

ACCCTGCCGGCGCCGTGGGCCCTGAGCCTACCGACAGGGGC

CTGCAGTGGAGGAGCTCCCCAGCCGCCGATGCCCAGGAGG

AGAATCTGTACGCCGCCGTGAAGCACACCCAGCCAGAGGA

CGGCGTGGAGATGGACACCCGCTCCCCACACGACGAGGATC

CACAGGCCGTGACCTACGCCGAGGTGAAGCACAGCCGCCCC

AGACGCGAGATGGCCAGCCCACCCAGCCCCCTGTCCGGCGA

GTTCCTGGACACCAAGGACAGGCAGGCCGAGGAGGACCGG

CAGATGGACACCGAGGCCGCCGCCTCCGAGGCCCCCCAGG

ACGTGACCTACGCCCAGCTGCACTCCCTGACCCTGCGGAGA

GAGGCCACCGAGCCCCCACCCAGCCAGGAGGGCCCCTCCCC

CGCCGTGCCTAGCATCTACGCCACCCTGGCCATCCACCGGA

GAAAGCGTGGATCCGGGGAAGGCCGAGGCTCCCTTCTAACA

TGTGGAGATGTCGAGGAAAACCCTGGCCCTATGGCGCTGCC

AGTCACTGCATTGTTATTGCCTCTGGCCCTGCTTCTCCATGC

GGCGCGCCCAGAAGTGCAGCTGGTCGAGAGCGGAGGCGGA

CTGGTTCAACCCGGAGGCAGCTTGAGACTGTCCTGCGCGGC

CAGCGGCTTCAACATCAAGGATACCTATATCCACTGGGTGA

GGCAGGCTCCAGGAAAGGGCCTGGAGTGGGTGGCAAGGAT

TTACCCTACTAATGGATATACACGCTACGCTGATTCCGTGA

AGGGACGCTTTACAATCTCAGCAGATACATCCAAAAACACG

GCCTATTTACAGATGAATAGTTTGCGGGCCGAAGACACGGC

TGTATACTATTGTTCTCGGTGGGGGGGCGATGGATTTTATGC

GATGGATTACTGGGGCCAGGGCACCCTGGTAACCGTGTCAA

GCGGCTCAACATCCGGGTCCGGTAAGCCGGGCTCCGGCGAG

GGGTCTACAAAGGGAGATATACAGATGACACAGTCCCCCA

GTTCCCTGTCCGCCTCAGTGGGAGACCGAGTGACGATTACC

TGTCGTGCCAGCCAGGACGTCAATACCGCCGTCGCTTGGTA

TCAGCAAAAACCAGGCAAGGCCCCGAAACTATTGATCTACA

GTGCCTCTTTTCTGTACTCCGGGGTGCCGAGCAGATTTAGTG

GCTCCAGGAGCGGAACCGATTTCACCCTAACCATTTCCAGT

TTGCAGCCAGAGGATTTCGCGACCTATTACTGCCAGCAACA

CTACACCACACCGCCAACTTTCGGACAAGGAACCAAGGTTG

AAATCAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACA

ATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAA

ACACCTTTGTCCAAGTCCCCTATTTCCCGGGCCTTCGAAGCC

CTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTA

TAGCTTGCTAGTAACAGTAGCGTTTATTATTTTCTGGGTGAA

ACGCGGCCGCAAGAAACTGCTCTACATCTTTAAACAGCCGT

TCATGAGGCCTGTGCAGACAACGCAGGAAGAGGATGGCTG

TAGTTGTCGGTTTCCGGAAGAGGAAGAGGGGGGCTGCGAGT

TGCGTGTCAAATTTTCTCGGTCTGCCGACGCCCCCGCGTACC

AGCAAGGGCAGAACCAGCTTTATAATGAGCTGAATCTTGGA

CGACGGGAGGAATATGACGTGCTTGACAAGAGGCGAGGTA

GGGACCCTGAGATGGGGGGAAAACCTCGGAGGAAAAACCC

ACAGGAAGGCCTGTATAACGAACTGCAGAAGGACAAGATG

GCTGAAGCCTACTCTGAGATTGGAATGAAAGGGGAACGCA

GACGCGGCAAGGGCCATGATGGCCTCTACCAAGGTCTAAGC

ACTGCCACCAAGGACACCTATGACGCACTCCACATGCAAGC

TCTACCTCCCCGTTGATAA

MC00296-
SEQ ID
MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKMS

BB7.2_CD8_
NO: 34
CKASGYTFTSYHIQWVKQRPGQGLEWIGWIYPGDGSTQYNEK

LIR1_HER2

FKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCAREGTYYAMD

protein

YWGQGTSVTVSSGGGGSGGGGSGGGGSDVLMTQTPLSLPVSL

Sequence

GDQVSISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNR

(VR296)

FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTF

GGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV

HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCLRHRRQGKH

WTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENL

YAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRRE

MASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYA

QLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIHRRKRGSGEGR

GSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPEVQLVES

GGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA

RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA

VYYCSRWGGDGFYAMDYWGQGTLVTVSSGSTSGSGKPGSGE

GSTKGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQ

QKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPED

FATYYCQQHYTTPPTFGQGTKVEIKIEVMYPPPYLDNEKSNGTI

IHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFII

FWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC

ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR

RGKGHDGLYQGLSTATKDTYDALHMQALPPR

MC00297-
SEQ ID
ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCT

BB7.2_CD8_
NO: 35
GCTGCTGCACGCAGCCAGACCCCAGGTGCAGCTGCAGCAGT

KIR2DL1_

CTGGACCTGAGCTGGTGAAGCCAGGAGCCTCCGTGAAGATG

HER2-

TCTTGCAAGGCCAGCGGCTACACCTTCACATCTTATCACATC

nucleic acid

CAGTGGGTGAAGCAGCGGCCCGGACAGGGCCTGGAGTGGA

(VR297)

TCGGATGGATCTACCCAGGCGACGGCTCCACACAGTATAAC

GAGAAGTTCAAGGGCAAGACCACACTGACCGCCGATAAGA

GCAGCAGCACCGCCTACATGCTGCTGAGCAGCCTGACCAGC

GAGGACAGCGCCATCTACTTTTGCGCCAGGGAGGGCACATA

CTATGCTATGGACTATTGGGGCCAGGGCACCAGCGTGACAG

TGTCTAGCGGAGGAGGAGGCTCCGGAGGAGGAGGCTCTGG

CGGCGGCGGCAGCGACGTGCTGATGACCCAGACACCACTG

AGCCTGCCCGTGAGCCTGGGCGATCAGGTGAGCATCTCCTG

TAGATCCTCTCAGAGCATCGTGCACTCCAACGGCAATACCT

ACCTGGAGTGGTATCTGCAGAAGCCAGGCCAGTCCCCCAAG

CTGCTGATCTATAAGGTGTCTAATCGGTTCAGCGGCGTGCCT

GACAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCT

GAAGATCAGCCGGGTGGAGGCAGAGGATCTGGGCGTGTAC

TATTGTTTCCAGGGCTCCCACGTGCCACGCACCTTTGGCGGC

GGTACCAAGCTGGAGATCAAGACTACGACCCCAGCACCTAG

ACCTCCCACCCCAGCTCCAACTATAGCTTCCCAGCCATTGTC

TCTCCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGGGCCG

TTCATACAAGAGGACTCGATTTCGCTTGCGATATCTACATAT

GGGCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCTAAGCT

TGGTTATTACCCTCTATTGCCATAGGTGGTGCTCAAACAAA

AAGAATGCTGCCGTCATGGACCAGGAGAGCGCGGGCAATC

GGACCGCAAACTCAGAGGACTCAGATGAACAAGATCCACA

GGAAGTGACCTACACTCAGCTGAACCATTGTGTGTTTACAC

AGCGCAAGATTACTCGTCCAAGCCAGCGTCCTAAGACCCCC

CCGACCGATATCATTGTGTATACCGAGCTTCCTAATGCCGA

ATCCCGCAGCAAGGTGGTCTCCTGCCCGCGGAGAAAGCGTG

GATCCGGGGAAGGCCGAGGCTCCCTTCTAACATGTGGAGAT

GTCGAGGAAAACCCTGGCCCTATGGCGCTGCCAGTCACTGC

ATTGTTATTGCCTCTGGCCCTGCTTCTCCATGCGGCGCGCCC

AGAAGTGCAGCTGGTCGAGAGCGGAGGCGGACTGGTTCAA

CCCGGAGGCAGCTTGAGACTGTCCTGCGCGGCCAGCGGCTT

CAACATCAAGGATACCTATATCCACTGGGTGAGGCAGGCTC

CAGGAAAGGGCCTGGAGTGGGTGGCAAGGATTTACCCTACT

AATGGATATACACGCTACGCTGATTCCGTGAAGGGACGCTT

TACAATCTCAGCAGATACATCCAAAAACACGGCCTATTTAC

AGATGAATAGTTTGCGGGCCGAAGACACGGCTGTATACTAT

TGTTCTCGGTGGGGGGGCGATGGATTTTATGCGATGGATTA

CTGGGGCCAGGGCACCCTGGTAACCGTGTCAAGCGGCTCAA

CATCCGGGTCCGGTAAGCCGGGCTCCGGCGAGGGGTCTACA

AAGGGAGATATACAGATGACACAGTCCCCCAGTTCCCTGTC

CGCCTCAGTGGGAGACCGAGTGACGATTACCTGTCGTGCCA

GCCAGGACGTCAATACCGCCGTCGCTTGGTATCAGCAAAAA

CCAGGCAAGGCCCCGAAACTATTGATCTACAGTGCCTCTTT

TCTGTACTCCGGGGTGCCGAGCAGATTTAGTGGCTCCAGGA

GCGGAACCGATTTCACCCTAACCATTTCCAGTTTGCAGCCA

GAGGATTTCGCGACCTATTACTGCCAGCAACACTACACCAC

ACCGCCAACTTTCGGACAAGGAACCAAGGTTGAAATCAAA

ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAA

GAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTT

GTCCAAGTCCCCTATTTCCCGGGCCTTCGAAGCCCTTTTGGG

TGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGC

TAGTAACAGTAGCGTTTATTATTTTCTGGGTGAAACGCGGC

CGCAAGAAACTGCTCTACATCTTTAAACAGCCGTTCATGAG

GCCTGTGCAGACAACGCAGGAAGAGGATGGCTGTAGTTGTC

GGTTTCCGGAAGAGGAAGAGGGGGGCTGCGAGTTGCGTGT

CAAATTTTCTCGGTCTGCCGACGCCCCCGCGTACCAGCAAG

GGCAGAACCAGCTTTATAATGAGCTGAATCTTGGACGACGG

GAGGAATATGACGTGCTTGACAAGAGGCGAGGTAGGGACC

CTGAGATGGGGGGAAAACCTCGGAGGAAAAACCCACAGGA

AGGCCTGTATAACGAACTGCAGAAGGACAAGATGGCTGAA

GCCTACTCTGAGATTGGAATGAAAGGGGAACGCAGACGCG

GCAAGGGCCATGATGGCCTCTACCAAGGTCTAAGCACTGCC

ACCAAGGACACCTATGACGCACTCCACATGCAAGCTCTACC

TCCCCGTTGATAA

MC00297:
SEQ ID
MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKMS

BB7.2_CD8_
NO: 36
CKASGYTFTSYHIQWVKQRPGQGLEWIGWIYPGDGSTQYNEK

KIR2DL1_

FKGKTTLTADKSSSTAYMLLSSLTSEDSAIYFCAREGTYYAMD

HER2

YWGQGTSVTVSSGGGGSGGGGSGGGGSDVLMTQTPLSLPVSL

protein

GDQVSISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNR

Sequence

FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTF

(VR297)

GGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV

HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCHRWCSNKKN

AAVMDQESAGNRTANSEDSDEQDPQEVTYTQLNHCVFTQRKI

TRPSQRPKTPPTDIIVYTELPNAESRSKVVSCPRRKRGSGEGRG

SLLTCGDVEENPGPMALPVTALLLPLALLLHAARPEVQLVESG

GGLLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA

RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA

VYYCSRWGGDGFYAMDYWGQGTLVTVSSGSTSGSGKPGSGE

GSTKGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQ

QKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPED

FATYYCQQHYTTPPTFGQGTKVEIKIEVMYPPPYLDNEKSNGTI

IHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFII

FWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC

ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR

RGKGHDGLYQGLSTATKDTYDALHMQALPPR

MC0421
SEQ ID
ATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCTGGCCCTG

HzBB7.2.2
NO: 275
CTTCTCCATGCGGCAAGGCCACAGGTGCAACTGGTTCAATC

LIR1(52)_

TGGTGCTGAGGTGAAAAAGCCCGGCGCATCCGTGAAAGTG

2A_HER2

AGCTGTAAGGCATCAGGGTACACCTTCACCAGCTATCACAT

nucleotide

ACAATGGGTCCGCCAGGCCCCCGGACAGAGGTTGGAATGG

sequence

ATTGGGTGGATTTACCCGGGTGACGGCTCAACCCAGTACAA

(VR421)

TGAGAAGTTCAAGGGCAGGGTGACTATCACACGCGATACCT

CCGCGAGCACAGCTTACATGGAGTTATCTAGCCTGAGATCC

GAAGATACGGCGGTGTATTACTGCGCGCGGGAAGGGACCT

ACTATGCCATGGACTATTGGGGACAAGGGACCCTGGTTACC

GTGAGTTCTGGGGGCGGGGGTTCCGGGGGAGGGGGATCTG

GGGGTGGAGGGAGCGATGTGGTAATGACCCAGACACCTTTG

TCTTTGAGTGTCACCCCCGGACAGCCGGCAAGTATATCCTG

TAGATCATCCCAATCAATCGTGCACTCCAACGGAAACACAT

ACTTGGAATGGTATCTCCAGAAACCTGGACAGTCCCCACAG

TTGCTCATCTACAAAGTGTCAAATCGCTTTTCAGGCGTGCCC

GATCGTTTCAGCGGCTCAGGCTCCGGGACAGACTTTACATT

GAAGATTAGCCGCGTAGAGGCAGAGGATGTGGGCGTTTACT

ATTGTTTTCAAGGGTCACACGTGCCACGCACATTCGGCGGC

GGTACCAAGGTGGAAATTAAGCACCCCAGCGACCCGCTGG

AGCTCGTTGTGTCCGGACCATCAGGGGGCCCGAGTAGCCCT

ACAACCGGCCCCACTTCTACCAGTGGACCGGAAGATCAACC

ACTTACACCAACGGGCAGCGACCCCCAGTCAGGCCTAGGGC

GCCACCTGGGTGTGGTCATCGGGATACTGGTCGCTGTCATC

CTGCTTCTGCTCCTTCTCTTGCTCCTATTCCTAATCCTGCGCC

ACAGGAGACAGGGCAAGCACTGGACCAGCACCCAGCGGAA

GGCCGACTTTCAGCACCCTGCCGGCGCCGTGGGCCCTGAGC

CTACCGACAGGGGCCTGCAGTGGAGGAGCTCCCCAGCCGCC

GATGCCCAGGAGGAGAATCTGTACGCCGCCGTGAAGCACA

CCCAGCCAGAGGACGGCGTGGAGATGGACACCCGCTCCCC

ACACGACGAGGACCCACAGGCCGTGACCTACGCCGAGGTG

AAGCACAGCCGCCCCAGACGCGAGATGGCCAGCCCACCCA

GCCCCCTGTCCGGCGAGTTCCTGGACACCAAGGACAGGCAG

GCCGAGGAGGACCGGCAGATGGACACCGAGGCCGCCGCCT

CCGAGGCCCCCCAGGACGTGACCTACGCCCAGCTGCACTCC

CTGACCCTGCGGAGAGAGGCCACCGAGCCCCCACCCAGCCA

GGAGGGCCCCTCCCCCGCCGTGCCTAGCATCTACGCCACCC

TGGCCATCCACCGGAGAAAGCGTGGATCCGGGGAAGGCCG

AGGCTCCCTTCTAACATGTGGAGATGTCGAGGAAAACCCTG

GCCCTATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCTGG

CCCTGCTTCTCCATGCGGCGCGCCCAGACATCCAGATGACC

CAATCCCCAAGCAGTCTCTCAGCCAGCGTGGGAGACAGGGT

TACAATCACGTGCCGCGCCAGCCAGGACGTCAACACCGCTG

TGGCTTGGTATCAGCAAAAGCCCGGGAAGGCACCAAAGCT

GCTTATTTATAGCGCCTCCTTCTTGTATTCTGGAGTGCCATC

CAGGTTTTCCGGGTCACGTAGCGGGACTGACTTTACCCTCA

CCATATCCAGCCTCCAGCCCGAGGATTTCGCCACCTATTACT

GTCAGCAACACTACACGACTCCACCGACTTTTGGACAGGGC

ACTAAAGTGGAGATTAAGGGCAGCACGAGTGGGAGTGGAA

AGCCCGGCAGCGGGGAGGGGTCTACCAAGGGAGAGGTCCA

GCTGGTTGAATCCGGAGGCGGGCTTGTGCAACCTGGAGGCT

CCCTGAGGCTTAGTTGTGCCGCGTCAGGATTCAACATTAAG

GATACCTATATTCATTGGGTCCGACAAGCCCCGGGCAAGGG

CTTGGAGTGGGTGGCCAGAATCTATCCGACCAACGGATATA

CAAGGTACGCCGATTCTGTGAAAGGACGCTTCACCATCAGC

GCGGACACATCCAAAAACACAGCCTATCTGCAGATGAACTC

CCTTCGCGCCGAGGATACAGCCGTGTACTATTGTAGTCGGT

GGGGAGGCGACGGCTTCTACGCGATGGACTATTGGGGACA

AGGAACACTGGTGACTGTCAGTAGCACTACGACCCCAGCAC

CTAGACCTCCCACCCCAGCTCCAACTATAGCTTCCCAGCCAT

TGTCTCTCCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGG

GCCGTTCATACAAGAGGACTCGATTTCGCTTGCGATATCTA

CATATGGGCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCT

AAGCTTGGTTATTACCCTCTATTGCAAAAGAGGACGAAAGA

AACTGCTTTATATATTCAAGCAACCTTTCATGCGCCCCGTAC

AGACCACGCAGGAGGAAGATGGGTGTAGCTGTCGCTTCCCT

GAGGAAGAGGAAGGTGGATGCGAGTTGCGGGTGAAGTTCA

GTCGATCCGCCGATGCGCCTGCCTATCAGCAAGGGCAGAAC

CAGCTTTATAACGAGTTAAACCTTGGCCGCCGGGAAGAGTA

TGACGTGTTGGACAAGCGTCGCGGGAGAGACCCTGAGATG

GGCGGAAAACCAAGGAGAAAAAATCCACAGGAAGGCTTAT

ATAACGAGTTGCAGAAAGACAAGATGGCCGAGGCATACTC

CGAAATCGGAATGAAGGGCGAGCGACGGCGCGGCAAAGGC

CACGATGGACTCTATCAGGGCTTAAGCACCGCCACCAAAGA

CACCTACGATGCACTTCATATGCAGGCACTCCCACCTAGAT

GATAA

MC0421
SEQ ID
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVS

HzBB7.2.2_
NO: 276
CKASGYTFTSYHIQWVRQAPGQRLEWIGWIYPGDGSTQYNEK

LIR1(52)_

FKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAM

2A_HER2

DYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLSV

Protein

TPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVS

Sequence

NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP

(VR421)

RTFGGGTKVEIKHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQ

PLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRR

QGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQ

EENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRP

RREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDV

TYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIHRRKRGS

GEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPDIQ

MTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPK

LLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ

HYTTPPTFGQGTKVEIKGSTSGSGKPGSGEGSTKGEVQLVESG

GGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR

IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV

YYCSRWGGDGFYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTI

ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGV

LLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP

EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD

VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI

GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

MC00428
SEQ ID
ATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCTGGCCCTG

HzBB7.2.1_
NO: 277
CTTCTCCATGCGGCAAGGCCACAGGTGCAACTGGTTCAATC

LIR1(52)_

TGGTGCTGAGGTGAAAAAGCCCGGCAGCTCTGTGAAAGTGA

(IRESL)_HER2

GCTGTAAGGCATCAGGGTATACCTTCACCAGCTATCACATA

nucleotide

CAATGGGTCCGCCAGGCCCCCGGACAGGGATTGGAATGGAT

sequence

GGGGTGGATTTACCCGGGTGACGGCTCAACCCAGTACAATG

(VR428)

AGAAGTTCAAGGGCAGGACAACTATCACAGCCGATAAGTC

CACGAGCACAGCTTACATGGAGTTATCTAGCCTGAGATCCG

AAGATACGGCGGTGTATTACTGCGCGCGGGAAGGGACCTAC

TATGCCATGGACTATTGGGGACAAGGGACCCTGGTTACCGT

GAGTTCTGGGGGCGGGGGTTCCGGGGGAGGGGGATCTGGG

GGTGGAGGGAGCGATGTGGTAATGACCCAGACACCTTTGTC

TTTGAGTGTCACCCCCGGACAGCCGGCAAGTATATCCTGTA

GATCATCCCAATCAATCGTGCACTCCAACGGAAACACATAC

TTGGAATGGTATCTCCAGAAACCTGGACAGTCCCCACAGTT

GCTCATCTACAAAGTGTCAAATCGCTTTTCAGGCGTGCCCG

ATCGTTTCAGCGGCTCAGGCTCCGGGACAGACTTTACATTG

AAGATTAGCCGCGTAGAGGCAGAGGATGTGGGCGTTTACTA

TTGTTTTCAAGGGTCACACGTGCCACGCACATTCGGCGGCG

GTACCAAGGTGGAAATTAAGCACCCCAGCGACCCGCTGGA

GCTCGTTGTGTCCGGACCATCAGGGGGCCCGAGTAGCCCTA

CAACCGGCCCCACTTCTACCAGTGGACCGGAAGATCAACCA

CTTACACCAACGGGCAGCGACCCCCAGTCAGGCCTAGGGCG

CCACCTGGGTGTGGTCATCGGGATACTGGTCGCTGTCATCCT

GCTTCTGCTCCTTCTCTTGCTCCTATTCCTAATCCTGCGCCAC

AGGAGACAGGGCAAGCACTGGACCAGCACCCAGCGGAAGG

CCGACTTTCAGCACCCTGCCGGCGCCGTGGGCCCTGAGCCT

ACCGACAGGGGCCTGCAGTGGAGGAGCTCCCCAGCCGCCG

ATGCCCAGGAGGAGAATCTGTACGCCGCCGTGAAGCACACC

CAGCCAGAGGACGGCGTGGAGATGGACACCCGCTCCCCAC

ACGACGAGGACCCACAGGCCGTGACCTACGCCGAGGTGAA

GCACAGCCGCCCCAGACGCGAGATGGCCAGCCCACCCAGC

CCCCTGTCCGGCGAGTTCCTGGACACCAAGGACAGGCAGGC

CGAGGAGGACCGGCAGATGGACACCGAGGCCGCCGCCTCC

GAGGCCCCCCAGGACGTGACCTACGCCCAGCTGCACTCCCT

GACCCTGCGGAGAGAGGCCACCGAGCCCCCACCCAGCCAG

GAGGGCCCCTCCCCCGCCGTGCCTAGCATCTACGCCACCCT

GGCCATCCACTGATAACCCCCCCCCCTAACGTTACTGGCCG

AAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGT

TATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCC

GGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGT

CTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTC

GTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAAC

AACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCAC

CTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAA

GATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGT

GAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAA

GCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACC

CCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTT

TACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCC

CGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGA

TAATATGATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCT

GGCCCTGCTTCTCCATGCGGCGCGCCCAGACATCCAGATGA

CCCAATCCCCAAGCAGTCTCTCAGCCAGCGTGGGAGACAGG

GTTACAATCACGTGCCGCGCCAGCCAGGACGTCAACACCGC

TGTGGCTTGGTATCAGCAAAAGCCCGGGAAGGCACCAAAG

CTGCTTATTTATAGCGCCTCCTTCTTGTATTCTGGAGTGCCA

TCCAGGTTTTCCGGGTCACGTAGCGGGACTGACTTTACCCTC

ACCATATCCAGCCTCCAGCCCGAGGATTTCGCCACCTATTA

CTGTCAGCAACACTACACGACTCCACCGACTTTTGGACAGG

GCACTAAAGTGGAGATTAAGGGCAGCACGAGTGGGAGTGG

AAAGCCCGGCAGCGGGGAGGGGTCTACCAAGGGAGAGGTC

CAGCTGGTTGAATCCGGAGGCGGGCTTGTGCAACCTGGAGG

CTCCCTGAGGCTTAGTTGTGCCGCGTCAGGATTCAACATTA

AGGATACCTATATTCATTGGGTCCGACAAGCCCCGGGCAAG

GGCTTGGAGTGGGTGGCCAGAATCTATCCGACCAACGGATA

TACAAGGTACGCCGATTCTGTGAAAGGACGCTTCACCATCA

GCGCGGACACATCCAAAAACACAGCCTATCTGCAGATGAAC

TCCCTTCGCGCCGAGGATACAGCCGTGTACTATTGTAGTCG

GTGGGGAGGCGACGGCTTCTACGCGATGGACTATTGGGGAC

AAGGAACACTGGTGACTGTCAGTAGCACTACGACCCCAGCA

CCTAGACCTCCCACCCCAGCTCCAACTATAGCTTCCCAGCC

ATTGTCTCTCCGGCCAGAGGCGTGTCGACCAGCCGCTGGAG

GGGCCGTTCATACAAGAGGACTCGATTTCGCTTGCGATATC

TACATATGGGCCCCTCTTGCCGGGACATGCGGTGTCCTGCTT

CTAAGCTTGGTTATTACCCTCTATTGCAAAAGAGGACGAAA

GAAACTGCTTTATATATTCAAGCAACCTTTCATGCGCCCCGT

ACAGACCACGCAGGAGGAAGATGGGTGTAGCTGTCGCTTCC

CTGAGGAAGAGGAAGGTGGATGCGAGTTGCGGGTGAAGTT

CAGTCGATCCGCCGATGCGCCTGCCTATCAGCAAGGGCAGA

ACCAGCTTTATAACGAGTTAAACCTTGGCCGCCGGGAAGAG

TATGACGTGTTGGACAAGCGTCGCGGGAGAGACCCTGAGAT

GGGCGGAAAACCAAGGAGAAAAAATCCACAGGAAGGCTTA

TATAACGAGTTGCAGAAAGACAAGATGGCCGAGGCATACT

CCGAAATCGGAATGAAGGGCGAGCGACGGCGCGGCAAAGG

CCACGATGGACTCTATCAGGGCTTAAGCACCGCCACCAAAG

ACACCTACGATGCACTTCATATGCAGGCACTCCCACCTAGA

TGATAA

MC0428
SEQ ID
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSVKVS

HzBB7.2.1_
NO: 278
CKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPGDGSTQYNE

LIR1(52)_

KFKGRTTITADKSTSTAYMELSSLRSEDTAVYYCAREGTYYA

(IRESL)_

MDYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLS

HER2 Protein

VTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKV

sequence

SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHV

(VR428)

PRTFGGGTKVEIKHPSDPLELVVSGPSGGPSSPTTGPTSTSGPED

QPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHR

RQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADA

QEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHS

RPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQD

VTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH*

MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITC

RASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSR

SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGST

SGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAASGEN

IKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS

ADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWG

QGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV

HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI

FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP

AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK

NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL

STATKDTYDALHMQALPPR*

MC0447
SEQ ID
ATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCTGGCCCTG

SN66E3.2
NO: 279
CTTCTCCATGCGGCAAGGCCAGATATAGTGATGACACAGTC

(LH)_LIR1

CCCCGACTCCCTGGCTGTCTCACTGGGAGAACGAGCGACGA

(30)_

TTAGTTGTAAGTCTAGCCAGAGCGTCCTGTATTCAAGCAAT

(IRESL)_HER2

AACAAGAATTACCTCGCCTGGTATCAGCAAAAGCCGGGACA

nucleotide

GCCACCCAAACTGTTGATTTACTGGGCCAGCACGAGAGAGA

Sequence

GCGGAGTGCCCGACCGCTTCAGCGGATCCGGGTCAGGCACA

(VR447)

GATTTTACCCTGACTATTAGCTCCCTTCAAGCGGAAGATGTC

GCCGTCTACTATTGCCAGCAATATTACGGAACTCCATTCAC

ATTCGGCGGTGGGACCAAAGTAGAGATAAAGGGTGGCGGG

GGATCCGGCGGTGGCGGTAGCGGGGGAGGCGGGTCCCAAG

TGCAACTAGTCCAATCAGGTGCCGAAGTCAAGAAACCAGGT

GCATCCGTGAAAGTGTCTTGCAAAGCCAGTGGCTACACTTT

TACTGACTACTATCTGCACTGGGTGCGTCAAGCACCCGGCC

AGGGGCTTGAATGGATGGGCTGGATTAACCCTTATACTGGA

GGGACAAATTACGCTCAGAAGTTCCAGGGACGCGTTACAAT

GACCCGAGACACCAGCATCAGCACAGCGTACATGGAGTTA

AGTGGGCTGACTTCCGACGATACCGCCGTGTATTACTGCGC

TCGGGCAGGGGCCTCTTACTATGATTTTTGGTCCGGTTGGGT

CTTCGATTACTGGGGGCAGGGAACCCTGGTGACAGTGTCCT

CAGGCCCCACTTCTACCAGTGGACCGGAAGATCAACCACTT

ACACCAACGGGCAGCGACCCCCAGTCAGGCCTAGGGCGCC

ACCTGGGTGTGGTCATCGGGATACTGGTCGCTGTCATCCTG

CTTCTGCTCCTTCTCTTGCTCCTATTCCTAATCCTGCGCCACA

GGAGACAGGGCAAGCACTGGACCAGCACCCAGCGGAAGGC

CGACTTTCAGCACCCTGCCGGCGCCGTGGGCCCTGAGCCTA

CCGACAGGGGCCTGCAGTGGAGGAGCTCCCCAGCCGCCGAT

GCCCAGGAGGAGAATCTGTACGCCGCCGTGAAGCACACCC

AGCCAGAGGACGGCGTGGAGATGGACACCCGCTCCCCACA

CGACGAGGACCCACAGGCCGTGACCTACGCCGAGGTGAAG

CACAGCCGCCCCAGACGCGAGATGGCCAGCCCACCCAGCCC

CCTGTCCGGCGAGTTCCTGGACACCAAGGACAGGCAGGCCG

AGGAGGACCGGCAGATGGACACCGAGGCCGCCGCCTCCGA

GGCCCCCCAGGACGTGACCTACGCCCAGCTGCACTCCCTGA

CCCTGCGGAGAGAGGCCACCGAGCCCCCACCCAGCCAGGA

GGGCCCCTCCCCCGCCGTGCCTAGCATCTACGCCACCCTGG

CCATCCACTGATAACCCCCCCCCCTAACGTTACTGGCCGAA

GCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTAT

TTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGA

AACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTT

CCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTG

AAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAAC

GTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTG

GCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGAT

ACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAG

TTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCG

TATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCAT

TGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTAC

ATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGA

ACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAA

TATGATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCTGGC

CCTGCTTCTCCATGCGGCGCGCCCAGACATCCAGATGACCC

AATCCCCAAGCAGTCTCTCAGCCAGCGTGGGAGACAGGGTT

ACAATCACGTGCCGCGCCAGCCAGGACGTCAACACCGCTGT

GGCTTGGTATCAGCAAAAGCCCGGGAAGGCACCAAAGCTG

CTTATTTATAGCGCCTCCTTCTTGTATTCTGGAGTGCCATCC

AGGTTTTCCGGGTCACGTAGCGGGACTGACTTTACCCTCAC

CATATCCAGCCTCCAGCCCGAGGATTTCGCCACCTATTACTG

TCAGCAACACTACACGACTCCACCGACTTTTGGACAGGGCA

CTAAAGTGGAGATTAAGGGCAGCACGAGTGGGAGTGGAAA

GCCCGGCAGCGGGGAGGGGTCTACCAAGGGAGAGGTCCAG

CTGGTTGAATCCGGAGGCGGGCTTGTGCAACCTGGAGGCTC

CCTGAGGCTTAGTTGTGCCGCGTCAGGATTCAACATTAAGG

ATACCTATATTCATTGGGTCCGACAAGCCCCGGGCAAGGGC

TTGGAGTGGGTGGCCAGAATCTATCCGACCAACGGATATAC

AAGGTACGCCGATTCTGTGAAAGGACGCTTCACCATCAGCG

CGGACACATCCAAAAACACAGCCTATCTGCAGATGAACTCC

CTTCGCGCCGAGGATACAGCCGTGTACTATTGTAGTCGGTG

GGGAGGCGACGGCTTCTACGCGATGGACTATTGGGGACAA

GGAACACTGGTGACTGTCAGTAGCACTACGACCCCAGCACC

TAGACCTCCCACCCCAGCTCCAACTATAGCTTCCCAGCCATT

GTCTCTCCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGGG

CCGTTCATACAAGAGGACTCGATTTCGCTTGCGATATCTAC

ATATGGGCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCTA

AGCTTGGTTATTACCCTCTATTGCAAAAGAGGACGAAAGAA

ACTGCTTTATATATTCAAGCAACCTTTCATGCGCCCCGTACA

GACCACGCAGGAGGAAGATGGGTGTAGCTGTCGCTTCCCTG

AGGAAGAGGAAGGTGGATGCGAGTTGCGGGTGAAGTTCAG

TCGATCCGCCGATGCGCCTGCCTATCAGCAAGGGCAGAACC

AGCTTTATAACGAGTTAAACCTTGGCCGCCGGGAAGAGTAT

GACGTGTTGGACAAGCGTCGCGGGAGAGACCCTGAGATGG

GCGGAAAACCAAGGAGAAAAAATCCACAGGAAGGCTTATA

TAACGAGTTGCAGAAAGACAAGATGGCCGAGGCATACTCC

GAAATCGGAATGAAGGGCGAGCGACGGCGCGGCAAAGGCC

ACGATGGACTCTATCAGGGCTTAAGCACCGCCACCAAAGAC

ACCTACGATGCACTTCATATGCAGGCACTCCCACCTAGATG

ATAA

MC0447
SEQ ID
MALPVTALLLPLALLLHAARPDIVMTQSPDSLAVSLGERATISC

SN66E3.2
NO: 280
KSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVP

(LH)_LIR1

DRESGSGSGTDFTLTISSLQAEDVAVYYCQQYYGTPFTFGGGT

(30)_

KVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS

(IRESL)_HER2

CKASGYTFTDYYLHWVRQAPGQGLEWMGWINPYTGGTNYA

Protein

QKFQGRVTMTRDTSISTAYMELSGLTSDDTAVYYCARAGASY

Sequence

YDFWSGWVFDYWGQGTLVTVSSGPTSTSGPEDQPLTPTGSDP

(VR447)

QSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTST

QRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAV

KHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPP

SPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSL

TLRREATEPPPSQEGPSPAVPSIYATLAIH*

MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITC

RASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSR

SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGST

SGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAASGEN

IKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS

ADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWG

QGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV

HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI

FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP

AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK

NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL

STATKDTYDALHMQALPPR*

MC0449
SEQ ID
ATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCTGGCCCTG

SN66E3.3
NO: 281
CTTCTCCATGCGGCAAGGCCAGATATAGTGATGACACAGTC

(LH)_LIR1

CCCCGACTCCCTGGCTGTCTCACTGGGAGAACGAGCGACGA

(26)_

TTAGTTGTAAGTCTAGCCAGAGCGTCCTGTATTCAAGCAAT

(IRESL)_HER2

AACAAGAATTACCTCGCCTGGTATCAGCAAAAGCCGGGACA

Nucleotide

GCCACCCAAACTGTTGATTTACTGGGCCAGCACGAGAGAGA

Sequence

GCGGAGTGCCCGACCGCTTCAGCGGATCCGGGTCAGGCACA

(VR449)

GATTTTACCCTGACTATTAGCTCCCTTCAAGCGGAAGATGTC

GCCGTCTACTATTGCCAGCAATATTACGGAACTCCATTCAC

ATTCGGCGGTGGGACCAAAGTAGAGATAAAGGGTGGCGGG

GGATCCGGCGGTGGCGGTAGCGGGGGAGGCGGGTCCCAAG

TGCAACTAGTCCAATCAGGTGCCGAAGTCAAGAAACCAGGT

GCATCCGTGAAAGTGTCTTGCAAAGCCAGTGGCTACACTTT

TACTGACTACTATCTGCACTGGGTGCGTCAAGCACCCGGCC

AGGGGCTTGAATGGATGGGCTGGATTAACCCTTATACTGGA

GGGACAAATTACGCTCAGAAGTTCCAGGGACGCGTTACAAT

GACCCGAGACACCAGCATCAGCACAGCGTACATGGAGTTA

AGTAGGCTGAGGTCCGAAGATACCGCCGTGTATTACTGCGC

TCGGGCAGGGGCCTCTTACTATGATTTTTGGTCCGGTTGGGT

CTTCGATTACTGGGGGCAGGGAACCCTGGTGACAGTGTCCT

CAACCAGTGGACCGGAAGATCAACCACTTACACCAACGGG

CAGCGACCCCCAGTCAGGCCTAGGGCGCCACCTGGGTGTGG

TCATCGGGATACTGGTCGCTGTCATCCTGCTTCTGCTCCTTC

TCTTGCTCCTATTCCTAATCCTGCGCCACAGGAGACAGGGC

AAGCACTGGACCAGCACCCAGCGGAAGGCCGACTTTCAGC

ACCCTGCCGGCGCCGTGGGCCCTGAGCCTACCGACAGGGGC

CTGCAGTGGAGGAGCTCCCCAGCCGCCGATGCCCAGGAGG

AGAATCTGTACGCCGCCGTGAAGCACACCCAGCCAGAGGA

CGGCGTGGAGATGGACACCCGCTCCCCACACGACGAGGAC

CCACAGGCCGTGACCTACGCCGAGGTGAAGCACAGCCGCCC

CAGACGCGAGATGGCCAGCCCACCCAGCCCCCTGTCCGGCG

AGTTCCTGGACACCAAGGACAGGCAGGCCGAGGAGGACCG

GCAGATGGACACCGAGGCCGCCGCCTCCGAGGCCCCCCAG

GACGTGACCTACGCCCAGCTGCACTCCCTGACCCTGCGGAG

AGAGGCCACCGAGCCCCCACCCAGCCAGGAGGGCCCCTCCC

CCGCCGTGCCTAGCATCTACGCCACCCTGGCCATCCACTGA

TAACCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAA

TAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATA

TTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCC

TGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGC

CAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCA

GTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGC

GACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGT

GCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCA

AAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGT

TGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACA

AGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGG

ATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTA

GTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGG

GACGTGGTTTTCCTTTGAAAAACACGATGATAATATGATGG

CGCTGCCAGTCACTGCATTGTTATTGCCTCTGGCCCTGCTTC

TCCATGCGGCGCGCCCAGACATCCAGATGACCCAATCCCCA

AGCAGTCTCTCAGCCAGCGTGGGAGACAGGGTTACAATCAC

GTGCCGCGCCAGCCAGGACGTCAACACCGCTGTGGCTTGGT

ATCAGCAAAAGCCCGGGAAGGCACCAAAGCTGCTTATTTAT

AGCGCCTCCTTCTTGTATTCTGGAGTGCCATCCAGGTTTTCC

GGGTCACGTAGCGGGACTGACTTTACCCTCACCATATCCAG

CCTCCAGCCCGAGGATTTCGCCACCTATTACTGTCAGCAAC

ACTACACGACTCCACCGACTTTTGGACAGGGCACTAAAGTG

GAGATTAAGGGCAGCACGAGTGGGAGTGGAAAGCCCGGCA

GCGGGGAGGGGTCTACCAAGGGAGAGGTCCAGCTGGTTGA

ATCCGGAGGCGGGCTTGTGCAACCTGGAGGCTCCCTGAGGC

TTAGTTGTGCCGCGTCAGGATTCAACATTAAGGATACCTAT

ATTCATTGGGTCCGACAAGCCCCGGGCAAGGGCTTGGAGTG

GGTGGCCAGAATCTATCCGACCAACGGATATACAAGGTACG

CCGATTCTGTGAAAGGACGCTTCACCATCAGCGCGGACACA

TCCAAAAACACAGCCTATCTGCAGATGAACTCCCTTCGCGC

CGAGGATACAGCCGTGTACTATTGTAGTCGGTGGGGAGGCG

ACGGCTTCTACGCGATGGACTATTGGGGACAAGGAACACTG

GTGACTGTCAGTAGCACTACGACCCCAGCACCTAGACCTCC

CACCCCAGCTCCAACTATAGCTTCCCAGCCATTGTCTCTCCG

GCCAGAGGCGTGTCGACCAGCCGCTGGAGGGGCCGTTCATA

CAAGAGGACTCGATTTCGCTTGCGATATCTACATATGGGCC

CCTCTTGCCGGGACATGCGGTGTCCTGCTTCTAAGCTTGGTT

ATTACCCTCTATTGCAAAAGAGGACGAAAGAAACTGCTTTA

TATATTCAAGCAACCTTTCATGCGCCCCGTACAGACCACGC

AGGAGGAAGATGGGTGTAGCTGTCGCTTCCCTGAGGAAGA

GGAAGGTGGATGCGAGTTGCGGGTGAAGTTCAGTCGATCCG

CCGATGCGCCTGCCTATCAGCAAGGGCAGAACCAGCTTTAT

AACGAGTTAAACCTTGGCCGCCGGGAAGAGTATGACGTGTT

GGACAAGCGTCGCGGGAGAGACCCTGAGATGGGCGGAAAA

CCAAGGAGAAAAAATCCACAGGAAGGCTTATATAACGAGT

TGCAGAAAGACAAGATGGCCGAGGCATACTCCGAAATCGG

AATGAAGGGCGAGCGACGGCGCGGCAAAGGCCACGATGGA

CTCTATCAGGGCTTAAGCACCGCCACCAAAGACACCTACGA

TGCACTTCATATGCAGGCACTCCCACCTAGATGATAA

MC0449
SEQ ID
MALPVTALLLPLALLLHAARPDIVMTQSPDSLAVSLGERATISC

SN66E3.3
NO: 282
KSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVP

(LH)_LIR1

DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYGTPFTFGGGT

(26)_

KVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS

(IRESL)_HER2

CKASGYTFTDYYLHWVRQAPGQGLEWMGWINPYTGGTNYA

Protein

QKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARAGASY

Sequence

YDFWSGWVFDYWGQGTLVTVSSTSGPEDQPLTPTGSDPQSGL

(VR449)

GRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKA

DFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQP

EDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGE

FLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREA

TEPPPSQEGPSPAVPSIYATLAIH*

MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITC

RASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRESGSR

SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGST

SGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAASGEN

IKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS

ADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWG

QGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV

HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI

FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP

AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK

NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL

STATKDTYDALHMQALPPR*

MC0515-
SEQ ID
ATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCTGGCCCTG

HzBB7.2(2)_
NO: 321
TGGTGCTGAGGTGAAAAAGCCCGGCGCATCCGTGAAAGTG

LIR1(30)_

CTTCTCCATGCGGCAAGGCCACAGGTGCAACTGGTTCAATC

2A_HER2

AGCTGTAAGGCATCAGGGTACACCTTCACCAGCTATCACAT

Nucleotide

ACAATGGGTCCGCCAGGCCCCCGGACAGAGGTTGGAATGG

Sequence

TGAGAAGTTCAAGGGCAGGGTGACTATCACACGCGATACCT

(VR515)

ATTGGGTGGATTTACCCGGGTGACGGCTCAACCCAGTACAA

CCGCGAGCACAGCTTACATGGAGTTATCTAGCCTGAGATCC

GAAGATACGGCGGTGTATTACTGCGCGCGGGAAGGGACCT

ACTATGCCATGGACTATTGGGGACAAGGGACCCTGGTTACC

GTGAGTTCTGGGGGCGGGGGTTCCGGGGGAGGGGGATCTG

GGGGTGGAGGGAGCGATGTGGTAATGACCCAGACACCTTTG

TCTTTGAGTGTCACCCCCGGACAGCCGGCAAGTATATCCTG

TAGATCATCCCAATCAATCGTGCACTCCAACGGAAACACAT

ACTTGGAATGGTATCTCCAGAAACCTGGACAGTCCCCACAG

TTGCTCATCTACAAAGTGTCAAATCGCTTTTCAGGCGTGCCC

GATCGTTTCAGCGGCTCAGGCTCCGGGACAGACTTTACATT

GAAGATTAGCCGCGTAGAGGCAGAGGATGTGGGCGTTTACT

ATTGTTTTCAAGGGTCACACGTGCCACGCACATTCGGCGGC

GGTACCAAGGTGGAAATTAAGGGCCCCACTTCTACCAGTGG

ACCGGAAGATCAACCACTTACACCAACGGGCAGCGACCCCC

AGTCAGGCCTAGGGCGCCACCTGGGTGTGGTCATCGGGATA

CTGGTCGCTGTCATCCTGCTTCTGCTCCTTCTCTTGCTCCTAT

TCCTAATCCTGCGCCACAGGAGACAGGGCAAGCACTGGACC

AGCACCCAGCGGAAGGCCGACTTTCAGCACCCTGCCGGCGC

CGTGGGCCCTGAGCCTACCGACAGGGGCCTGCAGTGGAGG

AGCTCCCCAGCCGCCGATGCCCAGGAGGAGAATCTGTACGC

CGCCGTGAAGCACACCCAGCCAGAGGACGGCGTGGAGATG

GACACCCGCTCCCCACACGACGAGGACCCACAGGCCGTGAC

CTACGCCGAGGTGAAGCACAGCCGCCCCAGACGCGAGATG

GCCAGCCCACCCAGCCCCCTGTCCGGCGAGTTCCTGGACAC

CAAGGACAGGCAGGCCGAGGAGGACCGGCAGATGGACACC

GAGGCCGCCGCCTCCGAGGCCCCCCAGGACGTGACCTACGC

CCAGCTGCACTCCCTGACCCTGCGGAGAGAGGCCACCGAGC

CCCCACCCAGCCAGGAGGGCCCCTCCCCCGCCGTGCCTAGC

ATCTACGCCACCCTGGCCATCCACGGATCCGGGGAAGGCCG

AGGCTCCCTTCTAACATGTGGAGATGTCGAGGAAAACCCTG

GCCCTATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCTGG

CCCTGCTTCTCCATGCGGCGCGCCCAGACATCCAGATGACC

CAATCCCCAAGCAGTCTCTCAGCCAGCGTGGGAGACAGGGT

TACAATCACGTGCCGCGCCAGCCAGGACGTCAACACCGCTG

TGGCTTGGTATCAGCAAAAGCCCGGGAAGGCACCAAAGCT

GCTTATTTATAGCGCCTCCTTCTTGTATTCTGGAGTGCCATC

CAGGTTTTCCGGGTCACGTAGCGGGACTGACTTTACCCTCA

CCATATCCAGCCTCCAGCCCGAGGATTTCGCCACCTATTACT

GTCAGCAACACTACACGACTCCACCGACTTTTGGACAGGGC

ACTAAAGTGGAGATTAAGGGCAGCACGAGTGGGAGTGGAA

AGCCCGGCAGCGGGGAGGGGTCTACCAAGGGAGAGGTCCA

GCTGGTTGAATCCGGAGGCGGGCTTGTGCAACCTGGAGGCT

CCCTGAGGCTTAGTTGTGCCGCGTCAGGATTCAACATTAAG

GATACCTATATTCATTGGGTCCGACAAGCCCCGGGCAAGGG

CTTGGAGTGGGTGGCCAGAATCTATCCGACCAACGGATATA

CAAGGTACGCCGATTCTGTGAAAGGACGCTTCACCATCAGC

GCGGACACATCCAAAAACACAGCCTATCTGCAGATGAACTC

CCTTCGCGCCGAGGATACAGCCGTGTACTATTGTAGTCGGT

GGGGAGGCGACGGCTTCTACGCGATGGACTATTGGGGACA

AGGAACACTGGTGACTGTCAGTAGCACTACGACCCCAGCAC

CTAGACCTCCCACCCCAGCTCCAACTATAGCTTCCCAGCCAT

TGTCTCTCCGGCCAGAGGCGTGTCGACCAGCCGCTGGAGGG

GCCGTTCATACAAGAGGACTCGATTTCGCTTGCGATATCTA

CATATGGGCCCCTCTTGCCGGGACATGCGGTGTCCTGCTTCT

AAGCTTGGTTATTACCCTCTATTGCAAAAGAGGACGAAAGA

AACTGCTTTATATATTCAAGCAACCTTTCATGCGCCCCGTAC

AGACCACGCAGGAGGAAGATGGGTGTAGCTGTCGCTTCCCT

GAGGAAGAGGAAGGTGGATGCGAGTTGCGGGTGAAGTTCA

GTCGATCCGCCGATGCGCCTGCCTATCAGCAAGGGCAGAAC

CAGCTTTATAACGAGTTAAACCTTGGCCGCCGGGAAGAGTA

TGACGTGTTGGACAAGCGTCGCGGGAGAGACCCTGAGATG

GGCGGAAAACCAAGGAGAAAAAATCCACAGGAAGGCTTAT

ATAACGAGTTGCAGAAAGACAAGATGGCCGAGGCATACTC

CGAAATCGGAATGAAGGGCGAGCGACGGCGCGGCAAAGGC

CACGATGGACTCTATCAGGGCTTAAGCACCGCCACCAAAGA

CACCTACGATGCACTTCATATGCAGGCACTCCCACCTAGAT

GATAA

MC0515
SEQ ID
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVS

HzBB7.2(2)_
NO: 322
CKASGYTFTSYHIQWVRQAPGQRLEWIGWIYPGDGSTQYNEK

LIR1(30)_

FKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGTYYAM

2A_HER2

DYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLSV

Protein

TPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVS

Sequence

NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP

(VR515)

RTFGGGTKVEIKGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVI

GILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAG

AVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEM

DTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKD

RQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPS

QEGPSPAVPSIYATLAIHGSGEGRGSLLTCGDVEENPGPMALPV

TALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCRASQD

VNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDF

TLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSGSGK

PGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTY

IHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTS

KNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTL

VTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG

LDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP

FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ

GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT

KDTYDALHMQALPPR

MC0516-
SEQ ID
ATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCTGGCCCTG

SN66E3.2
NO: 323
CTTCTCCATGCGGCAAGGCCAGATATAGTGATGACACAGTC

(LH)_LIR1

CCCCGACTCCCTGGCTGTCTCACTGGGAGAACGAGCGACGA

(30)_2A_HER2

TTAGTTGTAAGTCTAGCCAGAGCGTCCTGTATTCAAGCAAT

Nucleotide

AACAAGAATTACCTCGCCTGGTATCAGCAAAAGCCGGGACA

Sequence

GCCACCCAAACTGTTGATTTACTGGGCCAGCACGAGAGAGA

(VR516)

GCGGAGTGCCCGACCGCTTCAGCGGATCCGGGTCAGGCACA

GATTTTACCCTGACTATTAGCTCCCTTCAAGCGGAAGATGTC

GCCGTCTACTATTGCCAGCAATATTACGGAACTCCATTCAC

ATTCGGCGGTGGGACCAAAGTAGAGATAAAGGGTGGCGGG

GGATCCGGCGGTGGCGGTAGCGGGGGAGGCGGGTCCCAAG

TGCAACTAGTCCAATCAGGTGCCGAAGTCAAGAAACCAGGT

GCATCCGTGAAAGTGTCTTGCAAAGCCAGTGGCTACACTTT

TACTGACTACTATCTGCACTGGGTGCGTCAAGCACCCGGCC

AGGGGCTTGAATGGATGGGCTGGATTAACCCTTATACTGGA

GGGACAAATTACGCTCAGAAGTTCCAGGGACGCGTTACAAT

GACCCGAGACACCAGCATCAGCACAGCGTACATGGAGTTA

AGTGGGCTGACTTCCGACGATACCGCCGTGTATTACTGCGC

TCGGGCAGGGGCCTCTTACTATGATTTTTGGTCCGGTTGGGT

CTTCGATTACTGGGGGCAGGGAACCCTGGTGACAGTGTCCT

CAGGCCCCACTTCTACCAGTGGACCGGAAGATCAACCACTT

ACACCAACGGGCAGCGACCCCCAGTCAGGCCTAGGGCGCC

ACCTGGGTGTGGTCATCGGGATACTGGTCGCTGTCATCCTG

CTTCTGCTCCTTCTCTTGCTCCTATTCCTAATCCTGCGCCACA

GGAGACAGGGCAAGCACTGGACCAGCACCCAGCGGAAGGC

CGACTTTCAGCACCCTGCCGGCGCCGTGGGCCCTGAGCCTA

CCGACAGGGGCCTGCAGTGGAGGAGCTCCCCAGCCGCCGAT

GCCCAGGAGGAGAATCTGTACGCCGCCGTGAAGCACACCC

AGCCAGAGGACGGCGTGGAGATGGACACCCGCTCCCCACA

CGACGAGGACCCACAGGCCGTGACCTACGCCGAGGTGAAG

CACAGCCGCCCCAGACGCGAGATGGCCAGCCCACCCAGCCC

CCTGTCCGGCGAGTTCCTGGACACCAAGGACAGGCAGGCCG

AGGAGGACCGGCAGATGGACACCGAGGCCGCCGCCTCCGA

GGCCCCCCAGGACGTGACCTACGCCCAGCTGCACTCCCTGA

CCCTGCGGAGAGAGGCCACCGAGCCCCCACCCAGCCAGGA

GGGCCCCTCCCCCGCCGTGCCTAGCATCTACGCCACCCTGG

CCATCCACGGATCCGGGGAAGGCCGAGGCTCCCTTCTAACA

TGTGGAGATGTCGAGGAAAACCCTGGCCCTATGGCGCTGCC

AGTCACTGCATTGTTATTGCCTCTGGCCCTGCTTCTCCATGC

GGCGCGCCCAGACATCCAGATGACCCAATCCCCAAGCAGTC

TCTCAGCCAGCGTGGGAGACAGGGTTACAATCACGTGCCGC

GCCAGCCAGGACGTCAACACCGCTGTGGCTTGGTATCAGCA

AAAGCCCGGGAAGGCACCAAAGCTGCTTATTTATAGCGCCT

CCTTCTTGTATTCTGGAGTGCCATCCAGGTTTTCCGGGTCAC

GTAGCGGGACTGACTTTACCCTCACCATATCCAGCCTCCAG

CCCGAGGATTTCGCCACCTATTACTGTCAGCAACACTACAC

GACTCCACCGACTTTTGGACAGGGCACTAAAGTGGAGATTA

AGGGCAGCACGAGTGGGAGTGGAAAGCCCGGCAGCGGGGA

GGGGTCTACCAAGGGAGAGGTCCAGCTGGTTGAATCCGGA

GGCGGGCTTGTGCAACCTGGAGGCTCCCTGAGGCTTAGTTG

TGCCGCGTCAGGATTCAACATTAAGGATACCTATATTCATT

GGGTCCGACAAGCCCCGGGCAAGGGCTTGGAGTGGGTGGC

CAGAATCTATCCGACCAACGGATATACAAGGTACGCCGATT

CTGTGAAAGGACGCTTCACCATCAGCGCGGACACATCCAAA

AACACAGCCTATCTGCAGATGAACTCCCTTCGCGCCGAGGA

TACAGCCGTGTACTATTGTAGTCGGTGGGGAGGCGACGGCT

TCTACGCGATGGACTATTGGGGACAAGGAACACTGGTGACT

GTCAGTAGCACTACGACCCCAGCACCTAGACCTCCCACCCC

AGCTCCAACTATAGCTTCCCAGCCATTGTCTCTCCGGCCAGA

GGCGTGTCGACCAGCCGCTGGAGGGGCCGTTCATACAAGAG

GACTCGATTTCGCTTGCGATATCTACATATGGGCCCCTCTTG

CCGGGACATGCGGTGTCCTGCTTCTAAGCTTGGTTATTACCC

TCTATTGCAAAAGAGGACGAAAGAAACTGCTTTATATATTC

AAGCAACCTTTCATGCGCCCCGTACAGACCACGCAGGAGGA

AGATGGGTGTAGCTGTCGCTTCCCTGAGGAAGAGGAAGGTG

GATGCGAGTTGCGGGTGAAGTTCAGTCGATCCGCCGATGCG

CCTGCCTATCAGCAAGGGCAGAACCAGCTTTATAACGAGTT

AAACCTTGGCCGCCGGGAAGAGTATGACGTGTTGGACAAGC

GTCGCGGGAGAGACCCTGAGATGGGCGGAAAACCAAGGAG

AAAAAATCCACAGGAAGGCTTATATAACGAGTTGCAGAAA

GACAAGATGGCCGAGGCATACTCCGAAATCGGAATGAAGG

GCGAGCGACGGCGCGGCAAAGGCCACGATGGACTCTATCA

GGGCTTAAGCACCGCCACCAAAGACACCTACGATGCACTTC

ATATGCAGGCACTCCCACCTAGATGATAA

MC0516-
SEQ ID
MALPVTALLLPLALLLHAARPDIVMTQSPDSLAVSLGERATISC

SN66E3.2
NO: 324
KSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVP

(LH)_LIR1

DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYGTPFTFGGGT

(30)_2A_HER2

KVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS

Protein

CKASGYTFTDYYLHWVRQAPGQGLEWMGWINPYTGGTNYA

Sequence

QKFQGRVTMTRDTSISTAYMELSGLTSDDTAVYYCARAGASY

(VR516)

YDFWSGWVFDYWGQGTLVTVSSGPTSTSGPEDQPLTPTGSDP

QSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTST

QRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAV

KHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPP

SPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSL

TLRREATEPPPSQEGPSPAVPSIYATLAIHGSGEGRGSLLTCGDV

EENPGPMALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVG

DRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVP

SRESGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK

VEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLS

CAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS

VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY

AMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRP

AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRG

RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS

RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG

GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD

GLYQGLSTATKDTYDALHMQALPPR

MC0517-
SEQ ID
ATGGCGCTGCCAGTCACTGCATTGTTATTGCCTCTGGCCCTG

SN66E3.3_
NO: 325
CTTCTCCATGCGGCAAGGCCAGATATAGTGATGACACAGTC

(LH)_LIRI

CCCCGACTCCCTGGCTGTCTCACTGGGAGAACGAGCGACGA

(26)_2A_HER2

TTAGTTGTAAGTCTAGCCAGAGCGTCCTGTATTCAAGCAAT

Nuclotide

AACAAGAATTACCTCGCCTGGTATCAGCAAAAGCCGGGACA

Sequence

GCCACCCAAACTGTTGATTTACTGGGCCAGCACGAGAGAGA

(VR517)

GCGGAGTGCCCGACCGCTTCAGCGGATCCGGGTCAGGCACA

GATTTTACCCTGACTATTAGCTCCCTTCAAGCGGAAGATGTC

GCCGTCTACTATTGCCAGCAATATTACGGAACTCCATTCAC

ATTCGGCGGTGGGACCAAAGTAGAGATAAAGGGTGGCGGG

GGATCCGGCGGTGGCGGTAGCGGGGGAGGCGGGTCCCAAG

TGCAACTAGTCCAATCAGGTGCCGAAGTCAAGAAACCAGGT

GCATCCGTGAAAGTGTCTTGCAAAGCCAGTGGCTACACTTT

TACTGACTACTATCTGCACTGGGTGCGTCAAGCACCCGGCC

AGGGGCTTGAATGGATGGGCTGGATTAACCCTTATACTGGA

GGGACAAATTACGCTCAGAAGTTCCAGGGACGCGTTACAAT

GACCCGAGACACCAGCATCAGCACAGCGTACATGGAGTTA

AGTAGGCTGAGGTCCGAAGATACCGCCGTGTATTACTGCGC

TCGGGCAGGGGCCTCTTACTATGATTTTTGGTCCGGTTGGGT

CTTCGATTACTGGGGGCAGGGAACCCTGGTGACAGTGTCCT

CAACCAGTGGACCGGAAGATCAACCACTTACACCAACGGG

CAGCGACCCCCAGTCAGGCCTAGGGCGCCACCTGGGTGTGG

TCATCGGGATACTGGTCGCTGTCATCCTGCTTCTGCTCCTTC

TCTTGCTCCTATTCCTAATCCTGCGCCACAGGAGACAGGGC

AAGCACTGGACCAGCACCCAGCGGAAGGCCGACTTTCAGC

ACCCTGCCGGCGCCGTGGGCCCTGAGCCTACCGACAGGGGC

CTGCAGTGGAGGAGCTCCCCAGCCGCCGATGCCCAGGAGG

AGAATCTGTACGCCGCCGTGAAGCACACCCAGCCAGAGGA

CGGCGTGGAGATGGACACCCGCTCCCCACACGACGAGGAC

CCACAGGCCGTGACCTACGCCGAGGTGAAGCACAGCCGCCC

CAGACGCGAGATGGCCAGCCCACCCAGCCCCCTGTCCGGCG

AGTTCCTGGACACCAAGGACAGGCAGGCCGAGGAGGACCG

GCAGATGGACACCGAGGCCGCCGCCTCCGAGGCCCCCCAG

GACGTGACCTACGCCCAGCTGCACTCCCTGACCCTGCGGAG

AGAGGCCACCGAGCCCCCACCCAGCCAGGAGGGCCCCTCCC

CCGCCGTGCCTAGCATCTACGCCACCCTGGCCATCCACGGA

TCCGGGGAAGGCCGAGGCTCCCTTCTAACATGTGGAGATGT

CGAGGAAAACCCTGGCCCTATGGCGCTGCCAGTCACTGCAT

TGTTATTGCCTCTGGCCCTGCTTCTCCATGCGGCGCGCCCAG

ACATCCAGATGACCCAATCCCCAAGCAGTCTCTCAGCCAGC

GTGGGAGACAGGGTTACAATCACGTGCCGCGCCAGCCAGG

ACGTCAACACCGCTGTGGCTTGGTATCAGCAAAAGCCCGGG

AAGGCACCAAAGCTGCTTATTTATAGCGCCTCCTTCTTGTAT

TCTGGAGTGCCATCCAGGTTTTCCGGGTCACGTAGCGGGAC

TGACTTTACCCTCACCATATCCAGCCTCCAGCCCGAGGATTT

CGCCACCTATTACTGTCAGCAACACTACACGACTCCACCGA

CTTTTGGACAGGGCACTAAAGTGGAGATTAAGGGCAGCACG

AGTGGGAGTGGAAAGCCCGGCAGCGGGGAGGGGTCTACCA

AGGGAGAGGTCCAGCTGGTTGAATCCGGAGGCGGGCTTGTG

CAACCTGGAGGCTCCCTGAGGCTTAGTTGTGCCGCGTCAGG

ATTCAACATTAAGGATACCTATATTCATTGGGTCCGACAAG

CCCCGGGCAAGGGCTTGGAGTGGGTGGCCAGAATCTATCCG

ACCAACGGATATACAAGGTACGCCGATTCTGTGAAAGGACG

CTTCACCATCAGCGCGGACACATCCAAAAACACAGCCTATC

TGCAGATGAACTCCCTTCGCGCCGAGGATACAGCCGTGTAC

TATTGTAGTCGGTGGGGAGGCGACGGCTTCTACGCGATGGA

CTATTGGGGACAAGGAACACTGGTGACTGTCAGTAGCACTA

CGACCCCAGCACCTAGACCTCCCACCCCAGCTCCAACTATA

GCTTCCCAGCCATTGTCTCTCCGGCCAGAGGCGTGTCGACC

AGCCGCTGGAGGGGCCGTTCATACAAGAGGACTCGATTTCG

CTTGCGATATCTACATATGGGCCCCTCTTGCCGGGACATGC

GGTGTCCTGCTTCTAAGCTTGGTTATTACCCTCTATTGCAAA

AGAGGACGAAAGAAACTGCTTTATATATTCAAGCAACCTTT

CATGCGCCCCGTACAGACCACGCAGGAGGAAGATGGGTGT

AGCTGTCGCTTCCCTGAGGAAGAGGAAGGTGGATGCGAGTT

GCGGGTGAAGTTCAGTCGATCCGCCGATGCGCCTGCCTATC

AGCAAGGGCAGAACCAGCTTTATAACGAGTTAAACCTTGGC

CGCCGGGAAGAGTATGACGTGTTGGACAAGCGTCGCGGGA

GAGACCCTGAGATGGGCGGAAAACCAAGGAGAAAAAATCC

ACAGGAAGGCTTATATAACGAGTTGCAGAAAGACAAGATG

GCCGAGGCATACTCCGAAATCGGAATGAAGGGCGAGCGAC

GGCGCGGCAAAGGCCACGATGGACTCTATCAGGGCTTAAGC

ACCGCCACCAAAGACACCTACGATGCACTTCATATGCAGGC

ACTCCCACCTAGATGATAA

MC0517-
SEQ ID
MALPVTALLLPLALLLHAARPDIVMTQSPDSLAVSLGERATISC

SN66E3.3
NO: 326
KSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVP

(LH)_LIR1

DRESGSGSGTDFTLTISSLQAEDVAVYYCQQYYGTPFTFGGGT

(26)_2A_HER2

KVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS

Protein

CKASGYTFTDYYLHWVRQAPGQGLEWMGWINPYTGGTNYA

Sequence

QKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARAGASY

(VR517)

YDFWSGWVFDYWGQGTLVTVSSTSGPEDQPLTPTGSDPQSGL

GRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKA

DFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQP

EDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGE

FLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREA

TEPPPSQEGPSPAVPSIYATLAIHGSGEGRGSLLTCGDVEENPGP

MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITC

RASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSR

SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGST

SGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAASGEN

IKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS

ADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWG

QGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV

HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI

FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP

AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK

NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL

STATKDTYDALHMQALPPR

ii. Bicistronic iCAR Portion

In some embodiments, the bicistronic iCAR portions described below can be included as part of monocistronic iCAR constructs for use in co-transduction methods along with a described monocistronic aCAR construct.

1. iCAR Portion: scFv Component

In some embodiments, the bicistronic construct comprises an iCAR portion comprising a single chain variable fragment (scFv) component. In some embodiments, the iCAR portion comprises a single chain variable fragment (scFv) component. In some embodiments, the scFv targets an HLA antigen. In some embodiments, the HLA antigen is selected from the group consisting of HLA-A2, HLA-A3, HLA-A, HLA-B, HLA-C, HLA-G, HLA-E, HLA-F, HLA-DPA1, HLA-DQA1, HLA-DQB1, HLA-DQB2, HLA-DRB1, and HLA-DRB5. In some embodiments, the iCAR comprises an scFv. In some embodiments, the scFv is selected from the group consisting of BB7.2, 3PF12, 3PF12/C4, 3PF12/F12, 3PF12/B11, W6/32, BBM.1, SN66E3.1, SN66E3.2, SN66E.3, Ha5C2.A2, MWB1, MWB1-mod, Hz.BB7.2VH1-69_A18VK, Hz.BB7.2VH1-69 (27,30)_A18, HzBB7.2VH1-69 (27,30,48) A18, Hz.BB7.2 VH1-69 (27,30,67)_A18, Hz.BB7.2 VH1-69 (27,30,69)_A18, Hz.BB7.2 VH1-69 (27,30,67,69)_A18, Hz.BB7.2 VH1-3_A18, Hz.BB7.2 VH1-3(48)_A18, Hz.BB7.2 VH1-3(67)_A18, Hz.BB7.2 VH1-3(69)_A18, Hz.BB7.2 VH1-3(71)_A18, Hz.BB7.2 VH1-3(73)_A18, and MWB1.2. In some embodiments, the scFv has the VL and VH sequences of BB7.2 (SEQ ID NOs: 37 and 38). In some embodiments, the scFv has the VL and VH sequences of 3PF12/C4 (SEQ ID NOs: 39 and 40). In some embodiments, the scFv has the VL and VH sequences of 3PF12/F12 (SEQ ID NOs: 41 and 42). In some embodiments, the scFv has the VL and VH sequences of 3PF12/B11 (SEQ ID NOs: 43 and 44). In some embodiments, the scFv has the VL and VH sequences of W6/32 (SEQ ID NOs: 45 and 46). In some embodiments, the scFv has the VL and VH sequences of BBM.1 (SEQ ID NOs: 47 and 48). In some embodiments, the scFv has the VL and VH sequences of SN66E3 (SEQ ID NOs: 49 and 50). In some embodiments, the scFv has the VL and VH sequences of Ha5C2.A2 (SEQ ID NOs: 51 and 52). In some embodiments, the scFv has the VL and VH sequences of MWB1 (SEQ ID NOs: 53 and 54). In some embodiments, the scFv has the VL and VH sequences of MWB1-mod (SEQ ID NOs: 55 and 56). In some embodiments, the scFv has the VL and VH sequences of Hz.BB7.2 VH1-69_A18VK (SEQ ID NOs: 57 and 58). In some embodiments, the scFv has the VL and VH sequences of Hz.BB7.2 VH1-69 (27,30)_A18 (SEQ ID NOs: 59 and 60). In some embodiments, the scFv has the VL and VH sequences of Hz.BB7.2 VH1-69 (27,30,48)_A18 (SEQ ID NOs: 61 and 62). In some embodiments, the scFv has the VL and VH sequences of Hz.BB7.2 VH1-69 (27,30,67)_A18 (SEQ ID NOs: 63 and 64). In some embodiments, the scFv has the VL and VH sequences of Hz.BB7.2 VH1-69 (27,30,69) A18 (SEQ ID NOs: 65 and 66). In some embodiments, the scFv has the VL and VH sequences of Hz.BB7.2 VH1-69 (27,30,67,69)_A18 (SEQ ID NOs: 67 and 68). In some embodiments, the scFv has the VL and VH sequences of Hz.BB7.2 VH1-3_A18 (SEQ ID NOs: 69 and 70). In some embodiments, the scFv has the VL and VH sequences of Hz.BB7.2 VH1-3(48)_A18 (SEQ ID NOs: 71 and 72). In some embodiments, the scFv has the VL and VH sequences of Hz.BB7.2 VH1-3(67)_A18 (SEQ ID NOs: 73 and 74). In some embodiments, the scFv has the VL and VH sequences of Hz.BB7.2 VH1-3(69)_A18 (SEQ ID NOs: 75 and 76). In some embodiments, the scFv has the VL and VH sequences of Hz.BB7.2 VH1-3(71)_A18 (SEQ ID NOs: 77 and 78). In some embodiments, the scFv has the VL and VH sequences of Hz.BB7.2 VH1-3(73)_A18 (SEQ ID NOs: 79 and 80). In some embodiments, the scFv has the VL and VH sequences of MWB1.2 (SEQ ID NOs: 163 and 164). In some embodiments, the scFv has the VL and VH sequences of SN66E3.2 (SEQ ID NOs: 165 and 166). In some embodiments, the scFv has the VL and VH sequences of SN66E3.3 (SEQ ID NOs: 283 and 284) In some embodiments, the scFv is BB7.2 (SEQ ID NO:167). In some embodiments, the scFv is 3PF12 (SEQ ID NO:168). In some embodiments, the scFv is SN66E3.1 (SEQ ID NO:169). In some embodiments, the scFv is SN66E3.2 (SEQ ID NO:285). In some embodiments, the scFv is SN66E3.3 (SEQ ID NO:286). In some embodiments, the scFv is Hz BB7.2.1 (SEQ ID NO:287). In some embodiments, the scFv is HzBB7.2.2 (SEQ ID NO:288). In some embodiments, the scFv is MWB1.1 (SEQ ID NO:273). In some embodiments, the scFv is MWB1.2 (SEQ ID NO:274). In some embodiments, the scFv is 3PF12/C4. In some embodiments, the scFv is 3PF12/F12. In some embodiments, the scFv is 3PF12/B11. In some embodiments, the scFv is W6/32. In some embodiments, the scFv is BBM.1. In some embodiments, the scFv is Ha5C2.A2. In some embodiments, the scFv is MWB1. In some embodiments, the scFv is MWB1-mod. In some embodiments, the scFv is BB7.2. In some embodiments, the scFv is 3PF12. In some embodiments, the scFv is SN66E3.1. In some embodiments, the scFv is SN66E3.2. In some embodiments, the scFv is SN66E3.3. In some embodiments, the scFv is Hz BB7.2.1. In some embodiments, the scFv is HzBB7.2.2. In some embodiments, the scFv is MWB1.1. In some embodiments, the scFv is MWB1.2. In some embodiments, the scFv is Hz.BB7.2 VH1-69_A18VK. In some embodiments, the scFv is Hz.BB7.2 VH1-69 (27,30)_A18. In some embodiments, the scFv is Hz.BB7.2 VH1-69 (27,30,48)_A18. In some embodiments, the scFv is Hz.BB7.2 VH1-69 (27, 30, 67)_A18. In some embodiments, the scFv is Hz.BB7.2 VH1-69 (27, 30, 69)_A18. In some embodiments, the scFv is Hz.BB7.2 VH1-69 (27, 30, 67, 69)_A18. In some embodiments, the scFv is Hz.BB7.2VH1-3_A18. In some embodiments, the scFv is Hz.BB7.2 VH1-3(48)_A18. In some embodiments, the scFv is Hz.BB7.2-3(67)_A18. In some embodiments, the scFv is Hz.BB7.2 VH1-3(69)_A18. In some embodiments, the scFv is Hz.BB7.2 VH1-3(71)_A18. In some embodiments, the scFv is Hz.BB7.2 VH1-3(73)_A18. In some embodiments, the scFv is MWB1.2. In some embodiments, the scFv is SN66E3.2. In some embodiments, the scFv is MWB1.1. In some embodiments, the scFv is MWB1.2. In some embodiments, the scFv comprises Hz.BB7.2 heavy chain Hz.BB7.2VH1-69. In some embodiments, the scFv comprises Hz.BB7.2 Heavy chain Hz.BB7.2VH1-69 (H27Y, H30S. In some embodiments, the scFv comprises Hz.BB7.2 heavy chain HZ.BB7.2VH1-69 (H27Y, H30S, H48I). In some embodiments, the scFv comprises Hz.BB7.2 Heavy chain Hz.BB7.2VH1-69 (H27Y, H30S, H67T). In some embodiments, the scFv comprises Hz. BB7.2 Heavy chain Hz.BB7.2VH1-69 (H27Y, H30S, H69L). In some embodiments, the scFv comprises Hz.BB7.2 Heavy Chain HZ.BB7.2VH1-69 (H27Y, H30S, VH67T, H69L). In some embodiments, the scFv comprises Hz.BB7.2 Heavy Chain Hz.BB7.2 VH1-3. In some embodiments, the scFv comprises Hz.BB7.2 Heavy Chain Hz.BB7.2 VH1-3 (H48I). In some embodiments, the scFv comprises Hz.BB7.2 Heavy Chain VH1-3 (H67T). In some embodiments, the scFv comprises Hz.BB7.2 Heavy Chain Hz.BB7.2 VH1-3 (H69L). In some embodiments, the scFv comprises Hz.BB7.2 Heavy Chain Hz.BB7.2 VH1-3 (H71A). In some embodiments, the scFv comprises Hz.BB7.2 Heavy Chain Hz.BB7.2 VH1-3 (H73A). In some embodiments, the scFv comprises Hz.BB7.2 Light chain VKA18. The 6 CDR sequences for the variable heavy and variable light chains are shown in bold and underline in Table 2 for each sequence, also referred to as vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3. In some embodiments, the iCAR comprises the 6 CDR sequences for the variable heavy and variable light chains as show in bold and underline in Table 2 for each sequence, also referred to as vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3. In some embodiments, the iCAR comprises the 6 CDR sequences for the variable heavy and variable light chains as show in bold and underline in Table 2 for each sequence, also referred to as vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3, wherein each CDR individually optionally comprises one more substitutions. In some embodiments, the iCAR comprises the 6 CDR sequences for the variable heavy and variable light chains as show in bold and underline in Table 2 for each sequence, also referred to as vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3, wherein each CDR individually optionally comprises 1, 2, and/or 3 substitutions. In some embodiments, the iCAR comprises the 6 CDR sequences for the variable heavy and variable light chains as show in bold and underline in Table 2 for each sequence, also referred to as vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3, wherein each CDR individually comprises one more substitutions. In some embodiments, the iCAR comprises the 6 CDR sequences for the variable heavy and variable light chains as show in bold and underline in Table 2 for each sequence, also referred to as vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2, and vlCDR3, wherein each CDR individually comprises 1, 2, and/or 3 substitutions.

TABLE 2

iCAR vh, vl, and scFv sequences

Sequence
SEQ

Information
ID NO
Amino acid sequence

BB7.2 variable
37
DVLMTQTPLSLPVSLGDQVSISCRSSQSIVHSNGNTYLEWY

light chain

LQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRV

EAEDLGVYYCFQGSHVPRTFGGGTKLEIK

BB7.2 variable
38
QVQLQQSGPELVKPGASVKMSCKASGYTFTSYHIQWVKQR

heavy chain

PGQGLEWIGWIYPGDGSTQYNEKFKGKTTLTADKSSSTAY

MLLSSLTSEDSAIYFCAREGTYYAMDYWGQGTSVTVSS

3PF12/C4
39
DIVMTQSPSFLSASVGDRVTITCRASHGINNYLAWYQQKPG

variable light

KAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFA

chain

TYYCQQYDSYPPTFGRGTKVEIK

3PF12/C4
40
QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQ

variable heavy

APGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTV

chain

SLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGRGTL

VTVSS

3PF12/F12
41
DVVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKP

variable light

GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDF

chain

ATYYCQQYSSFPLTFGGGTKVDIK

3PF12/F12
42
QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQ

variable heavy

APGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTV

chain

SLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGRGTL

VTVSS

3PF12/B11
43
DVVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKP

variable light

GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDI

chain

ATYYCQQYDNLPPTFGGGTKLEIV

3PF12/B11
44
QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQ

variable heavy

APGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTV

chain

SLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGRGTL

VTVSS

W6/32 variable
45
SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKP

light chain

GQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAED

LAVYFCQQDYSSPPWTFGGGTKLEIR

W6/32 variable
46
QVQLKQSGPGLVQPSQSLSLTCTVSGFSLTSYGVHWVRQPP

heavy chain

GKGLEWLGVIWSGGSTDYNAAFISRLSIRKDNSKSQVFFK

MNSLQADDTAIYYCARTFTTSTSAWFAYWGQGTLVTVSA

BBM.1 variable
47
DIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPG

light chain

KSPQLLIYAATSLADGVPSRFSGSGSGTKFSLKIRTLQAEDF

VSYYCQQLYSKPYTFGGGTKLEIK

BBM.1 variable
48
EVQLQQSGAELVKPGASVKLSCTPSGFNVKDTYIHWVKQR

heavy chain

PKQGLEWIGRIDPSDGDIKYDPKFQGKATITADTSSNTVSL

QLSSLTSEDTAVYYCARWFGDYGAMNYWGQGTSVTVSS

SN66E3.1
49
DIVMTQSPDSLAVSLGERATISCKSSQSVLYSSNNKNYLAW

variable light

YQQKLGQPPKLLIYWASTRESGVPDRESGSGSGTNFTLTISS

chain

LQAENVAVYYCQQYYGTPFTFGGGTKVEIK

SN66E3.1
50
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYLHWVRQ

variable heavy

APGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDASIST

chain

VYMELSGLTSDDTAVHFCARAGASYYDFWSGWVFDYWG

QGTLVTVSS

Ha5C2.A2
51
DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPG

variable light

KAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA

chain

TYQCQQSYSTPFTFGGGTKVEIK

Ha5C2.A2
52
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAG

variable heavy

KGLEWIGRIYISGGTNYNPSLKSRVTMSVDTSKNQVSLKLS

chain

SVTAADTAVYYCARDILGGVSGWSHYGMDVWGQGTTVT

VSS

MWB1 variable
53
QSALTQPPSASGSPGQSVTISCTGTSSDVGGYKYVSWYQH

light chain

HPDKAPKLMIYEVNKRPSGVPDRESGSKSDNTASLTVSGLQ

AEDEADYYCSSYAGSNNWVFGGGTKLTVL

MWB1 variable
54
QVQLVESGGGVVQPGGSLRLSCAASGFTFSTYGMHWVRQ

heavy chain

APGKGLEWAASVSYDGSNKYYADSGQGRFTISRDTSMNSL

YLQVNSLRDETAVYYCAIGIYGAYSFDYWGQGTLVTVSS

MWB1.1
55
QSALTQPPSASGSPGQSVTISCTGTSSDVGGYKYVSWYQH

(MWB1.1)

HPDKAPKLMIYEVNKRPSGVPDRESGSKSDNTASLTVSGLQ

variable light

AEDEADYYCSSYAGSNNWVFGGGTKLTVL

chain

MWB1.1(MWB1.1)
56
QVQLVESGGGVVQPGGSLRLSCAASGFTFSTYGMHWVRQ

variable heavy

APGKGLEWVASISYDGSNKYYADSGQGRFTISRDTSKNSL

chain

YLQMNSLRAEDTAVYYCAIGIYGAYSFDYWGQGTLVTVSS

Hz.BB7.2_
57
DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWY

A18VK variable

LQKPGQSPQLLIYKVSNRFSGVPDRESGSGSGTDFTLKISRV

light chain

EAEDVGVYYCFQGSHVPRTFGGGTKVEIK

Hz.BB7.2 VH1-69
58
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYHIQWVRQA

variable heavy

PGQGLEWMGWIYPGDGSTQYNEKFKGRVTITADKSTSTA

chain

YMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTLVTVSS

Hz.BB7.2 VH1-69
59
DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWY

(27, 30)

LQKPGQSPQLLIYKVSNRFSGVPDRESGSGSGTDFTLKISRV

variable light

EAEDVGVYYCFQGSHVPRTFGGGTKVEIK

chain

Hz.BB7.2 Heavy
60
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYHIQWVRQA

chain VH1-69

PGQGLEWMGWIYPGDGSTQYNEKFKGRVTITADKSTSTA

(H27Y, H30S)

YMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTLVTVSS

HZ.BB7.2VH1-69
61
DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWY

(27, 30, 4)_

LQKPGQSPQLLIYKVSNRFSGVPDRESGSGSGTDFTLKISRV

A18 variable

EAEDVGVYYCFQGSHVPRTFGGGTKVEIK

light chain

Hz.BB7.2 heavy
62
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYHIQWVRQA

chain VH1-69

PGQGLEWIGWIYPGDGSTQYNEKFKGRVTITADKSTSTAY

(H27Y, H30S,

MELSSLRSEDTAVYYCAREGTYYAMDYWGQGTLVTVSS

H48I))

Hz.BB7.2 VH1-69
63
DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWY

(27, 30, 67)_A18

LQKPGQSPQLLIYKVSNRFSGVPDRESGSGSGTDFTLKISRV

variable light

EAEDVGVYYCFQGSHVPRTFGGGTKVEIK

chain

Hz.BB7.2 Heavy
64
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYHIQWVRQA

chain VH1-69

PGQGLEWMGWIYPGDGSTQYNEKFKGRTTITADKSTSTA

(H27Y, H30S,

YMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTLVTVSS

H67T))

HZ.BB7.2VH1-69
65
DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWY

(27, 30, 69)_

LQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRV

A18 variable

EAEDVGVYYCFQGSHVPRTFGGGTKVEIK

light chain

Hz. BB7.2 Heavy
66
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYHIQWVRQA

chain VH1-69

PGQGLEWMGWIYPGDGSTQYNEKFKGRVTLTADKSTSTA

(H27Y, H30S,

YMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTLVTVSS

H69L))

Hz.BB7.2 VH1-69
67
DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWY

(27, 30, 67, 69)_

LQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRV

A18 variable

EAEDVGVYYCFQGSHVPRTFGGGTKVEIK

light chain

Hz.BB7.2 Heavy
68
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYHIQWVRQA

Chain VH1-69

PGQGLEWMGWIYPGDGSTQYNEKFKGRTTLTADKSTSTA

(H27Y, H30S,

YMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTLVTVSS

VH67T, H69L))

Hz.BB7.2VH1-3_
69
DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWY

A18 variable

LQKPGQSPQLLIYKVSNRFSGVPDRESGSGSGTDFTLKISRV

light chain

EAEDVGVYYCFQGSHVPRTFGGGTKVEIK

Hz.BB7.2 Heavy
70
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQ

Chain VH1-3)

APGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDTSAST

AYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTLVTVS

S

Hz.BB7.2VH1-3
71
DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWY

(48)_A18

LQKPGQSPQLLIYKVSNRFSGVPDRESGSGSGTDFTLKISRV

variable light

EAEDVGVYYCFQGSHVPRTFGGGTKVEIK

chain

Hz.BB7.2 Heavy
72
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQ

Chain VH1-3

APGQRLEWIGWIYPGDGSTQYNEKFKGRVTITRDTSASTA

(H48I))

YMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTLVTVSS

Hz.BB7.2VH1-3
73
DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWY

(67)_A18

LQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRV

variable light

EAEDVGVYYCFQGSHVPRTFGGGTKVEIK

chain

(Hz.BB7.2 Light

chain VKA18)

Hz.BB7.2 Heavy
74
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQ

Chain VH1-3

APGQRLEWMGWIYPGDGSTQYNEKFKGRTTITRDTSAST

(H67T))

AYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTLVTVS

S

Hz.BB.2VH1-3
75
DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWY

(69)_A18

LQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRV

variable light

EAEDVGVYYCFQGSHVPRTFGGGTKVEIK

chain

Hz.BB7.2 Heavy
76
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQ

Chain VH1-3

APGQRLEWMGWIYPGDGSTQYNEKFKGRVTLTRDTSAST

(H69L))

AYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTLVTVS

S

Hz.BB7.2VH1-3
77
DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWY

(71)_A18

LQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRV

variable light

EAEDVGVYYCFQGSHVPRTFGGGTKVEIK

chain

Hz.BB7.2 VH1-3
78
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQ

(71)_variable

APGQRLEWMGWIYPGDGSTQYNEKFKGRVTITADTSAST

heavy chain

AYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTLVTVS

S

Hz.BB7.2VH1-3
79
DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWY

(73)_A18

LQKPGQSPQLLIYKVSNRFSGVPDRESGSGSGTDFTLKISRV

variable light

EAEDVGVYYCFQGSHVPRTFGGGTKVEIK

chain

Hz.BB7.2VH1-3
80
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQ

(73)_A18

APGQRLEWMGWIYPGDGSTQYNEKFKGRVTITRDKSAST

variable heavy

AYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTLVTVS

chain

S

MWB1.2 variable
163
QSALTQPPSASGSPGQSVTISCTGTSSDVGGYKYVSWYQQ

light chain

HPGKAPKLMIYEVNKRPSGVPDRFSGSKSGNTASLTVSGLQ

AEDEADYYCSSYAGSNNWVFGGGTKLTVL

MWB1.2 variable
164
QVQLVESGGGVVQPGGSLRLSCAASGFTFSTYGMHWVRQ

heavy chain

APGKGLEWVASISYDGSNKYYADSGQGRFTISRDTSKNSL

YLQMNSLRAEDTAVYYCAIGIYGAYSFDYWGQGTLVTVSS

SN66E3.2
165
DIVMTQSPDSLAVSLGERATISCKSSQSVLYSSNNKNYLAW

variable light

YQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISS

chain

LQAEDVAVYYCQQYYGTPFTFGGGTKVEIK

SN66E3.2
166
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYLHWVRQ

variable heavy

APGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSIST

chain

AYMELSGLTSDDTAVYYCARAGASYYDFWSGWVFDYWG

QGTLVTVSS

MWB1.1
273
QVQLVESGGGVVQPGGSLRLSCAASGFTFSTYGMHWVRQA

scFvVH_VL

PGKGLEWVASISYDGSNKYYADSGQGRFTISRDTSKNSLYL

QMNSLRAEDTAVYYCAIGIYGAYSFDYWGQGTLVTVSSGG

GGSGGGGSGGGGSQSALTQPPSASGSPGQSVTISCTGTSSDV

GGYKYVSWYQHHPDKAPKLMIYEVNKRPSGVPDRESGSKS

DNTASLTVSGLQAEDEADYYCSSYAGSNNWVFGGGTKLTV

L

MWB1.2scFvVH_
274
QVQLVESGGGVVQPGGSLRLSCAASGFTFSTYGMHWVRQ

VL

APGKGLEWVASISYDGSNKYYADSGQGRFTISRDTSKNSL

YLQMNSLRAEDTAVYYCAIGIYGAYSFDYWGQGTLVTVSS

GGGGSGGGGSGGGGSQSALTQPPSASGSPGQSVTISCTGTSS

DVGGYKYVS
WYQQHPGKAPKLMIYEVNKRPSGVPDRFSG

SKSGNTASLTVSGLQAEDEADYYCSSYAGSNNWVFGGGTK

LTVL

SN66E3.3
283
DIVMTQSPDSLAVSLGERATISCKSSQSVLYSSNNKNYLAW

Variable Light

YQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISS

chain

LQAEDVAVYYCQQYYGTPFTFGGGTKVEIK

SN66E3.3
284
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYLHWVRQ

variable Heavy

APGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSIST

chain

AYMELSRLRSEDTAVYYCARAGASYYDFWSGWVFDYWG

QGTLVTVSS

In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH.

In some embodiments, the iCAR scFv comprises a linker that covalently connects the VH and the VL to form the iCAR scFv.

In some embodiments, the heavy and light chains of the scFv are covalently connected via a linker. In some embodiments, the linker is a gly-ser polypeptide linker, i.e., a peptide that consists of glycine and serine residues. Exemplary gly-ser polypeptide linkers comprise the amino acid sequence Ser(Gly₄Ser)_n, as well as (Gly₄Ser)_nand/or (Gly₄Ser 3)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3, i.e., Ser(Gly₄Ser) 3. In some embodiments, n=4, i.e., Ser(Gly₄Ser) 4. In some embodiments, n=5. In some embodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. In some embodiments, n=9. In some embodiments, n=10. Another exemplary gly-ser polypeptide linker comprises the amino acid sequence Ser(Gly₄Ser)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In another embodiment, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly₄Ser)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly 3 Ser)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In another embodiment, n=5. In yet another embodiment, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly₄Ser 3)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly 3 Ser)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In another embodiment, n=5. In yet another embodiment, n=6.

In some embodiments, the iCAR comprises a GS based linker sequence, connecting the VH and VL or the VL and VH to form the scFv. In some embodiments, the GS linker comprises GGGGS (SEQ ID NO:153). In some embodiments, the iCAR comprises a Whitlow linker sequence, e.g., GSTSGSGKPGSGEGSTKG (SEQ ID NO:82). In some embodiments, the iCAR comprises the Vh and Vl sequences in the Vh-Vl orientation. In some embodiments, the iCAR comprises the Vh and Vl sequences in the Vl-Vh orientation. In some embodiments, the iCAR comprises a linker between the Vh and Vl sequences. In some embodiments, the iCAR does not comprise a linker between the Vh and Vl sequences.

TABLE 3

iCAR linkers

Sequence
SEQ ID

Information
NO
Amino acid sequence

(G4S)X3 linker
81
GGGGSGGGGSGGGGS

Whitlow linker
82
GSTSGSGKPGSGEGSTKG

PD1 linker
83
DFQWREKTPEPPVPCVPEQ

G4S
153
GGGGS

In some embodiments, the iCAR scFv comprises a linker. In some embodiments, the iCAR scFv is selected from the group consisting of BB7.2 scFv (SEQ ID NO: 167), 3PF12 scFv (SEQ ID NO: 168), SN66E3.1 scFv (SEQ ID NO: 169), SN66E3.2 scFv (SEQ ID NO: 285), SN66E3.3 scFv (SEQ ID NO: 286), Hz BB7.2.1 scFv (SEQ ID NO: 287), and Hz BB7.2.2 scFv (SEQ ID NO: 288). In some embodiments, the iCAR scFv is BB7.2 scFv (SEQ ID NO: 167). In some embodiments, the iCAR scFv is 3PF12 scFv (SEQ ID NO: 168). In some embodiments, the iCAR scFv is SN66E3.1 scFv (SEQ ID NO: 169). In some embodiments, the iCAR scFv is SN66E3.2 scFv (SEQ ID NO: 285). In some embodiments, the iCAR scFv is SN66E3.3 scFv (SEQ ID NO: 286). In some embodiments, the iCAR scFv is Hz BB7.2.1 scFv (SEQ ID NO: 287). In some embodiments, the iCAR scFv is Hz BB7.2.2 scFv (SEQ ID NO: 288).

TABLE 4

iCAR scFv sequences with linkers

Sequence
SEQ

Information
ID NO
Amino acid sequence

BB7.2 scFv
167
QVQLQQSGPELVKPGASVKMSCKASGYTFTSYHIQWVKQ

RPGQGLEWIGWIYPGDGSTQYNEKFKGKTTLTADKSSSTA

YMLLSSLTSEDSAIYFCAREGTYYAMDYWGQGTSVTVSS

GGGGSGGGGSGGGGSDVLMTQTPLSLPVSLGDQVSISCRS

SQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPD

RFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGG

GTKLEIK

3PF12 scFv
168
QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVR

QAPGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKK

TVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR

GTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSFLSASVGD

RVTITCRASHGINNYLAWYQQKPGKAPKLLIYAASTLQSG

VPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYDSYPPTFG

RGTKVEIK

SN66E3.1 scFv
169
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYLHWVR

QAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDASIS

TVYMELSGLTSDDTAVHFCARAGASYYDFWSGWVFDYW

GQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSL

GERATISCKSSQSVLYSSNNKNYLAWYQQKLGQPPKLLIY

WASTRESGVPDRFSGSGSGTNFTLTISSLQAENVAVYYCQ

QYYGTPFTFGGGTKVEIK

SN66E3.2 scFv
285
DIVMTQSPDSLAVSLGERATISCKSSQSVLYSSNNKNYLA

WYQQKPGQPPKLLIYWASTRESGVPDRESGSGSGTDFTLTI

SSLQAEDVAVYYCQQYYGTPFTFGGGTKVEIKGGGGSGG

GGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTD

YYLHWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRV

TMTRDTSISTAYMELSGLTSDDTAVYYCARAGASYYDFW

SGWVFDYWGQGTLVTVSS

SN66E3.3 scFv
286
DIVMTQSPDSLAVSLGERATISCKSSQSVLYSSNNKNYLA

WYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTI

SSLQAEDVAVYYCQQYYGTPFTFGGGTKVEIKGGGGSGG

GGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTD

YYLHWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRV

TMTRDTSISTAYMELSRLRSEDTAVYYCARAGASYYDFW

SGWVFDYWGQGTLVTVSS

Hz BB7.2.1 scFv
287
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYHIQWVRQ

APGQGLEWMGWIYPGDGSTQYNEKFKGRTTITADKSTST

AYMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTLVTV

SSGGGGSGGGGSGGGGSDVVMTQTPLSLSVTPGQPASISC

RSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVP

DRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFG

GGTKVEIK

HzBB7.2.2 scFV
288
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYHIQWVRQ

APGQRLEWIGWIYPGDGSTQYNEKFKGRVTITRDTSASTA

YMELSSLRSEDTAVYYCAREGTYYAMDYWGQGTLVTVS

SGGGGSGGGGSGGGGSDVVMTQTPLSLSVTPGQPASISCR

SSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVP

DRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFG

GGTKVEIK

In some embodiments, the iCAR scFv linker is a gly-ser polypeptide linker, i.e., a peptide that consists of glycine and serine residues. Exemplary gly-ser polypeptide linkers comprise the amino acid sequence Ser(Gly₄Ser)_n, as well as (Gly₄Ser)_nand/or (Gly₄Ser 3)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3, i.e., Ser(Gly₄Ser) 3. In some embodiments, n=4, i.e., Ser(Gly₄Ser) 4. In some embodiments, n=5. In some embodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. In some embodiments, n=9. In some embodiments, n=10. Another exemplary gly-ser polypeptide linker comprises the amino acid sequence Ser(Gly₄Ser)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In another embodiment, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly₄Ser)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly 3 Ser)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In another embodiment, n=5. In yet another embodiment, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly₄Ser 3)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly 3 Ser)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In another embodiment, n=5. In yet another embodiment, n=6.

2. iCAR Portion: Hinge Domain

In some embodiments, the bicistronic construct comprises an iCAR portion comprising a hinge domain component. In some embodiments, the hinge domain comprises a hinge selected from the group consisting of a PD-1 hinge domain, a CD28 hinge domain, and a CD8 hinge domain (including a CD8a hinge domain) a LIR1 Ig3-4 hinge domain, a LIR1 Ig-4 hinge domain, a LIR1 52 aa hinge domain, a LIR1 36 aa hinge domain, a LIR1 30 aa hinge domain, a LIR1 8 aa hinge domain, a CD33 hinge domain, and a KIR2DL1 hinge domain. In some embodiments, the hinge domain is a PD-1 hinge (SEQ ID NO: 86). In some embodiments, the hinge domain is a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the vector comprises a CD8 hinge domain. In some embodiments, the vector comprises a CD8a hinge domain (SEQ ID NO:84). In some embodiments, the vector comprises aLIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the vector comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the vector comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the vector comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the vector comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the vector comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the vector comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the vector comprises aKIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) (SEQ ID NO: 295).

TABLE 5

iCAR hinge sequences

Sequence
SEQ ID

Information
NO
Amino acid sequence

CD8 alpha
84
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR

GLDFACD

CD28
85
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

PD-1
86
TERRAEVPTAHPSPSPRPAGQFQTLV

LIR1 Ig3-4
87
VSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYK

DGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQY

RCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPG

PTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRL

RSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKP

YLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPL

TPTGSDPQSGLGRHLGV

LIR1 Ig-4
88
PLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQG

WMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMG

PVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPS

GGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV

LIR1 52 aa
89
HPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTG

SDPQSGLGRHLGV

LIR1 36 aa
90
PSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV

LIR1 30 aa
91
GPTSTSGPEDQPLTPTGSDPQSGLGRHLGV

LIR1 8 aa
92
GLGRHLGV

CD33
93
LNVTYVPQNPTTGIFPGDGSGKQETRAGVVH

KIR2DL1
94
PYEWSKSSDPLLVSVTGNPSNSWPSPTEPSSKTGNPRH

LH

LIR1 26 aa
289
TSGPEDQPLTPTGSDPQSGLGRHLGV

PD-1 (47)
290
GAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRP

AGQFQTLV

PD-1 (42)
291
APKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQF

QTLV

PD-1 (36)
292
KESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV

PD-1 (30)
293
ELRVTERRAEVPTAHPSPSPRPAGQFQTLV

PD-1 (26)
294
TERRAEVPTAHPSPSPRPAGQFQTLV

PD-1 (20)
295
VPTAHPSPSPRPAGQFQTLV

3. iCAR Portion: Transmembrane Domain

In some embodiments, the bicistronic construct comprises an iCAR portion comprising a transmembrane (TM) domain component. In some embodiments, the TM domain comprises a TM domain selected from the group consisting of a PD-1 TM domain, a CD28 TM domain, a CD8 TM domain (including a CD8a TM domain), a LIR1 TM domain, a CD33 TM domain, and a KIR2DL1 TM domain. In some embodiments, the TM domain is a PD-1 TM domain (SEQ ID NO:97). In some embodiments, the TM domain is a CD28 TM domain (SEQ ID NO:96). In some embodiments, the vector comprises a CD8 TM domain. In some embodiments, the vector comprises a CD8a TM domain (SEQ ID NO:95). In some embodiments, the vector comprises a LIR1 TM domain (SEQ ID NO:98). In some embodiments, the vector comprises a CD33 TM domain (SEQ ID NO:99). In some embodiments, the vector comprises a KIR2DL1 TM domain (SEQ ID NO:100).

TABLE 6

iCAR transmembrane sequences

Sequence
SEQ ID

Information
NO
Amino acid sequence

CD8 alpha
95
IYIWAPLAGTCGVLLLSLVITL

YC

CD28
96
FWVLVVVGGVLACYSLLVTV

AFIIFWV

PD-1
97
VGVVGGLLGSLVLLVWVLA

VI

LIR1
98
VIGILVAVILLLLLLLLLFLI

CD33
99
GAIGGAGVTALLALCLCLIFFI

V

KIR2DL1
100
ILIGTSVVIILFILLFFLL

4. iCAR Portion: Inhibitory Domain

In some embodiments, the bicistronic construct comprises an iCAR portion comprising an inhibitory domain component. In some embodiments, the iCAR portion comprises an inhibitory domain. In some embodiments, the inhibitory domain is selected from the group consisting of PD-1, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR3DL1, KIR3DL2, KIR3DL3, LAIR1, CD22, CD33, SIGLEC5, SIGLEC6, SIGLEC7, SIGLEC8, SIGLEC9, SIGLEC10, SIGLEC11, SIGLEC12, PECAM1/CD31, CD200R1, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, SLAMF1, SLAMF5, BTLA, LAGS, 2B4, CD160, CEACAM1, TIM3, VISTA, TIGIT, SIRPalpha, FcγRIIB, CD5, CD300a, CD300f, LIR1, LIR2, LIR3, LIR5, LIRE, Ly9, 2xPD1(G4S), 2xPD1(PD1), PVRIg, and AA2ARKIR2DL1, LIR1, and PD-1. In some embodiments, the inhibitory domain is KIR2DL1 (SEQ ID NO:102). In some embodiments, the inhibitory domain is LIR1 (SEQ ID NO:143). In some embodiments, the inhibitory domain is PD-1 (SEQ ID NO:101). In some embodiments, the inhibitory domain is KIR2DL2 (SEQ ID NO:103). In some embodiments, the inhibitory domain is KIR2DL3 (SEQ ID NO:104). In some embodiments, the inhibitory domain is KIR2DL4 (SEQ ID NO:105). In some embodiments, the inhibitory domain is KIR2DL5A (SEQ ID NO:106). In some embodiments, the inhibitory domain is KIR3DL1 (SEQ ID NO:107). In some embodiments, the inhibitory domain is KIR3DL2 (SEQ ID NO:108). In some embodiments, the inhibitory domain is KIR3DL3 (SEQ ID NO:109). In some embodiments, the inhibitory domain is LAIR1 (SEQ ID NO:110). In some embodiments, the inhibitory domain is CD22 (SEQ ID NO:111). In some embodiments, the inhibitory domain is CD33 (SEQ ID NO:112). In some embodiments, the inhibitory domain is SIGLEC5 (SEQ ID NO:113). In some embodiments, the inhibitory domain is SIGLEC6 (SEQ ID NO:114). In some embodiments, the inhibitory domain is SIGLEC7 (SEQ ID NO:115). In some embodiments, the inhibitory domain is SIGLEC8 (SEQ ID NO:116). In some embodiments, the inhibitory domain is SIGLEC9 (SEQ ID NO:117). In some embodiments, the inhibitory domain is SIGLEC10 (SEQ ID NO:118). In some embodiments, the inhibitory domain is SIGLEC11 (SEQ ID NO:119). In some embodiments, the inhibitory domain is SIGLEC12 (SEQ ID NO:120). In some embodiments, the inhibitory domain is PECAM1/CD31 (SEQ ID NO:121). In some embodiments, the inhibitory domain is CD200R1 (SEQ ID NO:122). In some embodiments, the inhibitory domain is FCRL1 (SEQ ID NO:123). In some embodiments, the inhibitory domain is FCRL2 (SEQ ID NO:124). In some embodiments, the inhibitory domain is FCRL3 (SEQ ID NO:125). In some embodiments, the inhibitory domain is FCRL4 (SEQ ID NO:126). In some embodiments, the inhibitory domain is FCRL5 (SEQ ID NO:127). In some embodiments, the inhibitory domain is SLAMF1 (SEQ ID NO:128). In some embodiments, the inhibitory domain is SLAMF5 (SEQ ID NO:129). In some embodiments, the inhibitory domain is BTLA (SEQ ID NO:130). In some embodiments, the inhibitory domain is LAG3 (SEQ ID NO:131). In some embodiments, the inhibitory domain is 2B4 (SEQ ID NO:132). In some embodiments, the inhibitory domain is CD160 (SEQ ID NO:133). In some embodiments, the inhibitory domain is CEACAM1 (SEQ ID NO:134). In some embodiments, the inhibitory domain is TIM3 (SEQ ID NO:135). In some embodiments, the inhibitory domain is VISTA (SEQ ID NO:136). In some embodiments, the inhibitory domain is TIGIT (SEQ ID NO:137). In some embodiments, the inhibitory domain is SIRPalpha (SEQ ID NO:138). In some embodiments, the inhibitory domain is FcγRIIB (SEQ ID NO:139). In some embodiments, the inhibitory domain is CD5 (SEQ ID NO:140). In some embodiments, the inhibitory domain is CD300a (SEQ ID NO:141). In some embodiments, the inhibitory domain is CD300f (SEQ ID NO:142). In some embodiments, the inhibitory domain is LIR2 (SEQ ID NO:144). In some embodiments, the inhibitory domain is LIR3 (SEQ ID NO:145). In some embodiments, the inhibitory domain is LIR5 (SEQ ID NO:146). In some embodiments, the inhibitory domain is LIR8 (SEQ ID NO:147). In some embodiments, the inhibitory domain is Ly9 (SEQ ID NO:148). In some embodiments, the inhibitory domain is 2xPD1(G4S) (SEQ ID NO:149). In some embodiments, the inhibitory domain is 2xPD1(PD1) (SEQ ID NO:150). In some embodiments, the inhibitory domain is PVRIg (SEQ ID NO:151). In some embodiments, the inhibitory domain is AA2AR (SEQ ID NO:152).

TABLE 7

iCAR inhibitory domain sequences

SEQ

Sequence
ID

Information
NO
Amino acid sequence

PD-1
101
CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWRE

KTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSA

QPLRPEDGHCSWPL

KIR2DL1
102
HRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQEVTYT

QLNHCVFTQRKITRPSQRPKTPPTDIIVYTELPNAESRSKVVS

CP

KIR2DL2
103
HRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQEVTYT

QLNHCVFTQRKITRPSQRPKTPPTDIIVYAELPNAESRSKVVS

CP

KIR2DL3
104
HRWCCNKKNAVVMDQEPAGNRTVNREDSDEQDPQEVTYA

QLNHCVFTQRKITRPSQRPKTPPTDIIVYTELPNAEP

KIR2DL4
105
RWCSKKKDAAVMNQEPAGHRTVNREDSDEQDPQEVTYAQ

LDHCIFTQRKITGPSQRSKRPSTDTSVCIELPNAEPRALSPAH

EHHSQALMGSSRETTALSQTQLASSNVPAAGI

KIR2DL5A
106
LHCCCSNKKNAAVMDQEPAGDRTVNREDSDDQDPQEVTY

AQLDHCVFTQTKITSPSQRPKTPPTDTTMYMELPNAKPRSLS

PAHKHHSQALRGSSRETTALSQNRVASSHVPAAGI

KIR3DL1
107
HLWCSNKKNAAVMDQEPAGNRTANSEDSDEQDPEEVTYA

QLDHCVFTQRKITRPSQRPKTPPTDTILYTELPNAKPRSKVV

SCP

KIR3DL2
108
YRWCSNKKNAAVMDQEPAGDRTVNRQDSDEQDPQEVTYA

QLDHCVFIQRKISRPSQRPKTPLTDTSVYTELPNAEPRSKVVS

CPRAPQSGLEGVF

KIR3DL3
109
HRWCANKKNAVVMDQEPAGNRTVNREDSDEQDPQEVTYA

QLNHCVFTQRKITRPSQRPKTPPTDTSV

LAIR1
110
HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATV

NGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQRTARA

VSPQSTKPMAESITYAAVARH

CD22
111
KLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSEGP

HSLGCYNPMMEDGISYTTLRFPEMNIPRTGDAESSEMQRPPP

DCDDTVTYSALHKRQVGDYENVIPDFPEDEGIHYSELIQFGV

GERPQAQENVDYVILKH

CD33
112
KTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETS

SCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ

SIGLEC5
113
KARRKQAAGRPEKMDDEDPIMGTITSGSRKKPWPDSPGDQ

ASPPGDAPPLEEQKELHYASLSFSEMKSREPKDQEAPSTTEY

SEIKTSK

SIGLEC6
114
RVKTRRKKAAQPVQNTDDVNPVMVSGSRGHQHQFQTGIVS

DHPAEAGPISEDEQELHYAVLHFHKVQPQEPKVTDTEYSEIK

IHK

SIGLEC7
115
RSCRKKSARPAADVGDIGMKDANTIRGSASQGNLTESWAD

DNPRHHGLAAHSSGEEREIQYAPLSFHKGEPQDLSGQEATN

NEYSEIKIPK

SIGLEC8
116
RSCRKKSARPAAGVGDTGMEDAKAIRGSASQGPLTESWKD

GNPLKKPPPAVAPSSGEEGELHYATLSFHKVKPQDPQGQEA

TDSEYSEIKIHKRETAETQACLRNHNPSSKEVRG

SIGLEC9
117
VRSCRKKSARPAAGVGDTGIEDANAVRGSASQGPLTEPWA

EDSPPDQPPPASARSSVGEGELQYASLSFQMVKPWDSRGQE

ATDTEYSEIKIHR

SIGLEC10
118
KILPKRRTQTETPRPRFSRHSTILDYINVVPTAGPLAQKRNQK

ATPNSPRTPLPPGAPSPESKKNQKKQYQLPSFPEPKSSTQAPE

SQESQEELHYATLNFPGVRPRPEARMPKGTQADYAEVKFQ

SIGLEC11
119
KICRKEARKRAAAEQDVPSTLGPISQGHQHECSAGSSQDHP

PPGAATYTPGKGEEQELHYASLSFQGLRLWEPADQEAPSTT

EYSEIKIHTGQPLRGPGFGLQLEREMSGMVPK

SIGLEC12
120
RSCRKKSARPAVGVGDTGMEDANAVRGSASQGPLIESPAD

DSPPHHAPPALATPSPEEGEIQYASLSFHKARPQYPQEQEAIG

YEYSEINIPK

PECAM1/CD31
121
KCYFLRKAKAKQMPVEMSRPAVPLLNSNNEKMSDPNMEA

NSHYGHNDDVRNHAMKPINDNKEPLNSDVQYTEVQVSSAE

SHKDLGKKDTETVYSEVRKAVPDAVESRYSRTEGSLDGT

CD200R1
122
KVNGCRKYKLNKTESTPVVEEDEMQPYASYTEKNNPLYDT

TNKVKASEALQSEVDTDLHTL

FCRL1
123
GLKRKIGRRSARDPLRSLPSPLPQEFTYLNSPTPGQLQPIYEN

VNVVSGDEVYSLAYYNQPEQESVAAETLGTHMEDKVSLDI

YSRLRKANITDVDYEDAM

FCRL2
124
HKISGESSATNEPRGASRPNPQEFTYSSPTPDMEELQPVYVN

VGSVDVDVVYSQVWSMQQPESSANIRTLLENKDSQVIYSSV

KKS

FCRL3
125
HYARARRKPGGLSATGTSSHSPSECQEPSSSRPSRIDPQEPTH

SKPLAPMELEPMYSNVNPGDSNPIYSQIWSIQHTKENSANCP

MMHQEHEELTVLYSELKKTHPDDSAGEASSRGRAHEEDDE

ENYENVPRVLLASDH

FCRL4
126
HCWRRRKSGVGFLGDETRLPPAPGPGESSHSICPAQVELQSL

YVDVHPKKGDLVYSEIQTTQLGEEEEANTSRTLLEDKDVSV

VYSEVKTQHPDNSAGKISSKDEES

FCRL5
127
LSRKAGRKPASDPARSPSDSDSQEPTYHNVPAWEELQPVYT

NANPRGENVVYSEVRIIQEKKKHAVASDPRHLRNKGSPIIYS

EVKVASTPVSGSLFLASSAPHR

SLAMF1
128
QLRRRGKTNHYQTTVEKKSLTIYAQVQKPGPLQKKLDSFPA

QDPCTTIYVAATEPVPESVQETNSITVYASVTLPES

SLAMF5
129
RLFKRRQGRIFPEGSCLNTFTKNPYAASKKTIYTYIMASRNT

QPAESRIYDEILQSKVLPSKEEPVNTVYSEVQFADKMGKAST

QDSKPPGTSSYEIVI

BTLA
130
RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVL

LSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLN

HSVIGPNSRLARNVKEAPTEYASICVRS

LAG3
131
HLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPE

PEPEPEPEPEQL

2B4
132
WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPG

GGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHS

PSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS

CD160
133
GCINITSSASQEGTRLNLICTVWHKKEEAEGFVVFLCKDRSG

DCSPETSLKQLRLKRDPGIDGVGEISSQLMFTISQVTPLHSGT

YQCCARSQKSGIRLQGHFFSILFTETGNYTVTGLKQRQHLEF

SHNEGTLS

CEACAM1
134
HFGKTGRASDQRDLTEHKPSVSNHTQDHSNDPPNKMNEVT

YSTLNFEAQQPTQPTSASPSLTATEIIYSEVKKQ

TIM3
135
FKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYT

IEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP

VISTA
136
YKQRQAASNRRAQELVRMDSNIQGIENPGFEASPPAQGIPE

AKVRHPLSYVAQRQPSESGRHLLSEPSTPLSPPGPGDVFFPSL

DPVPDSPNFEVI

TIGIT
137
LTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQA

EAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG

SIRPalpha
138
RIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPK

GKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVH

LNRTPKQPAPKPEPSFSEYASVQVPRK

FcγRIIB
139
VVALIYCRKKRISALPGYPECREMGETLPEKPANPTNPDEAD

KVGAENTITYSLLMHPDALEEPDDQNRI

CD5
140
KKLVKKFRQKKQRQWIGPTGMNQNMSFHRNHTATVRSHA

ENPTASHVDNEYSQPPRNSHLSAYPALEGALHRSSMQPDNS

SDSDYDLHGAQRL

CD300a
141
RMFQKWIKAGDHSELSQNPKQAATQSELHYANLELLMWPL

QEKPAPPREVEVEYSTVASPREELHYASVVFDSNTNRIAAQ

RPREEEPDSDYSVIRKT

CD300f
142
WRMMKYQQKAAGMSPEQVLQPLEGDLCYADLTLQLAGTS

PQKATTKLSSAQVDQVEVEYVTMASLPKEDISYASLTLGAE

DQEPTYCNMGHLSSHLPGRGPEEPTEYSTISRP

LIR1
143
LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSS

PAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTY

AEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEA

AASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYA

TLAIH

LIR2
144
LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSS

PAADAQEENLYAAVKDTQPEDGVEMDTRAAASEAPQDVT

YAQLHSLTLRRKATEPPPSQEREPPAEPSIYATLAIH

LIR3
145
RRQRHSKHRTSDQRKTDFQRPAGAAETEPKDRGLLRRSSPA

ADVQEENLYAAVKDTQSEDRVELDSQSPHDEDPQAVTYAP

VKHSSPRREMASPPSSLSGEFLDTKDRQVEEDRQMDTEAAA

SEASQDVTYAQLHSLTLRRKATEPPPSQEGEPPAEPSIYATL

AIH

LIR5
146
QHWRQGKHRTLAQRQADFQRPPGAAEPEPKDGGLQRRSSP

AADVQGENFCAAVKNTQPEDGVEMDTRQSPHDEDPQAVT

YAKVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTE

AAASEAPQDVTYAQLHSFTLRQKATEPPPSQEGASPAEPSV

YATLAIH

LIR8
147
RHRHQSKHRTSAHFYRPAGAAGPEPKDQGLQKRASPVADI

QEEILNAAVKDTQPKDGVEMDARAAASEAPQDVTYAQLHS

LTLRREATEPPPSQEREPPAEPSIYAPLAIH

Ly9
148
KRKGRCSVPAFCSSQAEAPADTPEPTAGHTLYSVLSQGYEK

LDTPLRPARQQPTPTSDSSSDSNLTTEEDEDRPEVHKPISGRY

EVFDQVTQEGAGHDPAPEGQADYDPVTPYVTEVESVVGEN

TMYAQVFNLQGKTPVSQKEESSATIYCSIRKPQVVPPPQQN

DLEIPESPTYENFT

2xPD1(G4S)
149
CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWRE

KTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSA

QPLRPEDGHCSWPLGGGGSGGGGSCSRAARGTIGARRTGQP

LKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYA

TIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

2xPD1(PD1)
150
CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWRE

KTPEPPVPCVPEQTEYATIDFQWREKTPEPPVPCVPEQVDYG

ELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRG

SADGPRSAQPLRPEDGHCSWPL

PVRIg
151
LRRHKHRPAPRLQPSRTSPQAPRARAWAPSQASQAALHVPY

ATINTSCRPATLDTAHPHGGPSWWASLPTHAAHRPQGPAA

WASTPIPARGSFVSVENGLYAQAGERPPHTGPGLTLFPDPRG

PRAMEGPLGVR

AA2AR
152
RIREFRQTFRKIIRSHVLRQQEPFKAAGTSARVLAAHGSDGE

QVSLRLNGHPPGVWANGSAPHPERRPNGYALGLVSGGSAQ

ESQGNTGLPDVELLSHELKGVCPEPPGLDDPLAQDGAGVS

5. Optional Synthetic PD-1

In some embodiments, the iCAR construct comprises an optional synthetic PD-1 sequence. In some embodiments, the iCAR comprises a synthetic PD-1 sequence shown in Table 8. In some embodiments, the iCAR construct comprises an optional synthetic LIR1 sequence. In some embodiments, the iCAR comprises a synthetic LIR1 sequence shown in Table 8.

TABLE 8

synthetic PD-1 and LIR1 sequences

SEQ

ID NO
Sequence intracellular synPD-1

243
CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELVFPSGMGTSSPARR

GSADGPRSAQPLRPEDGHCSWPL

244
CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP

CVPEQVDYGELVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

245
CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP

CVPEQVDYGELDFQWREKTPEPPVPCVPEQVDYGELVFPSGMGTSSPAR

RGSADGPRSAQPLRPEDGHCSWPL

246
CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP

CVPEQVDYGELDFQWREKTPEPPVPCVPEQVDYGELDFQWREKTPEPP

VPCVPEQVDYGELVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWP

L

247
CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP

CVPEQVDYGELDFQWREKTPEPPVPCVPEQVDYGELDFQWREKTPEPP

VPCVPEQVDYGELDFQWREKTPEPPVPCVPEQVDYGELVFPSGMGTSSP

ARRGSADGPRSAQPLRPEDGHCSWPL

248
CSRAARGTIGARRTGQPLKEDPSAVPVFSTEYATIVFPSGMGTSSPARRG

SADGPRSAQPLRPEDGHCSWPL

249
CSRAARGTIGARRTGQPLKEDPSAVPVFSTEYATIDFQWREKTPEPPVPC

VPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

250
CSRAARGTIGARRTGQPLKEDPSAVPVFSTEYATIDFQWREKTPEPPVPC

VPEQTEYATIDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRG

SADGPRSAQPLRPEDGHCSWPL

251
CSRAARGTIGARRTGQPLKEDPSAVPVFSTEYATIDFQWREKTPEPPVPC

VPEQTEYATIDFQWREKTPEPPVPCVPEQTEYATIDFQWREKTPEPPVPC

VPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

252
CSRAARGTIGARRTGQPLKEDPSAVPVESTEYATIDFQWREKTPEPPVPC

VPEQTEYATIDFQWREKTPEPPVPCVPEQTEYATIDFQWREKTPEPPVPC

VPEQTEYATIDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRG

SADGPRSAQPLRPEDGHCSWPL

253
CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP

CVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLG

GGGSGGGGSCSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQW

REKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPE

DGHCSWPL

254
CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP

CVPEQTEYATIDFQWREKTPEPPVPCVPEQVDYGELDFQWREKTPEPPV

PCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

296
LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQE

ENLYAAVKHTQPEDGVEMDTRSPHDEDPQANLYAAVKHSRPRREMAS

PPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDNLYAAVHSLTLRRE

ATEPPPSQEGPSPAVPNLYAAVAIH

297
LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQE

EVTYAEVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASP

PSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAEVHSLTLRREA

TEPPPSQEGPSPAVPVTYAEVAIH

298
LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQE

EVTYAQLKHTQPEDGVEMDTRSPHDEDPQAVTYAQLKHSRPRREMASP

PSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREA

TEPPPSQEGPSPAVPVTYAQLAIH

299
LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQE

ESIYATLKHTQPEDGVEMDTRSPHDEDPQASIYATLKHSRPRREMASPPS

PLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDSIYATLHSLTLRREATE

PPPSQEGPSPAVPSIYATLAIH

300
LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQE

EVTYAQLKHTQPEDGVEMDTRSPHDEDPQASIYATLKHSRPRREMASPP

SPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREAT

EPPPSQEGPSPAVPSIYATLAIH

301
LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQE

ETEYATIKHTQPEDGVEMDTRSPHDEDPQATEYATIKHSRPRREMASPPS

PLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREAT

EPPPSQEGPSPAVPSIYATLAIH

302
LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQE

EVTYAQLKHTQPEDGVEMDTRSPHDEDPQASIYATLKHSRPRREMASPP

SPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDTEYATIHSLTLRREAT

EPPPSQEGPSPAVPTEYATIAIH

304
LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQE

EVTYAQLKHTQPEDGVEMDTRSPHDEDPQATEYATIKHSRPRREMASPP

SPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDTEYATIHSLTLRREAT

EPPPSQEGPSPAVPSIYATLAIH

6. Exemplary iCARs

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of BB7.2 (SEQ ID NOs: 37 and 38). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G₄S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO: 112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:]122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306). In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of 3PF12/C4 (SEQ ID NOs: 39 and 40). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO: 114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of 3PF12/F12 (SEQ ID NOs: 41 and 42). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of 3PF12/B11 (SEQ ID NOs: 43 and 44). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO: 114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of W6/32 (SEQ ID NOs: 45 and 46). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO:117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of BBM.1 (SEQ ID NOs: 47 and 48). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO: 114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of SN66E3.1 (SEQ ID NOs: 49 and 50). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO:117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcβRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of Ha5C2.A2 (SEQ ID NOs: 51 and 52). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO: 114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of MWB1 (SEQ ID NOs: 53 and 54). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO: 111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of MWB1-mod (MWB1.1) (SEQ ID NOs: 55 and 56). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO: 114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of Hz.BB7.2 VH1-69_A18VK (SEQ ID NOs: 57 and 58). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of Hz.BB7.2 VH1-69 (27,30)_A18 (SEQ ID NOs: 59 and 60). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of Hz.BB7.2VH1-69 (27,30,48)_A18 (SEQ ID NOs: 61 and 62). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO:117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of Hz.BB7.2 VH1-69 (27,30,67)_A18 (SEQ ID NOs: 63 and 64). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of Hz.BB7.2 VH1-69 (27,30,69)_A18 (SEQ ID NOs: 65 and 66). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of Hz.BB7.2 VH1-69 (27,30,67,69)_A18 (SEQ ID NOs: 67 and 68). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of Hz.BB7.2 VH1-3_A18 (SEQ ID NOs: 69 and 70). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of Hz.BB7.2 VH1-3(48)_A18 (SEQ ID NOs: 71 and 72). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of Hz.BB7.2 VH1-3(67)_A18 (SEQ ID NOs: 73 and 74). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO:117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of Hz.Bb7.2 VH1-3(69)_A18 (SEQ ID NOs: 75 and 76). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO:117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the scFv has the VL and VH sequences of Hz.BB7.2 VH1-3(71)_A18 (SEQ ID NOs: 77 and 78). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR comprises an scFv component comprising the VL and VH sequences of Hz. BB7.2VH1-3(73)_A18 (SEQ ID NOs: 79 and 80). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the scFv has the VL and VH sequences of MWB1.2 (SEQ ID NOs: 163 and 164). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO: 139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the scFv has the VL and VH sequences of SN66E3.2 (SEQ ID NOs: 165 and 166). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the scFv has the VL and VH sequences of MWB1.1 (SEQ ID NOs: 273). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO:116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO: 117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO:119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the scFv has the VL and VH sequences of MWB1.2 (SEQ ID NOs: 274). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO:117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the scFv has the VL and VH sequences of SN66E3.3 (SEQ ID NOs: 283 and 284). In some embodiments, the orientation of the iCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the iCAR VH and VL regions is VL-VH. In some embodiments, the iCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the iCAR scFv. In some embodiments, the iCAR comprises a CD8 alpha hinge domain (SEQ ID NO:84). In some embodiments, the iCAR comprises a CD28 hinge domain (SEQ ID NO:85). In some embodiments, the iCAR comprises a PD-1 hinge domain (SEQ ID NO:86). In some embodiments, the iCAR comprises a LIR1 Ig3-4 hinge domain (SEQ ID NO:87). In some embodiments, the iCAR comprises a LIR1 Ig-4 hinge domain (SEQ ID NO:88). In some embodiments, the iCAR comprises a LIR1 52 aa hinge domain (SEQ ID NO:89). In some embodiments, the iCAR comprises a LIR1 36 aa hinge domain (SEQ ID NO:90). In some embodiments, the iCAR comprises a LIR1 30 aa hinge domain (SEQ ID NO:91). In some embodiments, the iCAR comprises a LIR1 8 aa hinge domain (SEQ ID NO:92). In some embodiments, the iCAR comprises a CD33 hinge domain (SEQ ID NO:93). In some embodiments, the iCAR comprises a KIR2DL1 hinge domain (SEQ ID NO:94). In some embodiments, the iCAR comprises aLIR1 26 aa hinge domain (SEQ ID NO: 289). In some embodiments, the iCAR comprises PD-1 (47) hinge domain (SEQ ID NO: 290). In some embodiments, the iCAR comprises PD-1 (42) hinge domain (SEQ ID NO: 291). In some embodiments, the iCAR comprises PD-1 (36) hinge domain (SEQ ID NO: 292). In some embodiments, the iCAR comprises PD-1 (30) hinge domain (SEQ ID NO: 293). In some embodiments, the iCAR comprises PD-1 (26) hinge domain (SEQ ID NO: 294). In some embodiments, the iCAR comprises PD-1 (20) hinge domain (SEQ ID NO: 295). In some embodiments, the iCAR comprises a CD8 alpha transmembrane domain (SEQ ID NO:95). In some embodiments, the iCAR comprises a CD28 transmembrane domain (SEQ ID NO:96). In some embodiments, the iCAR comprises a PD-1 transmembrane domain (SEQ ID NO:97). In some embodiments, the iCAR comprises a LIR1 transmembrane domain (SEQ ID NO:98). In some embodiments, the iCAR comprises a CD33 transmembrane domain (SEQ ID NO:99). In some embodiments, the iCAR comprises a KIR2DL1 transmembrane domain (SEQ ID NO:100). In some embodiments, the iCAR comprises a PD-1 inhibitory domain (SEQ ID NO:101). In some embodiments, the iCAR comprises a KIR2DL1 inhibitory domain (SEQ ID NO:102). In some embodiments, the iCAR comprises a KIR2DL2 inhibitory domain (SEQ ID NO:103). In some embodiments, the iCAR comprises a KIR2DL3 inhibitory domain (SEQ ID NO:104). In some embodiments, the iCAR comprises a KIR2DL4 inhibitory domain (SEQ ID NO:105). In some embodiments, the iCAR comprises a KIR2DL5A inhibitory domain (SEQ ID NO:106). In some embodiments, the iCAR comprises a KIR3DL1 inhibitory domain (SEQ ID NO:107). In some embodiments, the iCAR comprises a KIR3DL2 inhibitory domain (SEQ ID NO:108). In some embodiments, the iCAR comprises a KIR3DL3 inhibitory domain (SEQ ID NO:109). In some embodiments, the iCAR comprises a LAIR1 inhibitory domain (SEQ ID NO:110). In some embodiments, the iCAR comprises a CD22 inhibitory domain (SEQ ID NO:111). In some embodiments, the iCAR comprises a CD33 inhibitory domain (SEQ ID NO:112). In some embodiments, the iCAR comprises a SIGLEC5 inhibitory domain (SEQ ID NO: 113). In some embodiments, the iCAR comprises a SIGLEC6 inhibitory domain (SEQ ID NO:114). In some embodiments, the iCAR comprises a SIGLEC7 inhibitory domain (SEQ ID NO: 115). In some embodiments, the iCAR comprises a SIGLEC8 inhibitory domain (SEQ ID NO: 116). In some embodiments, the iCAR comprises a SIGLEC9 inhibitory domain (SEQ ID NO:117). In some embodiments, the iCAR comprises a SIGLEC10 inhibitory domain (SEQ ID NO: 118). In some embodiments, the iCAR comprises a SIGLEC11 inhibitory domain (SEQ ID NO: 119). In some embodiments, the iCAR comprises a SIGLEC12 inhibitory domain (SEQ ID NO:120). In some embodiments, the iCAR comprises a PECAM1/CD31 inhibitory domain (SEQ ID NO:121). In some embodiments, the iCAR comprises a CD200R1 inhibitory domain (SEQ ID NO:122). In some embodiments, the iCAR comprises a FCRL1 inhibitory domain (SEQ ID NO:123). In some embodiments, the iCAR comprises a FCRL2 inhibitory domain (SEQ ID NO:124). In some embodiments, the iCAR comprises a FCRL3 inhibitory domain (SEQ ID NO:125). In some embodiments, the iCAR comprises a FCRL4 inhibitory domain (SEQ ID NO:126). In some embodiments, the iCAR comprises a FCRL5 inhibitory domain (SEQ ID NO:127). In some embodiments, the iCAR comprises a SLAMF1 inhibitory domain (SEQ ID NO:128). In some embodiments, the iCAR comprises a SLAMF5 inhibitory domain (SEQ ID NO:129). In some embodiments, the iCAR comprises a BTLA inhibitory domain (SEQ ID NO:130). In some embodiments, the iCAR comprises a LAG3 inhibitory domain (SEQ ID NO:131). In some embodiments, the iCAR comprises a 2B4 inhibitory domain (SEQ ID NO:132). In some embodiments, the iCAR comprises a CD160 inhibitory domain (SEQ ID NO:133). In some embodiments, the iCAR comprises a CEACAM1 inhibitory domain (SEQ ID NO:134). In some embodiments, the iCAR comprises a TIM3 inhibitory domain (SEQ ID NO:135). In some embodiments, the iCAR comprises a VISTA inhibitory domain (SEQ ID NO:136). In some embodiments, the iCAR comprises a TIGIT inhibitory domain (SEQ ID NO:137). In some embodiments, the iCAR comprises a SIRPalpha inhibitory domain (SEQ ID NO:138). In some embodiments, the iCAR comprises a FcγRIIB inhibitory domain (SEQ ID NO:139). In some embodiments, the iCAR comprises a CD5 inhibitory domain (SEQ ID NO:140). In some embodiments, the iCAR comprises a CD300a inhibitory domain (SEQ ID NO:141). In some embodiments, the iCAR comprises a CD300f inhibitory domain (SEQ ID NO:142). In some embodiments, the iCAR comprises a LIR1 inhibitory domain (SEQ ID NO:143). In some embodiments, the iCAR comprises a LIR2 inhibitory domain (SEQ ID NO:144). In some embodiments, the iCAR comprises a LIR3 inhibitory domain (SEQ ID NO:145). In some embodiments, the iCAR comprises a LIR5 inhibitory domain (SEQ ID NO:146). In some embodiments, the iCAR comprises a LIR8 inhibitory domain (SEQ ID NO:147). In some embodiments, the iCAR comprises a Ly9 inhibitory domain (SEQ ID NO:148). In some embodiments, the iCAR comprises a 2xPD1(G4S) inhibitory domain (SEQ ID NO:149). In some embodiments, the iCAR comprises a 2xPD1(PD1) inhibitory domain (SEQ ID NO:150). In some embodiments, the iCAR comprises a PVRIg inhibitory domain (SEQ ID NO:151). In some embodiments, the iCAR comprises an AA2AR inhibitory domain (SEQ ID NO:152). In some embodiments, the iCAR comprises a signal peptide upstream of the iCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the iCAR has a set of components shown in Tables 9-10 and/or an amino acid sequence shown in Tables 11-12.

TABLE 9

iCAR constructs

VH
VL

SEQ
SEQ

ID
ID
Signal
scFv

Construct
scFv
NO
NO
peptide
Linker
Hinge
TM
Signaling

BB7.2
38
37
CD8alpha
(G4S) × 3
PD-1
PD-1
PD-1

MC0096
3PF12/C4
40
39
CD8alpha
(G4S) × 3
PD-1
PD-1
PD-1

(VR96)

MC0274
3PF12/F12
42
41
CD8alpha
(G4S) × 3
PD-1
PD-1
PD-1

(VR274)

MC0276
3PF12/B11
44
43
CD8alpha
(G4S) × 3
PD-1
PD-1
PD-1

(VR276)

MC0097
W6/32
46
45
CD8alpha
(G4S) × 3
PD-1
PD-1
PD-1

(VR97)

MC0098
BBM.1
48
47
CD8alpha
(G4S) × 3
PD-1
PD-1
PD-1

(VR98)

MC0099
SN66E3.1
50
49
CD8alpha
(G4S) × 3
PD-1
PD-1
PD-1

(VR99)

MC0100
Ha5C2.A2
52
51
CD8alpha
(G4S) × 3
PD-1
PD-1
PD-1

(VR100)

MC0101
MWB1
54
53
CD8alpha
(G4S) × 3
PD-1
PD-1
PD-1

(VR101)

MC0102
MWB1.1d
56
55
CD8alpha
(G4S) × 3
PD-1
PD-1
PD-1

(VR102)

MC0372
Hz.BB7.2 VH1-69_A18VK
58
57
CD8 alpha
(G4S) × 3
CD8 alpha
CD8 alpha
41BBz

(VR372)

MC0373
Hz.BB7.2 VH1-69 (27, 30)_A18
60
59
CD8 alpha
(G4S) × 3
CD8 alpha
CD8 alpha
41BBz

(VR373)

MC0374
Hz.BB7.2 VH1-69 (27, 30, 48)_A18
62
61
CD8 alpha
(G4S) × 3
CD8 alpha
CD8 alpha
41BBz

(VR374)

MC0375
Hz.BB7.2 VH1-69 (27, 30, 67)_A18
64
63
CD8 alpha
(G4S) × 3
CD8 alpha
CD8 alpha
41BBz

(VR375)

MC0376
Hz.BB7.2 VH1-69 (27, 30, 69)_A18
66
65
CD8 alpha
(G4S) × 3
CD8 alpha
CD8 alpha
41BBz

(VR376)

MC0377
Hz.BB7.2 VH1-69 (27, 30, 67, 69)_A18
68
67
CD8 alpha
(G4S) × 3
CD8 alpha
CD8 alpha
41BBz

(VR377)

MC0378
Hz.BB7.2 VH1-3_A18
70
69
CD8 alpha
(G4S) × 3
CD8alpha
CD8 alpha
41BBz

(VR378)

MC0379
Hz.BB7.2 VH1-3(48)_A18
72
71
CD8 alpha
(G4S) × 3
CD8 alpha
CD8 alpha
41BBz

(VR379)

MC0380
Hz.BB7.2 VH1-3(67)_A18
74
73
CD8 alpha
(G4S) × 3
CD8 alpha
CD8 alpha
41BBz

(VR380)

MC0381
Hz.BB7.2 VH1-3(69)_A18
76
75
CD8 alpha
(G4S) × 3
CD8 alpha
CD8 alpha
41BBz

(VR381)

MC0382
Hz.BB7.2 VH1-3(71)_A18
78
77
CD8 alpha
(G4S) × 3
CD8 alpha
CD8 alpha
41BBz

(VR382)

MC0383
Hz.BB7.2 VH1-3(73)_A18
80
79
CD8 alpha
(G4S) × 3
CD8 alpha
CD8 alpha
41BBz

(VR383)

MC0384
3PF12_274_LIR1_HER2_shRNA(A2)
40
41
CD8 alpha
3PF12_274
(G4S) × 3
PD-1
LIR-1

(VR384)

MC0385
3PF12_276_LIR1_HER2_shRNA(A2)
44
43
CD8 alpha
3PF12_276
(G4S) × 3
PD-1
LIR-1

(VR385)

MC0386
MWB1.1_HL_LIR1_HER2_shRNA(A2)
56
55
CD8 alpha
MWB1.1_HL
(G4S) × 3
PD-1
LIR-1

(VR386)

MC0387
MWB1.1_LH_LIR1_HER2_shRNA(A2)
56
55
CD8 alpha
MWB1.1_LH
(G4S) × 3
PD-1
LIR-1

(VR387)

MC0388
MWB1.2_HL_LIR1_HER2_shRNA(A2)
164
163
CD8 alpha
MWB1.2_HL
(G4S) × 3
PD-1
LIR-1

(VR388)

MC0389
MWB1.2_LH_LIR1_HER2_shRNA(A2)
164
163
CD8 alpha
MWB1.2_LH
(G4S) × 3
PD-1
LIR-1

(VR389)

MC0390
SN66E3.1_HL_LIR1_HER2_shRNA(A2)
50
49
CD8 alpha
SN66E3.1_HL
(G4S) × 3
PD-1
LIR-1

(VR390)

MC0391
SN66E3.1_LH_LIR1_HER2_shRNA(A2)
50
49
CD8 alpha
SN66E3.1_LH
(G4S) × 3
PD-1
LIR-1

(VR391)

MC0446
SN66E3.2_HL_LIR1_HER2_—
166
165
CD8 alpha
SN66E3.2_HL
(G4S) × 3
LIR1
LIR-1

(VR446)

MC0447
SN66E3.2_LH_LIR1_HER2_—
166
165
CD8 alpha
SN66E3.2_LH
(G4S) × 3
LIR1
LIR-1

(VR447)

MC0448
SN66E3.3(HL)_LIR1(26)_HER2
284
283
CD8 alpha
SN66E.3.3_HL

LIR1
LIR1

(VR448)

MC449
SN66E3.3(LH)_LIR1(26)_HER2
284
283
CD8 alpha
SN66E3.3_LH
(G4S) × 3
LIR1
LIR1

(VR449)

MC0428
HzBB7.2.1_H69_LIR1_H
64
63
CD8 alpha
HzBB7.2_H69
(G4S) × 3
LIR1
LIR-1

(VR428)

MC0421
HzBB7.2.2_H3_LIR1_)
72
71
CD8 alpha
HzBB7.2_H3
(G4S) × 3
LIR1
LIR-1

(VR421)

TABLE 10

iCAR constructs

Construct
Signal

scFv

Construct
Name
Peptide
scFv
Linker
Hinge
TM
Signaling

MC0058
1 × ITIM
CD8alpha
BB7.2
(G4S) × 3
PD-1
PD-1
1 × ITIM

(VR58)

VH VL

PD-1

MC0059
2 × ITIM
CD8alpha
BB7.2
(G4S) × 3
PD-1
PD-1
2 × ITIM

(VR59)

VH VL

PD-1

MC0060
3 × ITIM
CD8alpha
BB7.2
(G4S) × 3
PD-1
PD-1
3 × ITIM

(VR60)

VH VL

PD-1

MC0061
4 × ITIM
CD8alpha
BB7.2
(G4S) × 3
PD-1
PD-1
4 × ITIM

(VR61)

VH VL

PD-1

MC0062
5 × ITIM
CD8alpha
BB7.2
(G4S) × 3
PD-1
PD-1
5 × ITIM

(VR62)

VH VL

PD-1

MC0063
1 × ITSM
CD8alpha
BB7.2
(G4S) × 3
PD-1
PD-1
1 × ITSM

(VR63)

VH VL

PD-1

MC0064
2 × ITSM
CD8alpha
BB7.2
(G4S) × 3
PD-1
PD-1
2 × ITSM

(VR64)

VH VL

PD-1

MC0065
3 × ITSM
CD8alpha
BB7.2
(G4S) × 3
PD-1
PD-1
3 × ITSM

(VR65)

VH VL

PD-1

MC0066
4 × ITSM
CD8alpha
BB7.2
(G4S) × 3
PD-1
PD-1
4 × ITSM

(VR66)

VH VL

PD-1

MC0067
5 × ITSM
CD8alpha
BB7.2
(G4S) × 3
PD-1
PD-1
5 × ITSM

(VR67)

VH VL

PD-1

MC0068
2 × PD1(G4S)
CD8alpha
BB7.2
(G4S) × 3
PD-1
PD-1
2 × PD-1

(VR68)

VH VL

(G4S) × 2

MC0069
2 × PD1(PD1)
CD8alpha
BB7.2
(G4S) × 3
PD-1
PD-1
2 × PD-1

(VR69)

VH VL

(PD1 linker)

TABLE 11

iCAR constructs

SEQ

ID

Construct
scFv
NO
Amino acid sequence

MC0387
MWB1.1_
255
MALPVTALLLPLALLLHAARPQSALTQPPSASGSPGQSV

(VR387)
LH_LIR1_

TISCTGTSSDVGGYKYVSWYQHHPDKAPKLMIYEVNK

HER2_

RPSGVPDRFSGSKSDNTASLTVSGLQAEDEADYYCSSY

shRNA(A2)

AGSNNWVFGGGTKLTVLGGGGSGGGGSGGGGSQVQL

VESGGGVVQPGGSLRLSCAASGFTFSTYGMHWVRQAP

GKGLEWVASISYDGSNKYYADSGQGRFTISRDTSKNSL

YLQMNSLRAEDTAVYYCAIGIYGAYSFDYWGQGTLVT

VSSTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGS

LVLLVWVLAVILRHRRQGKHWTSTQRKADFQHPAGAV

GPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGV

EMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSG

EFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSL

TLRREATEPPPSQEGPSPAVPSIYATLAIH

MC0389
MWB1.2_
256
MALPVTALLLPLALLLHAARPQSALTQPPSASGSPGQSV

(VR389)
LH_LIR1_

TISCTGTSSDVGGYKYVSWYQQHPGKAPKLMIYEVNK

HER2_

RPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSY

shRNA(A2)

AGSNNWVFGGGTKLTVLGGGGSGGGGSGGGGSQVQL

VESGGGVVQPGGSLRLSCAASGFTFSTYGMHWVRQAP

GKGLEWVASISYDGSNKYYADSGQGRFTISRDTSKNSL

YLQMNSLRAEDTAVYYCAIGIYGAYSFDYWGQGTLVT

VSSTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGS

LVLLVWVLAVILRHRRQGKHWTSTQRKADFQHPAGAV

GPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGV

EMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSG

EFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSL

TLRREATEPPPSQEGPSPAVPSIYATLAIH

MC0391
SN66E3.1_
257
MALPVTALLLPLALLLHAARPDIVMTQSPDSLAVSLGE

(VR391)
LH_LIR1_

RATISCKSSQSVLYSSNNKNYLAWYQQKLGQPPKLLIY

HER2_

WASTRESGVPDRFSGSGSGTNFTLTISSLQAENVAVYYC

shRNA(A2)

QQYYGTPFTFGGGTKVEIKGGGGSGGGGSGGGGSQVQ

LVQSGAEVKKPGASVKVSCKASGYTFTDYYLHWVRQA

PGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDASIS

TVYMELSGLTSDDTAVHFCARAGASYYDFWSGWVED

YWGQGTLVTVSSTERRAEVPTAHPSPSPRPAGQFQTLV

VGVVGGLLGSLVLLVWVLAVILRHRRQGKHWTSTQRK

ADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAA

VKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRR

EMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQ

DVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH

MC0447
SN66E3.2
258
MALPVTALLLPLALLLHAARPDIVMTQSPDSLAVSLGE

(VR447)
(LH)_LIR1

RATISCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIY

(30)_HER2

WASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC

QQYYGTPFTFGGGTKVEIKGGGGSGGGGSGGGGSQVQ

LVQSGAEVKKPGASVKVSCKASGYTFTDYYLHWVRQA

PGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSIS

TAYMELSGLTSDDTAVYYCARAGASYYDFWSGWVED

YWGQGTLVTVSSGPTSTSGPEDQPLTPTGSDPQSGLGR

HLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQ

RKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLY

AAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRP

RREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEA

PQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATL

AIH

MC0449
SN66E3.3
305
MALPVTALLLPLALLLHAARPDIVMTQSPDSLAVSLGE

(VR449)
(LH)_LIR1

RATISCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIY

(26)_HER2

WASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC

QQYYGTPFTFGGGTKVEIKGGGGSGGGGSGGGGSQVQ

LVQSGAEVKKPGASVKVSCKASGYTFTDYYLHWVRQA

PGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSIS

TAYMELSRLRSEDTAVYYCARAGASYYDFWSGWVFD

YWGQGTLVTVSSTSGPEDQPLTPTGSDPQSGLGRHLGV

VIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADF

QHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKH

TQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMA

SPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVT

YAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH

MC0428
HzBB7.2.1_
259
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSS

(VR428)
_LIR1

VKVSCKASGYTFTSYHIQWVRQAPGQGLEWMGWIYPG

(52)_HER2

DGSTQYNEKFKGRTTITADKSTSTAYMELSSLRSEDTA

VYYCAREGTYYAMDYWGQGTLVTVSSGGGGSGGGGS

GGGGSDVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNG

NTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGS

GTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVE

IKHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPT

GSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRR

QGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPA

ADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVT

YAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQM

DTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSP

AVPSIYATLAIH

MC0421
HzBB7.2.2_
260
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGA

(VR421)
_H3_LIR1_

SVKVSCKASGYTFTSYHIQWVRQAPGQRLEWIGWIYPG

HER2_

DGSTQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTA

VYYCAREGTYYAMDYWGQGTLVTVSSGGGGSGGGGS

GGGGSDVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNG

NTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGS

GTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVE

IKHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPT

GSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRR

QGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPA

ADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVT

YAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQM

DTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSP

AVPSIYATLAIH

TABLE 12

iCAR constructs

SEQ

Construct
ID

Construct
Name
NO
full length iCAR sequence

MC0058
1xITIM
261
MALPVTALLLPLALLLHAARPEQKLISEEDLQVQLQQSGP

(VR58)

ELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWI

GWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLT

SEDSAIYFCAREGTYYAMDYWGQGTSVTVSSGGGGSGG

GGSGGGGSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHS

NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSG

SGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEI

KTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV

LLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSV

DYGELVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCS

WPL

MC0059
2xITIM
262
MALPVTALLLPLALLLHAARPEQKLISEEDLQVQLQQSGP

(VR59)

ELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWI

GWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLT

SEDSAIYFCAREGTYYAMDYWGQGTSVTVSSGGGGSGG

GGSGGGGSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHS

NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSG

SGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEI

KTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV

LLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSV

DYGELDFQWREKTPEPPVPCVPEQVDYGELVFPSGMGTS

SPARRGSADGPRSAQPLRPEDGHCSWPL

MC0060
3xITIM
263
MALPVTALLLPLALLLHAARPEQKLISEEDLQVQLQQSGP

(VR60)

ELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWI

GWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLT

SEDSAIYFCAREGTYYAMDYWGQGTSVTVSSGGGGSGG

GGSGGGGSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHS

NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSG

SGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEI

KTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV

LLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSV

DYGELDFQWREKTPEPPVPCVPEQVDYGELDFQWREKTP

EPPVPCVPEQVDYGELVFPSGMGTSSPARRGSADGPRSA

QPLRPEDGHCSWPL

MC0061
4xITIM
264
MALPVTALLLPLALLLHAARPEQKLISEEDLQVQLQQSGP

(VR61)

ELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWI

GWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLT

SEDSAIYFCAREGTYYAMDYWGQGTSVTVSSGGGGSGG

GGSGGGGSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHS

NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSG

SGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEI

KTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV

LLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSV

DYGELDFQWREKTPEPPVPCVPEQVDYGELDFQWREKTP

EPPVPCVPEQVDYGELDFQWREKTPEPPVPCVPEQVDYG

ELVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

MC0062
5xITIM
265
MALPVTALLLPLALLLHAARPEQKLISEEDLQVQLQQSGP

(VR62)

ELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWI

GWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLT

SEDSAIYFCAREGTYYAMDYWGQGTSVTVSSGGGGSGG

GGSGGGGSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHS

NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSG

SGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEI

KTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV

LLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSV

DYGELDFQWREKTPEPPVPCVPEQVDYGELDFQWREKTP

EPPVPCVPEQVDYGELDFQWREKTPEPPVPCVPEQVDYG

ELDFQWREKTPEPPVPCVPEQVDYGELVFPSGMGTSSPA

RRGSADGPRSAQPLRPEDGHCSWPL

MC0063
1xITSM
266
MALPVTALLLPLALLLHAARPEQKLISEEDLQVQLQQSGP

(VR63)

ELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWI

GWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLT

SEDSAIYFCAREGTYYAMDYWGQGTSVTVSSGGGGSGG

GGSGGGGSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHS

NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSG

SGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEI

KTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV

LLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSTE

YATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWP

L

MC0064
2xITSM
267
MALPVTALLLPLALLLHAARPEQKLISEEDLQVQLQQSGP

(VR64)

ELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWI

GWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLT

SEDSAIYFCAREGTYYAMDYWGQGTSVTVSSGGGGSGG

GGSGGGGSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHS

NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSG

SGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEI

KTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV

LLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSTE

YATIDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSP

ARRGSADGPRSAQPLRPEDGHCSWPL

MC0065
3xITSM
268
MALPVTALLLPLALLLHAARPEQKLISEEDLQVQLQQSGP

(VR65)

ELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWI

GWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLT

SEDSAIYFCAREGTYYAMDYWGQGTSVTVSSGGGGSGG

GGSGGGGSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHS

NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSG

SGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEI

KTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV

LLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSTE

YATIDFQWREKTPEPPVPCVPEQTEYATIDFQWREKTPEP

PVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPL

RPEDGHCSWPL

MC0066
4xITSM
269
MALPVTALLLPLALLLHAARPEQKLISEEDLQVQLQQSGP

(VR66)

ELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWI

GWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLT

SEDSAIYFCAREGTYYAMDYWGQGTSVTVSSGGGGSGG

GGSGGGGSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHS

NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSG

SGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEI

KTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV

LLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSTE

YATIDFQWREKTPEPPVPCVPEQTEYATIDFQWREKTPEP

PVPCVPEQTEYATIDFQWREKTPEPPVPCVPEQTEYATIVF

PSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

MC0067
5xITSM
270
MALPVTALLLPLALLLHAARPEQKLISEEDLQVQLQQSGP

(VR67)

ELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWI

GWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLT

SEDSAIYFCAREGTYYAMDYWGQGTSVTVSSGGGGSGG

GGSGGGGSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHS

NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSG

SGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEI

KTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV

LLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSTE

YATIDFQWREKTPEPPVPCVPEQTEYATIDFQWREKTPEP

PVPCVPEQTEYATIDFQWREKTPEPPVPCVPEQTEYATIDF

QWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSA

DGPRSAQPLRPEDGHCSWPL

MC0068
2xPD1(G4S)
271
MALPVTALLLPLALLLHAARPEQKLISEEDLQVQLQQSGP

(VR68)

ELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWI

GWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLT

SEDSAIYFCAREGTYYAMDYWGQGTSVTVSSGGGGSGG

GGSGGGGSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHS

NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSG

SGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEI

KTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV

LLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSV

DYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSS

PARRGSADGPRSAQPLRPEDGHCSWPLGGGGSGGGGSCS

RAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWRE

KTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRS

AQPLRPEDGHCSWPL

MC0069
2xPD1(PD1)
272
MALPVTALLLPLALLLHAARPEQKLISEEDLQVQLQQSGP

(VR69)

ELVKPGASVKMSCKASGYTFTSYHIQWVKQRPGQGLEWI

GWIYPGDGSTQYNEKFKGKTTLTADKSSSTAYMLLSSLT

SEDSAIYFCAREGTYYAMDYWGQGTSVTVSSGGGGSGG

GGSGGGGSDVLMTQTPLSLPVSLGDQVSISCRSSQSIVHS

NGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRESGSG

SGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEI

KTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLV

LLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSV

DYGELDFQWREKTPEPPVPCVPEQTEYATIDFQWREKTP

EPPVPCVPEQVDYGELDFQWREKTPEPPVPCVPEQTEYAT

IVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

MC0456
LIR1(ITIM1)
327
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASV

(VR456)
X4

KVSCKASGYTFTSYHIQWVRQAPGQRLEWIGWIYPGDGS

TQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYC

AREGTYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGS

DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEW

YLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIS

RVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKTERRAEVP

TAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVI

LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWR

SSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQ

ANLYAAVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDR

QMDTEAAASEAPQDNLYAAVHSLTLRREATEPPPSQEGP

SPAVPNLYAAVAIH

MC0457
LIR1(ITIM2)
328
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASV

(VR457)
X4

KVSCKASGYTFTSYHIQWVRQAPGQRLEWIGWIYPGDGS

TQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYC

AREGTYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGS

DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEW

YLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIS

RVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKTERRAEVP

TAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVI

LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWR

SSPAADAQEEVTYAEVKHTQPEDGVEMDTRSPHDEDPQ

AVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDR

QMDTEAAASEAPQDVTYAEVHSLTLRREATEPPPSQEGP

SPAVPVTYAEVAIH

MC0458
LIR1(ITIM3)
329
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASV

(VR458)
X4

KVSCKASGYTFTSYHIQWVRQAPGQRLEWIGWIYPGDGS

TQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYC

AREGTYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGS

DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEW

YLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIS

RVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKTERRAEVP

TAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVI

LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWR

SSPAADAQEEVTYAQLKHTQPEDGVEMDTRSPHDEDPQ

AVTYAQLKHSRPRREMASPPSPLSGEFLDTKDRQAEEDR

QMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGP

SPAVPVTYAQLAIH

MC0459
LIR1(ITM4)
330
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASV

(VR459)
X4

KVSCKASGYTFTSYHIQWVRQAPGQRLEWIGWIYPGDGS

TQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYC

AREGTYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGS

DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEW

YLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIS

RVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKTERRAEVP

TAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVI

LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWR

SSPAADAQEESIYATLKHTQPEDGVEMDTRSPHDEDPQA

SIYATLKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQM

DTEAAASEAPQDSIYATLHSLTLRREATEPPPSQEGPSPAV

PSIYATLAIH

MC0460
LIR1ITIM
331
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASV

(VR460)
(3-4)

KVSCKASGYTFTSYHIQWVRQAPGQRLEWIGWIYPGDGS

TQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYC

AREGTYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGS

DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEW

YLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIS

RVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKTERRAEVP

TAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVI

LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWR

SSPAADAQEEVTYAQLKHTQPEDGVEMDTRSPHDEDPQ

ASIYATLKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQ

MDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSP

AVPSIYATLAIH

MC0461
PD-1ITSM
332
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASV

(VR461)
LIR1

KVSCKASGYTFTSYHIQWVRQAPGQRLEWIGWIYPGDGS

(ITIM3-4)

TQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYC

AREGTYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGS

DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEW

YLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIS

RVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKTERRAEVP

TAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVI

LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWR

SSPAADAQEETEYATIKHTQPEDGVEMDTRSPHDEDPQA

TEYATIKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQM

DTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPA

VPSIYATLAIH

MC0462
LIR1
333
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASV

(VR462)
(ITIM3-4)

KVSCKASGYTFTSYHIQWVRQAPGQRLEWIGWIYPGDGS

PD-

TQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYC

1ITSMX2

AREGTYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGS

DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEW

YLQKPGQSPQLLIYKVSNRFSGVPDRESGSGSGTDFTLKIS

RVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKTERRAEVP

TAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVI

LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWR

SSPAADAQEEVTYAQLKHTQPEDGVEMDTRSPHDEDPQ

ASIYATLKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQ

MDTEAAASEAPQDTEYATIHSLTLRREATEPPPSQEGPSP

AVPTEYATIAIH

MC0463
LIR1
334
MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASV

(VR463)
ITIM3,

KVSCKASGYTFTSYHIQWVRQAPGQRLEWIGWIYPGDGS

PD-1

TQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYC

(ITSM)X2,

AREGTYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGS

LIR1 ITIM4

DVVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEW

YLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIS

RVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKTERRAEVP

TAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVI

LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWR

SSPAADAQEEVTYAQLKHTQPEDGVEMDTRSPHDEDPQ

ATEYATIKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQ

MDTEAAASEAPQDTEYATIHSLTLRREATEPPPSQEGPSP

AVPSIYATLAIH

In some embodiments, the iCAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:305, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:327, SEQ ID NO:328, SEQ ID NO:329, SEQ ID NO:330, SEQ ID NO:331, SEQ ID NO:332, SEQ ID NO:333, and SEQ ID NO:334.

7. iCAR Portion/aCAR Portion: Linker

In some embodiments, the iCAR portion is covalently linked to the aCAR portion via a linker. In a certain embodiment, the linker is a gly-ser polypeptide linker, i.e., a peptide that consists of glycine and serine residues. Exemplary gly-ser polypeptide linkers comprise the amino acid sequence Ser(Gly₄Ser)_n, as well as (Gly₄Ser)_nand/or (Gly₄Ser₃)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3, i.e., Ser(Gly₄Ser)₃. In some embodiments, n=4, i.e., Ser(Gly₄Ser)₄. In some embodiments, n=5. In some embodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. In some embodiments, n=9. In some embodiments, n=10. Another exemplary gly-ser polypeptide linker comprises the amino acid sequence Ser(Gly₄Ser)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In another embodiment, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly₄Ser)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly₃Ser)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In another embodiment, n=5. In yet another embodiment, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly₄Ser₃)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly₃Ser)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In another embodiment, n=5. In yet another embodiment, n=6.

In some embodiments, the bicistronic construct comprises a linker that covalently connects the iCAR portion and the aCAR portion. In some embodiments, the bicistronic construct comprises a viral self-cleaving 2A peptide between the nucleic acid sequence encoding the iCAR portion and the nucleic acid sequence encoding the aCAR portion of the construct. In some embodiments, the viral self-cleaving 2A peptide includes T2A from Thosea asigna virus (TaV). In some embodiments, the iCAR portion is covalently linked to the aCAR portion via a linker. In some embodiments, the iCAR portion is covalently linked to the aCAR portion via a GSG. In some embodiments, the iCAR portion is covalently linked to the aCAR portion via a GGGGS linker (SEQ ID NO: 153). In some embodiments, the iCAR portion is covalently linked to the aCAR portion via a GGGGSGGGGSGGGGS linker (SEQ ID NO:154). In some embodiments, the iCAR is covalently linked to the aCAR portion via a T2A linker (SEQ ID NO: 155). In some embodiments, the iCAR is covalently linked to the aCAR portion via a F2A linker (SEQ ID NO: 156). In some embodiments, the iCAR is covalently linked to the aCAR portion via a P2A linker (SEQ ID NO:157). In some embodiments, the GAR is covalently linked to the aCAR portion via a E2A linker (SEQ ID NO:158). In some embodiments, the AR is covalently linked to the aCAR portion via a IRES long linker (SEQ ID NO:159). In some embodiments, the iCAR is covalently linked to the aCAR portion via a IRES short linker (SEQ ID NO: 160).

TABLE 13

iCAR portion/aCAR portion linker sequences

Sequence

Information
SEQ ID NO
Amino acid sequence

G₄S
153
GGGGS

(G₄S)X3
154
GGGGSGGGGSGGGGS

T2A
155
GSGEGRGSLLTCGDVEENPGP

F2A
156
GSGVKQTLNFDLLKLAGDVESNPGP

P2A
157
GSGATNFSLLKQAGDVEENPGP

E2A
158
GSGQCTNYALLKLAGDVESNPGP

IRES long
159
CCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTT

GGAATAAGGCCGGTGTGCGTTTGTCTATATGTTA

TTTTCCACCATATTGCCGTCTTTTGGCAATGTGAG

GGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGC

ATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAA

TGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGT

TCCTCTGGAAGCTTCTTGAAGACAAACAACGTCT

GTAGCGACCCTTTGCAGGCAGCGGAACCCCCCAC

CTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACG

TGTATAAGATACACCTGCAAAGGCGGCACAACCC

CAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAA

GAGTCAAATGGCTCTCCTCAAGCGTATTCAACAA

GGGGCTGAAGGATGCCCAGAAGGTACCCCATTGT

ATGGGATCTGATCTGGGGCCTCGGTGCACATGCT

TTACATGTGTTTAGTCGAGGTTAAAAAAACGTCT

AGGCCCCCCGAACCACGGGGACGTGGTTTTCCTT

TGAAAAACACGATGATAATATG

IRES short
160
CCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAA

TGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCT

TGAAGACAAACAACGTCTGTAGCGACCCTTTGCA

GGCAGCGGAACCCCCCACCTGGCGACAGGTGCCT

CTGCGGCCAAAAGCCACGTGTATAAGATACACCT

GCAAAGGCGGCACAACCCCAGTGCCACGTTGTG

AGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCT

CCTCAAGCGTATTCAACAAGGGGCTGAAGGATGC

CCAGAAGGTACCCCATTGTATGGGATCTGATCTG

GGGCCTCGGTGCACATGCTTTACATGTGTTTAGT

CGAGGTTAAAAAAACGTCTAGGCCCCCCGAACC

ACGGGGACGTGGTTTTCCTTTGAAAAACACGATG

ATAATATG

8. iCAR Portion/aCAR Portion: Signal Peptide

In some embodiments, the bicistronic construct comprises a signal peptide upstream of the iCAR and aCAR portions. In some embodiments, the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161). In some embodiments, the signal peptide is a GM-CSF signal peptide (SEQ ID NO: 162). In some embodiments, the signal peptide is a mIgK signal peptide (SEQ ID NO: 306).

TABLE 14

iCAR/aCAR signal peptide sequences

Sequence

Information
SEQ ID NO
Amino acid sequence

CD8 alpha
161
MALPVTALLLPLALLLHAARP

GM-CSF
162
MLLLVTSLLLCELPHPAFLLIP

mIgK
306
MSVPTQVLGLLLLWLTDARC

9. aCAR Portion: aCAR Scfv

In some embodiments, the bicistronic construct comprises an aCAR portion comprising a single chain variable fragment (scFv) component. In some embodiments, the iCAR portion comprises an scFv component. In some embodiments, the scFv targets Her2, Mesothelin, or EGFR. In some embodiments, the scFv targets Her2. In some embodiments, the scFv targets Mesothelin. In some embodiments, the scFv targets EGFR. In some embodiments, the scFv is an scFv based on trastuzumab (anti-Her2 antibody, also referred to as HERCEPTIN*), pertuzumab (anti-Her2 antibody, also referred to as PERJETA*), another commercial anti-Her2 antibody including, but not limited to, FRP5, A21, XMT1517, XMT1518, XMT1519, FWP51, bioequivalents thereof, or biosimilars thereof. In some embodiments, the scFv has the VH and VL domains of trastuzumab, pertuzumab, FRP5, A21, XMT1517, XMT1518, XMT1519, FWP51, bioequivalents thereof, or biosimilars thereof. In some embodiments, the scFv is an scFv based on cetuximab (anti-EGFR antibody, also referred to as ERBITUX*), panitumumab (anti-EGFR antibody, also referred to as VECTIBIX*), another commercial anti-EGFR antibody including, but not limited to, Imgatuzumab, Nimotuzumab, Necitumumab, ICR62, Matuzumab, C10, Zalutumumab, PIX, P2X, P3X, EGFR-1a1-VHH, bioequivalents thereof, or biosimilars thereof. In some embodiments, the scFv has the VH and VL domains of cetuximab, panitumumab, Imgatuzumab, Nimotuzumab, Necitumumab, ICR62, Matuzumab, C10, Zalutumumab, PIX, P2X, P3X, EGFR-1a1-VHH, bioequivalents thereof, or biosimilars thereof. In some embodiments, the scFv is an scFv based on a commercial anti-Mesothelin antibody including, but not limited to, Amatuximab, P4, SS1, SD1, SD2, 1H7, 3C02, bioequivalents thereof, or biosimilars thereof. In some embodiments, the scFv has the VH and VL domains of Amatuximab, P4, SS1, SD1, SD2, 1H7, 3C02, bioequivalents thereof, or biosimilars thereof.

In some embodiments, the scFv targets Her2. In some embodiments, the Her2 scFv is based on the Vh and Vl from trastuzumab or pertuzumab. In some embodiments, the Her2 scFv is based on the Vh and Vl from trastuzumab. In some embodiments, the Her2 scFv is based on the Vh and Vl from pertuzumab. The Vh and VI chains for trastuzumab and pertuzumab are provided below in Tables 15 and 16. In some embodiments, the Her2 scFv is based on the Vh and Vl from FRP5. In some embodiments, the Her2 scFv is based on the Vh and Vl from A21. In some embodiments, the Her2 scFv is based on the Vh and Vl from XMT1517. In some embodiments, the Her2 scFv is based on the Vh and Vl from XMT1518. In some embodiments, the Her2 scFv is based on the Vh and Vl from XMT1519. In some embodiments, the Her2 scFv is based on the Vh and Vl from FWP51. In some embodiments, the Her2 scFv is based on the Vh and Vl from trastuzumab F9G.

TABLE 15

anti-Her2 sequences

SEQ

Sequence
ID

Information
NO
Amino acid sequence

trastuzumab
170
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP

Variable heavy

GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQ

chain

MNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

trastuzumab
171
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPG

Variable light

KAPKLLIYSASFLYSGVPSRESGSRSGTDFTLTISSLQPEDFAT

chain

YYCQQHYTTPPTFGQGTKVEIK

trastuzumab
172
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP

scFv

GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQ

MNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

GSTSGSGKPGSGEGSTKGDIQMTQSPSSLSASVGDRVTITC

RASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSG

SRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK

Trastuzumab
307
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPG

F9G variable

KAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFAT

Heavy chain

YYCQQHYTTPPTFGQGTKVEIK

Trastuzumab
308
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP

F9G variable

GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQ

Light chain

MNSLRAEDTAVYYCSRWGGDGGYAMDYWGQGTLVTVSS

pertuzumab
173
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQA

Variable heavy

PGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYL

chain

QMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS

pertuzumab
174
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPG

Variable light

KAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFA

chain

TYYCQQYYIYPYTFGQGTKVEIK

pertuzumab scFv
175
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPG

KAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFA

TYYCQQYYIYPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKG

EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQA

PGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYL

QMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS

FRP5 variable
176
QVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQA

heavy vhain

PGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYL

QINNLKSEDMATYFCARWEVYHGYVPYWGQGTTVTVSS

FRP5 variable
177
DIQLTQSHKFLSTSVGDRVSITCKASQDVYNAVAWYQQKPG

light vhain

QSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLA

VYFCQQHFRTPFTFGSGTKLEIK

A21 variable
178
EVQLQQSGPEVVKTGASVKISCKASGYSFTGYFINWVKKNS

heavy vhain

GKSPEWIGHISSSYATSTYNQKFKNKAAFTVDTSSSTAFMQL

NSLTSEDSAVYYCVRSGNYEEYAMDYWGQGTSVTVSS

A21 variable
179
DIVLTQTPSSLPVSVGEKVTMTCKSSQTLLYSNNQKNYLAW

light vhain

YQQKPGQSPKLLISWAFTRKSGVPDRFTGSGSGTDFTLTIGS

VKAEDLAVYYCQQYSNYPWTFGGGTKLEIK

XMT1517
180
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQA

variable heavy

PGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLY

vhain

LQMNSLRAEDTAVYYCAKEAPYYAKDYMDVWGKGTTVT

VSS

XMT1517
181
EIVLTQSPGTLSLSPGERATLSCRASQSVSSDYLAWYQQKPG

variable light

QAPRLLIYGASSRATGIPDRESGSGSGTDFTLTISRLEPEDFA

vhain

VYYCQQYVSYWTFGGGTKVEIK

XMT1518
182
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQA

variable heavy

PGKGLEWVAGIWWDGSNEKYADSVKGRFTISRDNSKNTLY

vhain

LQMNSLRAEDTAVYYCAKEAPYYAKDYMDVWGKGTTVT

VSS

XMT1518
183
EIVLTQSPGTLSLSPGERATLSCRASQSVSSDYLAWYQQKPG

variable light

QAPRLLIYGASRRATGIPDRESGSGSGTDFTLTISRLEPEDFA

vhain

VYYCQQYVSYWTFGGGTKVEIK

XMT1519
184
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQA

variable heavy

PGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQ

vhain

MNSLRAEDTAVYYCARGGHGYFDLWGRGTLVTVSS

XMT1519
185
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPG

variable light

QAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA

vhain

VYYCQQYHHSPLTFGGGTKVEIK

FWP51 variable
186
QVQLQQSGAELVRPGTSVKLSCKASDYTFTSYWMNWVKQ

heavy vhain

RPGQGLEWIGMIDPSDSETQYNQMFKDKAALTVDKSSNTA

YMQLSSLTSEDSAVYYCAKGGASGDWYFDVWGQGTTVT

FWP51 variable
187
DIQLTQSPSSLSASLGGEVTITCKASQDIKKYIAWYQHKPGK

light vhain

SPRLLIHYTSVLQPGIPSRESGSGSGRDYSFSIHNLEPEDIATY

YCLHYDYLYTFGGGTKLEI

FWP51 VL VH
188
MLLLVTSLLLCELPHPAFLLIPDYKDDDDKQVQLQQSGAEL

VRPGTSVKLSCKASDYTFTSYWMNWVKQRPGQGLEWIGMI

DPSDSETQYNQMFKDKAALTVDKSSNTAYMQLSSLTSEDS

AVYYCAKGGASGDWYFDVWGQGTTVTGSTSGSGKPGSGE

GSTKGDIQLTQSPSSLSASLGGEVTITCKASQDIKKYIAWYQ

HKPGKSPRLLIHYTSVLQPGIPSRFSGSGSGRDYSFSIHNLEPE

DIATYYCLHYDYLYTFGGGTKLEI

Anti HER2 VHH
309
QVQLVQSGGGLVQAGGSLRLSCAASGRTESSYAMAWFRQA

PGKEREFVAAISWSGANIYVADSVKGRFTISRDNAKDTVYL

QMNSLKPEDTAVYYCAVKLGFAPVEERQYDYWGQGTQVT

VSS

In some embodiments, the scFv targets EGFR. In some embodiments, the EGFR scFv is based on the Vh and Vl from cetuximab, panitumumab, Imgatuzumab, Nimotuzumab, Necitumumab, ICR62, Matuzumab, C10, Zalutumumab, P1X, P2X, P3X, or EGFR-1a1-VHH. In some embodiments, the EGFR scFv is based on the Vh and Vl from cetuximab. In some embodiments, the EGFR scFv is based on the Vh and Vl from panitumumab. In some embodiments, the EGFR scFv is based on the Vh and Vl from Imgatuzumab. In some embodiments, the EGFR scFv is based on the Vh and Vl from Nimotuzumab. In some embodiments, the EGFR scFv is based on the Vh and Vl from Nimotuzumab (K5). In some embodiments, the EGFR scFv is based on the Vh and VI from Necitumumab. In some embodiments, the EGFR scFv is based on the Vh and VI from ICR62. In some embodiments, the EGFR scFv is based on the Vh and Vl from Matuzumab. In some embodiments, the EGFR scFv is based on the Vh and Vl from C10. In some embodiments, the EGFR scFv is based on the Vh and Vl from Zalutumumab. In some embodiments, the EGFR scFv is based on the Vh and Vl from PIX. In some embodiments, the EGFR scFv is based on the Vh and Vl from P2X. In some embodiments, the EGFR scFv is based on the Vh and Vl from P3X. In some embodiments, the EGFR scFv is based on EGFR-1a1-VHH. In some embodiments, the EGFR scFv is based on EGFR-VHH.

TABLE 16

anti-EGFR sequences

Sequence
SEQ

Information
ID NO
Amino acid sequence

cetuximab
189
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPG

Variable heavy

KGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMN

chain

SLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVS

cetuximab
190
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSP

Variable light

RLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQ

chain

QNNNWPTTFGAGTKLELK

cetuximab scFv
191
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPG

(SEQ ID NO:)

KGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMN

SLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSGSTSGS

GKPGSGEGSTKGDILLTQSPVILSVSPGERVSFSCRASQSIGTN

IHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINS

VESEDIADYYCQQNNNWPTTFGAGTKLELK

panitumumab
192
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQS

Variable heavy

PGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSS

chain

VTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS

panitumumab
193
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGK

Variable light

APKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATY

chain

FCQHFDHLPLAFGGGTKVEIK

panitumumab
194
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGK

scFv

APKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATY

FCQHFDHLPLAFGGGTKVEIKGSTSGSGKPGSGEGSTKGQVQ

LQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGK

GLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTA

ADTAIYYCVRDRVTGAFDIWGQGTMVTVSS

Imgatuzuma
195
QVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYKIHWVRQAP

variable heavy

GQGLEWMGYFNPNSGYSTYAQKFQGRVTITADKSTSTAYM

chain

ELSSLRSEDTAVYYCARLSPGGYYVMDAWGQGTTVTVSS

Imgatuzumab
196
DIQMTQSPSSLSASVGDRVTITCRASQGINNYLNWYQQKPGK

variable light

APKRLIYNTNNLQTGVPSRFSGSGSGTEFTLTISSLQPEDFATY

chain

YCLQHNSFPTFGQGTKLEIK

Nimotuzumab
197
QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAP

variable heavy

GQGLEWIGGINPTSGGSNFNEKFKTRVTITVDESTNTAYMEL

chain

SSLRSEDTAFYFCARQGLWFDSDGRGFDFWGQGSTVTVSS

Nimotuzumab
198
DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQ

variable light

QTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPE

chain

DIATYYCFQYSHVPWTFGQGTKLQIT

Nimotuzumab
310
DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQ

(K5) variable

QTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPE

light chain

DIATYYCFQYSHVPWTFGQGTKLQIT

Nimotuzumab
311
QVQLQQSGAEVKKPGSSVKVSCKASGYTFTDYYIYWVRQAP

(K5) variable

GQGLEWIGGINPVTQRPVFNEKFKTRVTITVDESTNTAYMEL

Heavy chain

SSLRSEDTAFYFCARQGLWFDSDGRGFDFWGQGSTVTVSS

Necitumumab
199
QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQP

variable heavy

PGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKV

chain

NSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS

Necitumumab
200
EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQ

variable light

APRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVY

chain

YCHQYGSTPLTFGGGTKAEIK

ICR62 variable
201
QVNLLQSGAALVKPGASVKLSCKGSGFTFTDYKIHWVKQSH

heavy chain

GKSLEWIGYFNPNSGYSTYNEKFKSKATLTADKSTDTAYME

LTSLTSEDSATYYCTRLSPGGYYVMDAWGQGASVTVSS

ICR62 variable
202
DIQMTQSPSFLSASVGDRVTINCKASQNINNYLNWYQQKLG

light chain

EAPKRLIYNTNNLQTGIPSRFSGSGSGTDYTLTISSLQPEDFAT

YFCLQHNSFPTFGAGTKLELK

ICR62 VL VH
203
MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSFLSASVGDRVTIN

CKASQNINNYLNWYQQKLGEAPKRLIYNTNNLQTGIPSRFSG

SGSGTDYTLTISSLQPEDFATYFCLQHNSFPTFGAGTKLELKG

STSGSGKPGSGEGSTKGQVNLLQSGAALVKPGASVKLSCKG

SGFTFTDYKIHWVKQSHGKSLEWIGYFNPNSGYSTYNEKFKS

KATLTADKSTDTAYMELTSLTSEDSATYYCTRLSPGGYYVM

DAWGQGASVTVSS

Matuzumab
204
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQ

variable heavy

APGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAY

chain

MELSSLRSEDTAVYYCASRDYDYDGRYFDYWGQGTLVTVS

S

Matuzumab
205
DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGK

variable light

APKLLIYDTSNLASGVPSRESGSGSGTDYTFTISSLQPEDIATY

chain

YCQQWSSHIFTFGQGTKVEIK

C10 variable
206
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIGWVRQAP

heavy chain

GQGLEWMGGIIPIFGIANYAQKFQGRVTITADESTSSAYMELS

SLRSEDTAVYYCAREEGPYCSSTSCYAAFDIWGQGTLVTLSS

C10 variable
207
QSVLTQDPAVSVALGQTVKITCQGDSLRSYFASWYQQKPGQ

light chain

APTLVMYARNDRPAGVPDRESGSKSGTSASLSAISGLQPEDE

AYYCAAWDDSLNGYLFGAGTKLTVL

Zalutumumab
208
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQA

variable heavy

PGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYL

chain

QMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTL

VTVSS

Zalutumumab
209
AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGK

variable light

APKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATY

chain

YCQQFNSYPLTFGGGTKVEIK

P1X variable
210
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP

heavy chain

GQGLEWMGSIIPIFGTVNYAQKFQGRVTITADESTSTAYMEL

SSLRSEDTAVYYCARDPSVNLYWYFDLWGRGTLVTVSS

P1X variable
211
DIQMTQSPSTLSASVGDRVTITCRASQSISSWWAWYQQKPG

light chain

KAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFAT

YYCQQYHAHPTTFGGGTKVEIK

P2X variable
212
QVQLVQSGAEVKKPGSSVKVSCKASGGTFGSYAISWVRQAP

heavy chain

GQGLEWMGSIIPIFGAANPAQKSQGRVTITADESTSTAYMEL

SSLRSEDTAVYYCAKMGRGKVAFDIWGQGTMVTVSS

P2X variable
213
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSPNNKNYLAWY

light chain

QQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQ

AEDVAVYYCQQYYGSPITFGGGTKVEIK

P3X variable
214
QVQLVQSGAEVKKPGASVKVSCKASGYAFTSYGINWVRQA

heavy chain

PGQGLEWMGWISAYNGNTYYAQKLRGRVTMTTDTSTSTAY

MELRSLRSDDTAVYYCARDLGGYGSGSVPFDPWGQGTLVT

VSS

P3X variable
215
EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQ

light chain

APRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVY

YCQDYRTWPRRVFGGGTKVEIK

EGFR-la1-
216
QVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAP

VHH variable

GKEREFVAAINWSGGSTSYADSVKGRFTISRDNTKNTVYLQ

heavy chain

MNSLKPEDTAAFYCAATYNPYSRDHYFPRMTTEYDYWGQG

TQVTVSS

EGFR-VHH
312
EVQQASGGGLVQAGGSLRLSCAASGRTETTSAIAWFRQAPG

variable heavy

KEREFVAQISASGLGINYSGTVKGRFTISRDADKTTVYLQMN

chain

SLTPEDTAVYYCAAGFHYIAAIRRTTDFHFWGPGTLVTVSS

In some embodiments, the scFv targets Mesothelin. In some embodiments, the Mesothelin scFv is based on the Vh and VI from Amatuximab, P4, SS1, SD1, SD2, 1H7, or 3C02. In some embodiments, the Mesothelin scFv is based on the Vh and VI from Amatuximab. In some embodiments, the Mesothelin scFv is based on the Vh and VI from P4. In some embodiments, the Mesothelin scFv is based on the Vh and VI from SS1. In some embodiments, the Mesothelin scFv is based on SD1. In some embodiments, the Mesothelin scFv is based on SD2. In some embodiments, the Mesothelin scFv is based on the Vh and VI from 1H7. In some embodiments, the Mesothelin scFv is based on the Vh and VI from 3C02.

TABLE 17

anti-Mesothelin sequences

Sequence
SEQ

Information
ID NO
Amino acid sequence

Amatuximab
217
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWV

variable heavy

RQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTS

chain

TSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGT

LVTVSS

Amatuximab
218
DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKS

variable light

GKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPE

chain

DFATYYCQQWSKHPLTFGQGTKLEIK

P4 variable
219
QVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIR

heavy chain

QSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTS

KNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQ

GTTVTVSS

P4 variable light
220
QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQ

chain

KPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAG

VLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVL

P4 VL VH
221
MLLLVTSLLLCELPHPAFLLIPQPVLTQSSSLSASPGASASL

TCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQ

QGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMI

WHSSAAVFGGGTQLTVLGSTSGSGKPGSGEGSTKGQVQL

QQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPS

RGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQF

SLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTV

TVSS

SS1 variable
222
QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVK

heavy chain

QSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSST

AYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVT

VSS

SS1 variable
223
DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKS

light chain

GTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAE

DDATYYCQQWSKHPLTFGSGTKVEIK

SS1 VL VH
224
MLLLVTSLLLCELPHPDIELTQSPAIMSASPGEKVTMTCSA

SSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRESGS

GSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKV

EIKGSTSGSGKPGSGEGSTKGQVQLQQSGPELEKPGASVK

ISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGAS

SYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCA

RGGYDGRGFDYWGSGTPVTVSS

SD1 VHH
225
QVQLVQSGGGLVQPGGSLRLSCAASDFDFAAYEMSWVR

QSAPGQGLEWVAIISHDGIDKYYTDSVKGRFTISRDNSKN

TLYLQMNTLRAEDTATYYCLRLGAVGQGTLVTVSSS

SD2 VHH
226
QVQLVQSGGGLVQPGGSLRLSCAASDFAFDDYEMSWVR

QAPGKALEWIGDINHSGTTIYNPSLKSRVTISRDNSKNTL

YLQMNTLRAEDTAIYYCARPHYGDYSDAFDIWGQGTMV

TVSS

1H7 variable
227
EVQLQQSGTVLARPGASVKMSCKASGYSFTNYRMNWV

heavy chain

KQRPGQGLEWIGGIYPGNRDTTYNQKFKDKAKLTAVTSA

NTAYMELSSLTNEDSAVYYCTRGVIGIYFDYWGQGTTLT

VSS

1H7 variable
228
DIVMTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNW

light chain

YQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIH

PVEEEDAATYYCQQNNEAPLTFGAGTKLELK

1H7 VL VH
229
MLLLVTSLLLCELPHPAFLLIPDIVMTQSPASLAVSLGQRA

TISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNL

ESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQNNEA

PLTFGAGTKLELKGSTSGSGKPGSGEGSTKGEVQLQQSGT

VLARPGASVKMSCKASGYSFTNYRMNWVKQRPGQGLE

WIGGIYPGNRDTTYNQKFKDKAKLTAVTSANTAYMELSS

LTNEDSAVYYCTRGVIGIYFDYWGQGTTLTVSS

3C02 variable
230
QVQLQQSGTVLARPGASVKMSCKASGYSFTNYRMYWV

heavy chain

KQRPGQGLEWIGAIYPGNSDTTYKQKFKGKAKLTAVTSA

STAYMELSSLTNEDSAVYYCTRGIRGSYFDVWGAGTTVT

VSS

3C02 variable
231
DIVMTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNW

light chain

YQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIH

PVEEEDAATYYCQQSNEDPYTFGGGTKLEIK

3C02 VL VH
232
MLLLVTSLLLCELPHPAFLLIPDIVMTQSPASLAVSLGQRA

TISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNL

ESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDP

YTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGT

VLARPGASVKMSCKASGYSFTNYRMYWVKQRPGQGLE

WIGAIYPGNSDTTYKQKFKGKAKLTAVTSASTAYMELSS

LTNEDSAVYYCTRGIRGSYFDVWGAGTTVTVSS

M1 variable
313
EIVLTQSPATLSLSPGERATISCRASQSVSSNFAWYQQRPG

Ligh Chain

QAPRLLIYDASNRATGIPPRFSGSGSGTDFTLTISSLEPEDF

AAYYCHQRSNWLYTFGQGTKVDIK

M1 variable
314
QVQLQQSGAEVKKPGASVKVSCKASGYTFTGYYMHWV

Heavy Chain

RQAPGQGLEWMGRINPNSGGTNYAQKFQGRVTMTRDTS

ISTAYMELSRLRSEDTAVYYCARGRYYGMDVWGQGTM

VTVSS

M5 Variable
315
DIVMTQSPSSLSASVGDRVTITCRASQSIRYYLSWYQQKP

Light chain

GKAPKLLIYTASILQNGVPSRESGSGSGTDFTLTISSLQPED

FATYYCLQTYTTPDFGPGTKVEIK

M5 Variable
316
QVQLVQSGAEVEKPGASVKVSCKASGYTFTDYYMHWV

Heavy chain

RQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDT

SISTAYMELSRLRSDDTAVYYCASGWDFDYWGQGTLVT

VSS

VD9.V3
317
DIQMTQSPSSLSASVGDRVTITCKSSQSVLYSSNQKNYLA

Variable light

WFQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLT

chain

ISSLQPEDFATYFCHQYLSSYTFGQGTKVEIK

VD9.V3
318
EVQLVESGGGLVQPGGSLRLSCAASGYTFTTYWMHWVR

Variable Heavy

QAPGKGLEWVGYIRPSTGYTEYNQKFKDRFTISADTSKN

chain

TAYLQMNSLRAEDTAVYYCARSRWLLDYWGQGTLVTV

SS

In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH.

In some embodiments, the aCAR scFv comprises a linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a GS based linker sequence, connecting the VH and VL to form the scFv. In some embodiments, the GS linker comprises GGGGS (SEQ ID NO:81). In some embodiments, the aCAR comprises a Whitlow linker sequence, e.g., GSTSGSGKPGSGEGSTKG (SEQ ID NO:82).

10. aCAR Portion: Hinge and Transmembrane Domain

In some embodiments, the bicistronic construct comprises an aCAR portion comprising a hinge transmembrane (TM) domain component. In some embodiments, the aCAR portion comprises a hinge TM domain. In some embodiments, the hinge TM domain comprises a hinge TM domain selected from the group consisting of a CD28 hinge TM domain and a CD8 hinge TM domain (including a CD8a hinge TM domain). In some embodiments, the hinge TM domain is a CD28 hinge TM domain. In some embodiments, the vector comprises a CD8 hinge TM domain. In some embodiments, the vector comprises a CD8a hinge TM domain. In some embodiments, the hinge domain comprises a hinge domain selected from the group consisting of a CD28 hinge domain and a CD8 hinge domain (including a CD8a hinge domain). In some embodiments, the hinge domain is a CD28 hinge domain. In some embodiments, the vector comprises a CD8 hinge domain. In some embodiments, the vector comprises a CD8a hinge domain. In some embodiments, the TM domain comprises a TM domain selected from the group consisting of a CD28 TM domain and a CD8 TM domain (including a CD8a TM domain). In some embodiments, the TM domain is a CD28 TM domain. In some embodiments, the vector comprises a CD8 TM domain. In some embodiments, the vector comprises a CD8a TM domain. In some embodiments, the hinge domain is a CD28 hinge domain of SEQ ID NO:85. In some embodiments, the vector comprises a CD8a hinge domain of SEQ ID NO:84. In some embodiments, the TM domain is a CD28 TM domain of SEQ ID NO:319. In some embodiments, the vector comprises a CD8a TM domain of SEQ ID NO:320.

TABLE 18

aCAR hinge and TM domain sequences

Sequence
SEQ ID

Information
NO
Amino acid sequence

CD28 hinge
85
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSP

LFPGPSKP

CD28 TM
319
FWVLVVVGGVLACYSLLVTVAFIIFWV

CD8alpha hinge
84
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAG

GAVHTRGLDFACD

CD8alpha TM
320
IYIWAPLAGTCGVLLLSLVITLYC

11. aCAR Portion: Co-Stimulatory and Activation Signaling Domain

In some embodiments, the bicistronic construct comprises an aCAR portion comprising co-stimulatory domain component. In some embodiments, the aCAR portion comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain is selected from the group consisting of CD137 (4-1BB) or CD28 or both 4-1BB and CD28 (28BB). In some embodiments, the co-stimulatory domain is a CD137 (4-1BB) co-stimulatory domain. In some embodiments, the co-stimulatory domain is a CD28 co-stimulatory domain. In some embodiments, the activation signaling domain is CD3z domain. In some embodiments, the co-stimulatory domain is a 28BB co-stimulatory domain. In some embodiments, the co-stimulatory domain is 4-1BB (SEQ ID NO:233). In some embodiments, the co-stimulatory domain is CD28 (SEQ ID NO:234). In some embodiments, the activation signaling domain is CD3z (SEQ ID NO:235).

TABLE 19

aCAR co-stimulatory and activation signaling domain sequences

Sequence
SEQ

Information
ID NO
Amino acid sequence

4-1BB costim
233
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG

GCEL

CD28 costim
234
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA

AY

CD3z activation
235
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD

signaling

KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS

EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA

LPPR

12. aCAR Portion: Immunoreceptor Tyrosine-Based Activation Motif (ITAM)

In some embodiments, the aCAR portion comprises an Immunoreceptor Tyrosine-Based Activation Motif (ITAM). In some embodiments, the ITAM is a CD3 zeta domain. In some embodiments, the ITAM is a CD3 zeta domain of SEQ ID NO:236. In some embodiments, the ITAM is a CD3 zeta 3F domain of SEQ ID NO:237. In some embodiments, the ITAM is a CD3 zeta 4F domain of SEQ ID NO:238. In some embodiments, the ITAM is a CD3 zeta 40F domain of SEQ ID NO:239.

TABLE 20

aCAR ITAM domain sequences

Sequence
SEQ

Information
ID NO
Amino acid sequence

CD3 zeta domain
236
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD

KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY

SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ

ALPPR

CD3 Zeta 3F
237
RVKFSRSADAPAYQQGQNQLFNELNLGRREEYDVLD

KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAFS

EIGMKGERRRGKGHDGLFQGLSTATKDTYDALHMQA

LPPR

CD3 Zeta 4F
238
RVKFSRSADAPAYQQGQNQLFNELNLGRREEFDVLD

KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAFS

EIGMKGERRRGKGHDGLFQGLSTATKDTYDALHMQA

LPPR

CD3 Zeta 4OF
239
RVKFSRSADAPAYQQGQNQLFNELNLGRREEFDVLD

KRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAYS

EIGMKGERRRGKGHDGLYQGLSTATKDTFDALHMQA

LPPR

13. Exemplary aCARs

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of trastuzumab (SEQ ID NOs: 170 and 171). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z activation signaling domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of trastuzumab F9G (SEQ ID NOs: 307 and 308). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z activation signaling domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of pertuzumab (SEQ ID NOs: 173 and 174). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of FRP5 (SEQ ID NOs: 176 and 177). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of A21 (SEQ ID NOs: 178 and 179). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of XMT1517 (SEQ ID NOs: 180 and 181). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of XMT1518 (SEQ ID NOs: 182 and 183). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of XMT1519 (SEQ ID NOs: 184 and 185). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of FWP51 (SEQ ID NOs: 186 and 187). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the anti-HER2 VHH (SEQ ID NO: 309). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of Cetuximab (SEQ ID NOs: 189 and 190). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of Panitumumab (SEQ ID NOs: 192 and 193). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of Imgatuzumab (SEQ ID NOs: 195 and 196). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of Nimotuzumab (SEQ ID NOs: 197 and 198). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of Nimotuzumab (K5) (SEQ ID NOs: 310 and 311). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of Necitumumab (SEQ ID NOs: 199 and 200). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of ICR62 (SEQ ID NOs: 201 and 202). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of Matuzumab (SEQ ID NOs: 204 and 205). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of C10 (SEQ ID NOs: 206 and 207). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of Zalutumumab (SEQ ID NOs: 208 and 209). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of P1X (SEQ ID NOs: 210 and 211). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of P2X (SEQ ID NOs: 212 and 213). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of P3X (SEQ ID NOs: 214 and 215). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VHH sequence of EGFR-1a1-VHH (SEQ ID NO: 216). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VHH sequence of EGFR-VHH (SEQ ID NO: 312). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of Amatuximab (SEQ ID NOs: 217 and 218). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of P4 (SEQ ID NOs: 219 and 220). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of SS1 (SEQ ID NOs: 222 and 223). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VHH sequence of SD1 (SEQ ID NO: 225). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VHH sequence of SD2 (SEQ ID NO: 226). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of 1H7 (SEQ ID NOs: 227 and 228). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR comprises an scFv component comprising the VL and VH sequences of 3C02 (SEQ ID NOs: 230 and 231). In some embodiments, the orientation of the aCAR VH and VL regions is VH-VL. In some embodiments, the orientation of the aCAR VH and VL regions is VL-VH. In some embodiments, the aCAR scFv comprises a (G4S)X3 linker (SEQ ID NO:81) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR scFv comprises a Whitlow linker (SEQ ID NO:82) linker that covalently connects the VH and the VL to form the aCAR scFv. In some embodiments, the aCAR comprises a CD8 alpha hinge TM domain (SEQ ID NO:84). In some embodiments, the aCAR comprises a CD28 hinge TM domain (SEQ ID NO:85). In some embodiments, the aCAR comprises a 4-1BB costimulatory domain (SEQ ID NO: 233). In some embodiments, the aCAR comprises a CD28 costimulatory domain (SEQ ID NO: 234). In some embodiments, the aCAR comprises a CD3z costimulatory domain (SEQ ID NO: 235). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta domain (SEQ ID NO: 236). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 3F domain (SEQ ID NO: 237). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 4F domain (SEQ ID NO: 238). In some embodiments, the aCAR comprises an ITAM comprising a CD3 zeta 40F domain (SEQ ID NO: 239). In some embodiments, the aCAR comprises a signal peptide upstream of the aCAR portion, wherein the signal peptide is a CD8 alpha signal peptide (SEQ ID NO: 161), a GM-CSF signal peptide (SEQ ID NO: 162), or a mIgK signal peptide (SEQ ID NO: 306).

In some embodiments, the aCAR has a set of components shown in Table 21.

TABLE 21

aCAR constructs

Signal

scFv

Co-

Construct
Peptide
scFv
Linker
Hinge
TM
stimulatory
Signaling

Anti-EGFR

MC0001
CD8
Imgatuzumab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR1)
alpha
VL_VH

alpha
alpha

MC0002
CD8
Cextuximab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR2)
alpha
VL_VH

alpha
alpha

MC0003
CD8
Panitumumab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR3)
alpha
VL_VH

alpha
alpha

MC0004
CD8
Nimotuzumab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR4)
alpha
VL_VH

alpha
alpha

MC0005
CD8
Necitumumab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR5)
alpha
VL_VH

alpha
alpha

MC0163
GM-
ICR62 VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR163)
CSF
VL

alpha
alpha

MC0164
GM-
ICR62 VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR164)
CSF
VH

alpha
alpha

MC0165
GM-
Matuzumab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR165)
CSF
VH VL BBz

alpha
alpha

MC0166
GM-
Matuzumab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR166)
CSF
VL VH BBz

alpha
alpha

MC0167
GM-
C10 VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR167)
CSF
BBz

alpha
alpha

MC0168
GM-
C10 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR168)
CSF
BBz

alpha
alpha

MC0169
GM-
Zalutumumab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR169)
CSF
VH VL BBz

alpha
alpha

MC0170
GM-
Zalutumumab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR170)
CSF
VL VH BBz

alpha
alpha

MC0171
GM-
P1X VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR171)
CSF
BBz

alpha
alpha

MC0172
GM-
P1X VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR172)
CSF
BBz

alpha
alpha

MC0173
GM-
P2X VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR173)
CSF
BBz

alpha
alpha

MC0174
GM-
P2X VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR174)
CSF
BBz

alpha
alpha

MC0175
GM-
P3X VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR175)
CSF
BBz

alpha
alpha

MC0176
GM-
P3X VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR176)
CSF
BBz

alpha
alpha

MC0177
GM-
EGFR-la1-
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR177)
CSF
VHH BBz

alpha
alpha

N/A
CD8
ICR62
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
VH_VL

alpha
alpha

N/A
CD8
ICR62
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
VL_VH

alpha
alpha

N/A
CD8
Matuzumab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
VH_VL

alpha
alpha

N/A
CD8
Matuzumab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
VL_VH

alpha
alpha

N/A
CD8
C10 VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
BBz

alpha
alpha

N/A
CD8
C10 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
BBz

alpha
alpha

N/A
CD8
Zalutumumab
whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
VH VL

alpha
alpha

MC0483
CD8
Zalutumumab
whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR483)
alpha
VL_VH

alpha
alpha

N/A
CD8
P1X VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
BBz

alpha
alpha

N/A
CD8
P1X VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
BBz

alpha
alpha

N/A
CD8
P2X VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
BBz

alpha
alpha

N/A
CD8
P2X VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
BBz

alpha
alpha

N/A
CD8
P3X VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
BBz

alpha
alpha

N/A
CD8
P3X VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
BBz

alpha
alpha

MC0484
CD8
EGFR-l1a-
whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR484)
alpha
VHH

alpha
alpha

Anti-HER2

MC0006
CD8
Trastuzumab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR6)
alpha
VL_VH

alpha
alpha

MC0007
CD8
Pertuzumab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR7)
alpha
VL_VH

alpha
alpha

MC0008
CD8
FRP5 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR8)
alpha

alpha
alpha

MC0009
CD8
A21 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR9)
alpha

alpha
alpha

MC0178
GM-
XMT1517
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR178)
CSF
VH VL

alpha
alpha

MC0179
GM-
XMT1517
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR179)
CSF
VL VH

alpha
alpha

MC0180
GM-
XMT1518
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR180)
CSF
VH VL

alpha
alpha

MC0181
GM-
XMT1518
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR181)
CSF
VL VH

alpha
alpha

MC0182
GM-
XMT1519
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR182)
CSF
VH VL

alpha
alpha

MC0183
GM-
XMT1519
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR183)
CSF
VL VH

alpha
alpha

MC0184
GM-
FWP51
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR184)
CSF
VH VL

alpha
alpha

MC0185
GM-
FWP51
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR185)
CSF
VL VH

alpha
alpha

N/A
GM-
Trastuzumab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

CSF
VL_VH

alpha
alpha

N/A
GM-
Pertuzumab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

CSF
VL_VH

alpha
alpha

N/A
GM-
FRP5 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

CSF

alpha
alpha

N/A
GM-
A21 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

CSF

alpha
alpha

N/A
CD8
XMT1517
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
VH VL

alpha
alpha

N/A
CD8
XMT1517
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
VL VH

alpha
alpha

N/A
CD8
XMT1518
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
VH VL

alpha
alpha

N/A
CD8
XMT1518
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
VL VH

alpha
alpha

N/A
CD8
XMT1519
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
VH VL

alpha
alpha

N/A
CD8
XMT1519
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
VL VH

alpha
alpha

N/A
CD8
FWP51
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
VH VL

alpha
alpha

N/A
CD8
FWP51
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
VL VH

alpha
alpha

Anti-Mesothelin

MC0159
GM-
Amatuximab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR159)
CSF
VH VL

alpha
alpha

MC0160
GM-
Amatuximab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR160)
CSF
VL VL

alpha
alpha

MC0161
GM-
P4 VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR161)
CSF

alpha
alpha

MC0162
GM-
P4 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR162)
CSF

alpha
alpha

MC0186
GM-
SS1 VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR186)
CSF

alpha
alpha

MC0187
GM-
SS1 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR187)
CSF

alpha
alpha

MC0188
GM-
SD1 VHH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR188)
CSF

alpha
alpha

MC0189
GM-
SD2 VHH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR189)
CSF

alpha
alpha

MC0190
GM-
1H07 VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR190)
CSF

alpha
alpha

MC0191
GM-
1H07 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR191)
CSF

alpha
alpha

MC0192
GM-
3C02 VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR192)
CSF

alpha
alpha

MC0193
GM-
3C02 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR193)
CSF

alpha
alpha

N/A
CD8
Amatuximab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha
VH VL

alpha
alpha

MC0485
CD8
Amatuximab
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR485)
alpha
VL VH

alpha
alpha

N/A
CD8
P4 VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha

alpha
alpha

MC0487
CD8
P4 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR487)
alpha

alpha
alpha

N/A
CD8
SS1 VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha

alpha
alpha

MC0488
CD8
SS1 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR488)
alpha

alpha
alpha

N/A
CD8
SD1 VHH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha

alpha
alpha

N/A
CD8
SD2 VHH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha

alpha
alpha

N/A
CD8
1H07 VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha

alpha
alpha

MC0490
CD8
1H07 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR490)
alpha

alpha
alpha

N/A
CD8
3C02 VH VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha

alpha
alpha

N/A
CD8
3C02 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

alpha

alpha
alpha

MC0486
CD8
M1 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR486)
alpha

alpha
alpha

MC0498
CD8
M5 VL VH
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR498)
alpha

alpha
alpha

MC0489
CD8
7D9.V3 VL
Whitlow
CD8
CD8
4-1BB
CD3 zeta

(VR489)
alpha
VH

alpha
alpha

14. Optional shRNA

In some embodiments, the bicistronic construct comprises an optional short hairpin RNA (shRNA). In some embodiments, the bicistronic construct comprises an HLA-A2 shRNA. In some embodiments, the bicistronic construct comprises an HLA-A2 shRNA having a sequence of SEQ ID NO:240. In some embodiments, the bicistronic construct comprises an HLA-A2 shRNA having a sequence of SEQ ID NO:241. In some embodiments, the bicistronic construct comprises an HLA-beta2 shRNA. In some embodiments, the bicistronic construct comprises an HLA-beta2 shRNA having a sequence of SEQ ID NO:242. In some embodiments, the bicistronic construct comprises an HLA-A2 shRNA having both sequences of SEQ ID NO:240 and SEQ ID NO:242. In some embodiments, the bicistronic construct comprises an HLA-A2 shRNA having both sequences of SEQ ID NO:241 and SEQ ID NO:242.

TABLE 22

shRNA sequences

SEQ

Sequence
ID

Information
NO
Amino acid sequence

HLA-A2-shRNA
240
GGATTACATCGCCCTGAAAGTTCAAGAGACTTTCAGGGC

1

GATGTAATCCTTTTTT

HLA-A2-shRNA
241
CACCTGCCATGTGCAGCATGATTTGTGTAGTCATGCTGC

2

ACATGGCAGGTG

HLA-beta2-
242
GAATGGAGAGAGAATTGAATTCAAGAGATTCAATTCTCT

shRNA

CTCCATTC

15. Monocistronic Constructs

In some embodiments, the iCAR and aCAR constructs are expressed by separate vectors, and the iCAR/aCAR pairs are co-expressed in cells. Methods of co-expressing multiple constructs in the same cell are well known in the art and include, e.g., co-transfection of two or more expression vectors, integration of the constructs into the same or different loci within a cell, optionally followed by enrichment for co-expression.

III. CAR-T Bicistronic ICAR/aCAR Vector Construction

In some embodiments, the bicistronic construct or co-transduction of monocistronic aCAR and iCAR constructs allows for the iCAR and the aCAR to be encoded by a single nucleic acid vector. In some embodiments, the present invention provides a vector comprising a nucleic acid molecule of the invention as defined in any one of the above embodiments, and at least one control element, such as a promoter, operably linked to the nucleic acid molecule.

In some embodiments, the vector is a lentiviral (LV) vector. In some embodiments, the LV vector is a commercially available LV vector. In some embodiments, the LV vector includes but is not limited to pLenti, pLVX-Puro, pLVX-IRES-Puro/Neo/Hygro, pLVx-EF1a-IRES (TAKARA), and/or pcLV-EF1a (Sirion). In some embodiments, the LV vector is pLVX-Puro. In some embodiments, the LV vector is pLVX-IRES-Puro/Neo/Hygro. In some embodiments, the LV vector is pLVx-EF1a-IRES (TAKARA). In some embodiments, the LV vector is pcLV-EF1a (Sirion).

In some embodiments, the vector comprises an EF1 promoter. In some embodiments, the vector comprises a CMV promoter. In some embodiments, the vector comprises a PGK promoter.

In some embodiments, the nucleotide sequence of the vector comprises an internal ribosome entry site (IRES) between the nucleotide sequence encoding for the aCAR and the nucleotide sequence encoding for the iCAR. In general, the nucleotide sequence encoding for the aCAR and the nucleotide sequence encoding for the iCAR can be in any sequential order, but in particular embodiments, the nucleotide sequence encoding for the aCAR is downstream of the nucleotide sequence encoding for the iCAR.

In some embodiments, the nucleotide sequences encoding for the aCAR and the iCAR are encoded on a single vector. In some embodiments, the vector comprises an internal ribosome entry site (IRES) between the nucleotide sequence encoding for the aCAR and the nucleotide sequence encoding for the iCAR. In some embodiments, the nucleotide sequence encoding for the aCAR is downstream of the nucleotide sequence encoding for the iCAR. In some embodiments, the nucleotide sequence comprises a viral self-cleaving 2A peptide located between the nucleotide sequence encoding for the aCAR and the nucleotide sequence encoding for the iCAR. In some embodiments, the nucleotide sequence of the vector comprises a viral self-cleaving 2A peptide between the nucleotide sequence encoding for the aCAR and the nucleotide sequence encoding for the iCAR. In some embodiments, the viral self-cleaving 2A peptide includes is the T2A from Thosea asigna virus (TaV). In some embodiments, the vector comprises a nucleotide sequence encoding the constitutive aCAR linked via a flexible linker to said iCAR.

The immune cells may be transfected with the appropriate nucleic acid molecule described herein by e.g., RNA transfection or by incorporation in a plasmid fit for replication and/or transcription in a eukaryotic cell or a viral vector. In some embodiments, the vector is selected from a retroviral or lentiviral vector.

Combinations of retroviral vector and an appropriate packaging line can also be used, where the capsid proteins will be functional for infecting human cells. Several amphotropic virus-producing cell lines are known, including PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Nati. Acad. Sci. USA 85:6460-6464). Alternatively, non-amphotropic particles can be used, such as, particles pseudotyped with VSVG, RD 114 or GAL V envelope and in some embodiments produced in a PG13 cell line. Cells can further be transduced by direct co-culture with producer cells, e.g., by the method of Bregni, et al. (1992) Blood 80: 1418-1422, or culturing with viral supernatant alone or concentrated vector stocks, e.g., by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J Clin. Invest. 89: 1817.

In some embodiments, the iCAR and aCAR are encoded by different constructs, for example as separate monocistronic aCAR and iCAR constructs. In some embodiments, the iCAR and aCAR are encoded by a single construct, for example as separate monocistronic aCAR and iCAR constructs within a single expression vector.

In some embodiments, the iCAR and aCAR are encoded by the same expression vector. In some embodiments, the expression vector comprises a nucleic acid sequence that encodes a bicistronic iCAR/aCAR selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the expression vector comprises a bicistronic iCAR/aCAR nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the expression vector comprises a bicistronic iCAR/aCAR nucleic acid that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:322, SEQ ID NO:324, and SEQ ID NO:326.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits at least 75% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits at least 80% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits at least 85% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits at least 91% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits at least 92% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits at least 93% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits at least 94% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits at least 96% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits at least 97% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits at least 98% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits at least 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the nucleic acid sequence that encodes a bicistronic iCAR/aCAR exhibits 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

As used herein, sequence identity can include the identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in, for example but not limited to Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site. For example, BLASTN can be used to compare nucleic acid sequences, while BLASTP can be used to compare amino acid sequences. To compare two nucleic acid sequences, the options can be set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (such as C:\seq1.txt); --j is set to a file containing the second nucleic acid sequence to be compared (such as C:\seq2.txt); --p is set to blastn; --o is set to any desired file name (such as C:\output.txt); --q is set to --1; --r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\B12seq --i c:\seq1.txt --j c:\seq2.txt --p blastn --o c:\output.txt --q --1 --r 2.

IV. Construction of Effector Cells

In still another aspect, the present invention provides a method for preparing a safe effector immune cell comprising: (i) transfecting an effector immune cell directed to a tumor-associated antigen with a nucleic acid molecule comprising a nucleotide sequence encoding a bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction as defined herein above or transducing the cells with a vector or (ii) transfecting a naïve effector immune cell with a nucleic acid molecule comprising a nucleotide sequence encoding a bicistronic iCAR/aCAR construct as defined herein above; or transducing an effector immune cell with a vector as defined herein above. In some embodiments, the bicistronic iCAR/aCAR construct is encoded a single vector.

In still another aspect, the present invention provides a method for preparing a safe effector immune cell comprising: (i) transfecting a TCR-engineered effector immune cell directed to a tumor-associated antigen with a nucleic acid molecule comprising a nucleotide sequence encoding a bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction as defined herein above or transducing the cells with a vector or (ii) transfecting a naïve effector immune cell with a nucleic acid molecule comprising a nucleotide sequence encoding a bicistronic iCAR/aCAR construct as defined herein above; or transducing an effector immune cell with a vector as defined herein above. In some embodiments, the bicistronic iCAR/aCAR construct is encoded a single vector. In some embodiments, the bicistronic iCAR and aCAR constructs are encoded on different/separate vectors. In some embodiments, the monocistronic aCAR and iCAR constructs for co-transduction are encoded on a single vector. In some embodiments, the monocistronic aCAR and iCAR constructs for co-transduction are encoded on different/separate vectors.

In some embodiments, the immune cell for use in engineering includes but is not limited to a T-cell, a natural killer cell, or a cytokine-induced killer cell. In some embodiments, the immune cell for use in engineering includes but is not limited to a Jurkat T-cell, a Jurkat-NFAT T-cell, and/or a peripheral blood mononuclear cell (PBMC).

In some embodiments, the immune cell is modified such that is a safe effector immune cell. In yet another aspect, the present invention provides a safe effector immune cell obtained by the method of the present invention as described above. The safe effector immune cell may be a redirected T cell expressing an exogenous T cell receptor (TCR) and a bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction, wherein the exogenous TCR is directed to a non-polymorphic cell surface epitope of an antigen or a single allelic variant of a polymorphic cell surface epitope, wherein said epitope is a tumor-associated antigen or is shared at least by cells of related tumor and normal tissue, and a bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction is as defined above; or the safe effector immune cell is a redirected effector immune cell such as a natural killer cell or a T cell expressing a bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction as defined above.

In some embodiments, the safe effector immune cell expresses on its surface an aCAR comprising an extracellular domain that specifically binds to a non-polymorphic cell surface epitope of an antigen and an iCAR comprising an extracellular domain that specifically binds a single allelic variant of a polymorphic cell surface epitope of a different antigen to which the extracellular domain of said aCAR binds. In some embodiments, the extracellular domain of the iCAR specifically binds a single allelic variant of a different polymorphic cell surface epitope are of the same antigen to which the extracellular domain of said aCAR binds; or the extracellular domain of the iCAR specifically binds a different single allelic variant of the same polymorphic cell surface epitope area to which the extracellular domain of said aCAR binds.

In some embodiments, the aCAR and the iCAR are present on the cell surface as separate proteins. In some embodiments, the expression level on the cell surface of the iCAR is greater than or equal to the expression level of the aCAR. In some embodiments, the extracellular domain of the iCAR expressed on the cell surface is directed against or specifically binds to a single allelic variant of an at least one extracellular polymorphic epitope.

In some embodiments, the extracellular domain of the iCAR expressed on the cell surface is directed against or specifically binds to a single allelic variant of HLA-A2. In some embodiments, the iCAR will be directed toward HLA-A2. In some embodiments, the aCAR with be directed toward EGFR. In some embodiments, the aCAR with be directed toward HER2. In some embodiments, the iCAR/aCAR set will be HLA-A2 and EGFR respectively. In some embodiments, the iCAR/aCAR set will be HLA-A2 and HER2 respectively.

In some embodiments, the safe effector immune cell comprises a bicistronic iCAR/aCAR nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the safe effector immune cell comprises and expression vector comprising a bicistronic iCAR/aCAR nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the safe effector immune cell comprises a bicistronic iCAR/aCAR nucleic acid that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:322, SEQ ID NO:324, and SEQ ID NO:326.

In some embodiments, the safe effector immune cell comprises expression vector comprises a bicistronic iCAR/aCAR nucleic acid that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:322, SEQ ID NO:324, and SEQ ID NO:326.

In some embodiments, EGFR is the aCAR target and HLA is the iCAR target. In some embodiments, HER2 is the aCAR target and HLA is the iCAR target. In some embodiments, the safe effector immune cells used for treating cancer as defined comprises an expression vector. In some embodiments, the iCAR and aCAR are encoded by a bicistronic nucleic acid based expression vector. In some embodiments, the expression vector comprises a nucleic acid sequence a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:3, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325. In some embodiments, the expression vector comprises a nucleic acid sequence that codes for an amino sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:322, SEQ ID NO:324, and SEQ ID NO:326.

In some embodiments, the safe effector immune cells used for treating cancer comprises an expression vector that comprises a bicistronic iCAR/aCAR nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the safe effector immune cells used for treating cancer comprises a bicistronic iCAR/aCAR nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325.

In some embodiments, the safe effector immune cells used for treating cancer as comprises a bicistronic iCAR/aCAR nucleic acid that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:322, SEQ ID NO:324, and SEQ ID NO:326.

In some embodiments, the safe effector immune cells used for treating cancer as comprises an expression vector that comprises a bicistronic iCAR/aCAR nucleic acid that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:322, SEQ ID NO:324, and SEQ ID NO:326.

A. In Vitro Assays

In some embodiments, the bicistronic iCAR/aCAR constructs will be tested for activity effects, including effectiveness and ability to inhibit, using a variety of assays. In some embodiments, the bicistronic iCAR/aCAR constructs or monocistronic aCAR and iCAR constructs for co-transduction will be tested in-vitro and/or in-vivo. In some embodiments, the bicistronic iCAR/aCAR constructs or monocistronic aCAR and iCAR constructs for co-transduction will be tested in-vitro. In some embodiments, the bicistronic iCAR/aCAR constructs or monocistronic aCAR and iCAR constructs for co-transduction will be tested in-vivo. In some embodiments, the in vitro assays measure cytokine secretion and/or cytotoxicity effects. In some embodiments, the in vivo assays will evaluate the bicistronic iCAR/aCAR constructs or monocistronic aCAR and iCAR constructs for co-transduction inhibition and protection to on-target off tumor xenografts. In some embodiments, the in vivo assays will evaluate the bicistronic iCAR/aCAR constructs or monocistronic aCAR and iCAR constructs for co-transduction inhibition and protection to on-target off tumor tissue and/or viral organs.

i. Luciferase Cytotoxicity Assay

In some embodiments, bicistronic iCAR/aCAR constructs or monocistronic aCAR and iCAR constructs for co-transduction are evaluated using a luciferase cytotoxicity assay. Generally, for a luciferase cytotoxic assay, target tumor cells (which can be referred to as “T”) are engineered to express firefly luciferase. In some embodiments, commercially available ATCC cell lines are used. In some embodiments, H1703 cells were used. In some embodiments, H1650 cells were used. In some embodiments, H1792 cells were used. In some embodiments, H292 cells were used. The in vitro luciferase assay can be performed according to the Bright-Glo Luciferase assay (commercially available from Promega or BPS Biosciences or other commercial vendors). Transduced effector (E) T cells (which have been transduced with bicistronic iCAR/aCAR constructs or mock/control construct) can be incubated for 18-48 hrs with recombinant target cells expressing the iCAR or aCAR target to be tested in different effector to target ratios. In some embodiments, the iCAR/aCAR pair comprises any of aCAR and/or iCAR with the components as described above. In some embodiments, the bicistronic iCAR/aCAR constructs described above are to be tested. In some embodiments, the bicistronic iCAR/aCAR comprises an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325. In some embodiments, the bicistronic iCAR/aCAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:322, SEQ ID NO:324, and SEQ ID NO:326. Cell killing can be quantified indirectly by estimating the number of live cells with the Bright-Glo Luciferase system. Cell killing can also be measured using an IncuCyte cytotoxicity assay.

In some embodiments, the ‘off-tumor’ cytotoxicity can be manipulated by sorting transduced T cell populations according to iCAR/aCAR expression level or by selecting a sub population of recombinant target cells according to their target expression, including for example, expression of the gene product encoding for at least one extracellular polymorphic epitope. In some embodiments, the aCAR and iCAR target is any target with an extracellular domain. In some embodiments, the sorting is based on EGFR, HER2, or HLA-A2 expression level.

In some embodiments, the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction is examined to determine whether the iCAR transduced T cells can discriminate between the ‘on-tumor’ cells (e.g., tumor cells) and ‘off-tumor’ cells (e.g., non-tumor cells) in vitro. Generally, this is tested by examining the killing effect of transduced T cells incubated with a mix of ‘on-tumor’ and ‘off-tumor’ cells at a ratio of 1:1 to 1:10. In some embodiments, the ratio Target cells to Effector T cells (T:E ratio) is 1:0.02, 1:0.04, 1:0.06, 1:0.08, 1:0.1, 1:0.12, 1:0.12, 1:0.14, 1:0.16, 1:0.18, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In some embodiments, the E:T ratio (Effector T cells to Target cells) is 0.02:1, 0.04:1, 0.06:1, 0.08:1, 0.1:1, 0.12:1, 0.12:1, 0.14:1, 0.16:1, 0.18:1, 2:1, 3:1, 4:1, 5:1:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1. The on tumor recombinant cells can be distinguished from the ‘off-tumor’ recombinant cells by luciferase expression in embodiments where only one cell population will be engineered to express the luciferase gene at a time). Killing can be quantified after 24-48 hrs of co-incubation using the Bright-Glo Luciferase assay (Promega). Killing can also be quantified using an IncCyte cytotoxicity assay. In some embodiments, transduced cells were only used in the assay of transduction efficiency was greater than 10% and expression was observed for both aCAR and iCAR.

In some embodiments, the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction transduced T cells exhibit about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and/or about 95% less off-tumor cell killing as compared to T cells transduced with aCAR (or other control) but not transduced with the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction. In some embodiments, the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction transduced T cells exhibit about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, or about 10-fold less off-tumor cell killing as compared to T cells transduced with aCAR (or other control) but not transduced with the bicistronic iCAR/aCAR construct.

ii. Caspase 3

In some embodiments, caspase 3-detection assays are employed to determine the level of apoptosis of the ‘on-tumor’ cells (e.g., tumor cells) and ‘off-tumor’ cells (e.g., non-tumor cells) in vitro. In some embodiments, caspase 3-detection of cytotoxic lymphocyte (CTL) induced apoptosis by an antibody to activated cleaved caspase 3 is examined.

Generally, one of the pathways by which CTLs kill target cells is by inducing apoptosis through the Fas ligand. The CASP3 protein is a member of the cysteine-aspartic acid protease (caspase) family. Typically, sequential activation of caspases plays a significant role in the execution-phase of cell apoptosis and as such, cleavage of pro-caspase 3 to caspase 3 results in conformational change and expression of catalytic activity. The cleaved activated form of caspase 3 can be recognized specifically by a monoclonal antibody.

In some embodiments, transduced T cells can be incubated with either ‘on-tumor’ (e.g., mimicking tumor) and ‘off-tumor’ cells (e.g., mimicking non-tumor) recombinant cells. In some embodiments, the ‘on-tumor’ (e.g., tumor) and ‘off-tumor’ cells (e.g., non-tumor) recombinant cells have been previously labeled with CFSE ((5(6)-Carboxyfluorescein N-hydroxysuccinimidyl ester)) or other cell tracer dye (e.g., CellTrace Violet). In some embodiments, co-incubation of target cells with effector cells occurs for about 1 hour to 6 about hours, about 2 hours to about 5 hours, or about 2 to about 4 hrs. In some embodiments, target cell apoptosis is quantified by flow cytometry. Cells can be permeabilized and fixed by an inside staining kit (Miltenyi or BD bioscience) and stained with an antibody for activated caspase 3 (BD bioscience).

In some embodiments, the bicistronic iCAR/aCAR construct transduced T cells induce about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and/or about 95% less off-tumor cell apoptosis as compared to T cells transduced with the bicistronic iCAR/aCAR construct but not transduced with the iCAR (or other appropriate control). In some embodiments, the bicistronic iCAR/aCAR construct transduced T cells induce about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, or about 10-fold less off-tumor cell apoptosis as compared to T cells transduced with aCAR (or other control) but not transduced with the bicistronic iCAR/aCAR construct.

iii. Time-Lapse Microscopy

Time lapse microscopy of the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction transduced T cells can be employed in order to discern target binding. In some embodiments, target cells will be labeled with a reporter gene (for example but not limited to a fluorescent protein such as mCherry). In some embodiments, transduced T cells are incubated with either ‘on-tumor’ or ‘off-tumor’ cells for up to 5 days. In some embodiments, time lapse microscopy can be used to visualize killing. In some embodiments, flow cytometry analysis using viable cell number staining and CountBright™ beads (commercially available from Thermofisher/Invitrogen) for determining target cell number at end-point time will be conducted.

In some embodiments, in order to determine if the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction transduced T cells can discern targets in vitro, each recombinant target cells (‘on-tumor’ or ‘off-tumor’) is labeled with a different reporter protein (for example GFP and mCherry). In some embodiments, any report protein pair would work, so long as the reporter pair contains two reporters which are easily distinguishable. In some embodiments, transduced T cells (Effector cells) will be co-incubated with the recombinant cells (target cells) at a 1:1 ratio of E/T. In some embodiments, the ration of effector to target (E/T) includes but is not limited to 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 6:1, 4:1, 2:1, or 1:1. In some embodiments, the cell fate is then examined by microscopy imaging.

iv. Cytokine Expression Intra Cellular Staining

Cytokine expression and/or release can be examined in order to determine T cells activation. In some embodiments, a bicistronic iCAR/aCAR construct transduced T cells are incubated with the recombinant target cells and cytokine production for one or more cytokines is quantified, for example, either by measuring cytokine secretion in cell culture supernatant according to or by flow cytometry analysis, or by Luminex and/or MSD. For the flow cytometry analysis, a Golgi stop can be employed to prevent the secretion of the cytokines. In some embodiments, following a 6 hour and 18 hour to 24 hour incubation of the transduced T cells with target cells, T cells will be permeabilized and fixed by an intracellular staining kit (Miltenyi) and stained with antibodies for the T cell markers (CD3 and CD8) and for one or more cytokines. In some embodiments, the cytokines include but are not limited to IL-2, INFγ, and/or TNFα. In some embodiments, the cytokines are secreted and include but are not limited to IL-2, INFγ, and/or TNFα. In some embodiments, the cytokines are intracellular and include but are not limited to IL-2, INFγ, and/or TNFα.

v. T Cell Degranulation Assay Measured by CD107a Staining

Staining for CD107a can also be examined as a surrogate for cytolytic activity of the transduced T cells. Generally, degranulating of T cells can be identified by the surface expression of CD107a, a lysosomal associated membrane protein (LAMP-1), and surface expression of LAMP-1 has been shown to correlate with CD8 T cell cytotoxicity. Further, this molecule is located on the luminal side of lysosomes. Typically, upon activation, CD107a is transferred to the cell membrane surface of activated lymphocytes. Moreover, CD107a is expressed on the cell surface transiently and is rapidly re-internalized via the endocytic pathway. Therefore, while not being bound by theory, CD107a detection is maximized by antibody staining during cell stimulation and by the addition of monensin (for example, to prevent acidification and subsequent degradation of endocytosed CD107a antibody complexes).

In some embodiments, the bicistronic iCAR/aCAR construct transduced T cells are incubated with the target cells for about 6 hours to about 24 hours and CD107a expression on the CD8 T cells is examined. In some embodiments, the target cells expresso only one target protein recognized by aCAR (as in tumor cells) or target cells expressing both target proteins recognized by aCAR and iCAR (as in normal cells). In some embodiments, the bicistronic iCAR/aCAR construct transduced T cells are incubated with the target cells for about 6 ours to about 24 hrs in the presence of monensin and CD107a expression on the CD8 T cells is followed by flow cytometry using conjugated antibodies against the T cell surface markers (for example, CD3 and CD8) and a conjugated antibody for CD107a.

vi. Quantitation of Secreted Cytokines by ELISA/Luminex

In some embodiments, following co-cultivation of bicistronic iCAR/aCAR construct transduced T-cells (Jurkat, or primary T-cells) expressing iCAR or aCAR or both aCAR and iCAR with modified target cells, expressing iCAR or aCAR or both aCAR and iCAR antigens on their cell surface, conditioned medium will be collected, and cytokine's concentration will be measured by cytokine ELISA or by Luminex xMAP Multiplex Assay technology (Luminex). In some embodiments, the cytokine is selected from the group consisting of IL-2, INFγ and/or TNF□. In some embodiments, the cytokine is selected from the group consisting of IL-2. In some embodiments, the cytokine is selected from the group consisting of INFγ. In some embodiments, the cytokine is selected from the group consisting of TNF□. In some embodiments, a decrease of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% is demonstrated with bicistronic iCAR/aCAR construct transduced cells.

vii. Cytokines Secretion Measured by Cytometric Bead Array (CBA) Assay

Cytometric Bead Array (CBA) is used to measure a variety of soluble and intracellular proteins, including cytokines, chemokines and growth factors. In some embodiments, T-cells (primary T-cells or Jurkat cells) transduced with aCAR or both aCAR and iCAR constructs (Effector cells) are stimulated with modified target cells expressing both iCAR and aCAR or aCAR or iCAR target antigens on their cell surface. In some embodiments, the effector to target ratio ranges from 20:1 up to 1:1. In some embodiments, the effector to target ratio ranges from 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, following several hours of co-incubation the effector cells produce and secrete cytokines which indicate their effector state. In some embodiments, the supernatant of the reaction is collected, and secreted IL-2, IFN-γ, and/or TNFα were measured and quantified by multiplex CBA assay.

In some embodiments, a decrease of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% is demonstrated with dual CAR (aCAR/iCAR) transduced cells were co-incubated with target cells expressing both target antigens as compared to IL-2, IFN-γ, and/or TNFα secretion resulted from co-incubation of the same effector cells with target cells expressing only one target. In some embodiments, a decrease of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% in IL-2 IFN-γ, and/or TNFα secretion was demonstrated when bicistronic iCAR/aCAR construct transduced cells were co-incubated with target cells expressing both target antigens as compared to IL-2 IFN-γ, and/or TNFα secretion resulted from co-incubation of the same effector cells with target cells expressing only one target. In some embodiments, a decrease of 86%.

B. In Vivo Assays

In some embodiments, the bicistronic iCAR/aCAR construct are tested for effectiveness in vivo. In some embodiments, NOD/SCID/γc- or similar mice are inoculated subcutaneously or orthotopically with tumor cells. In some embodiments, the tumor cells are EGFR and HER2 positive cells lines A549, A431, Fadu, SK-OV-3, U-87, MCF7, NCI-H460, NCI-H1703, NCI-H1650, NCI-H1975, NCI-H292 (ATCC cell lines) cells. In some embodiments, for establishment of and/or differentiation between ‘on-target’ cells and ‘off-tumor’ cells, A549, A431, Fadu, SK-OV-3, U-87, MCF7, NCI-H460 NCI-H1703, NCI-H1650, NCI-H1975, NCI-H292 can be engineered to be deficient or express the iCAR epitope, thereby representing the healthy cells. In some embodiments, the iCAR epitope comprises at least one extracellular polymorphic epitope. In some embodiments, the iCAR epitope is from HLA (including, for example, HLA-A2, HLA-A3, HLA-A, HLA-B, HLA-C, HLA-G, HLA-E, HLA-F, HLA-DPA1, HLA-DQA1, HLA-DQB1, HLA-DQB2, HLA-DRB1, or HLA-DRB5). In some embodiments, the iCAR epitope is from HLA-A2. Other cells that could be employed in these assays include but are not limited to Raji or any other recombinant cell lines. In some embodiments, such assays can be in a PDX (patient derived xenograft) model.

For the assay, mice will be divided into study groups; one cohort will be injected with the A549, A431, Fadu, SK-OV-3, U-87, MCF7, NCI-H460 NCI-H1703, NCI-H1650, NCI-H1975, and/or NCI-H292 cells not expressing the iCAR epitope, while the other will be injected with the corresponding A549, A431, Fadu, SK-OV-3, U-87, MCF7, NCI-H460 NCI-H1703, NCI-H1650, NCI-H1975, NCI-H292 cells expressing the iCAR epitope. Following staging, mice will be infused intravenously with T cells transduced with aCAR, aCAR/iCAR and a control group of untransduced T cells or no T cells. Tumor burden will be measured by through measurement of the subcutaneous tumor volume.

According to one embodiment of the assay, in order to test whether the T cells expressing the bicistronic iCAR/aCAR constructs could discriminate between the target cells and off target cells in vivo within the same organism, mice are injected with a 1:1 mixture of the ‘on-tumor’/‘off-tumor A549, A431, Fadu, SK-OV-3, U-87, MCF7, NCI-H460 NCI-H1703, NCI-H1650, NCI-H1975, and/or NCI-H292 cells, followed by injection of transduced T cells expressing either the aCAR alone or both aCAR and iCAR (including as the bicistronic iCAR/aCAR constructs as described herein) after staging. With this embodiment, upon sacrifice of the mice the presence of the ‘on-tumor’ and ‘off-tumor cells Will be evaluated by immunohistochemical staining

According to one embodiment of the assay, in order to test whether the T cells expressing the bicistronic iCAR/aCAR constructs could discriminate between the target cells and off target cells in vivo within the same organism, mice are injected with a 1:10 mixture of the ‘on-tumor’/‘off-tumor NALM-6, A549, A431, Fadu, SK-OV-3, U-87, MCF7, and/or NCI-H460 NCI-H1703, NCI-H1650, NCI-H1975, NCI-H292 cells, followed by injection of transduced T cells expressing either the aCAR alone or both aCAR and iCAR. With this embodiment, upon sacrifice of the mice the presence of the ‘on-tumor’ and ‘off-tumor cells in the spleen and bone marrow will be analyzed by flow cytometry for iCAR and aCAR markers.

i. Tumor Growth Kinetics in Human Xenograft Mouse Models

In some embodiments, the tumor cells express either the iCAR target, aCAR target or both. In some embodiments, an aCAR tumor cell line could be the EGFR or HER2 positive cells lines A549, A431, Fadu, SK-OV-3 U-87, MCF7, and/or NCI-H460 (ATCC cell lines). In some embodiments, tumor cells that express both the aCAR and iCAR (i.e. ‘off-tumor’ cells) are NALM 6, A549, A431, Fadu, SK-OV-3, U-87, MCF7, MDA-MB-231, and/or NCI-H460 engineered to express the iCAR epitope (for example, HLA-A2) thereby representing the healthy cells. In some embodiments, NALM 6 and NALM 6-HLA-A2 can also be engineered to express a reporter gene (e.g., firefly luciferase, GFP, mCherry), for easy detection. In some embodiments, A549 and A549-HLA-A2 can also be engineered to express a reporter gene (e.g., firefly luciferase), for easy detection. In some embodiments, A431 and A431-HLA-A2 can also be engineered to express a reporter gene (e.g., firefly luciferase), for easy detection. In some embodiments, Fadu and Fadu-HLA-A2 can also be engineered to express a reporter gene (e.g., firefly luciferase), for easy detection. In some embodiments, SK-OV-3 and SK-OV-3-HLA-A2 can also be engineered to express a reporter gene (e.g., firefly luciferase), for easy detection. In some embodiments, NCI-H460 and NCI-H460-HLA-A2 can also be engineered to express a reporter gene (e.g., firefly luciferase), for easy detection. In some embodiments, U-87 and U-87-HLA-A2 can also be engineered to express a reporter gene (e.g., firefly luciferase), for easy detection. In some embodiments, MCF7 and MCF7-HLA-A2 can also be engineered to express a reporter gene (e.g., firefly luciferase), for easy detection. In some embodiments, can also be engineered to express a reporter gene (e.g., firefly luciferase), for easy detection. In some embodiments, NCI-H460 and NCI-H460-HLA-A2 can also be engineered to express a reporter gene (e.g., firefly luciferase), for easy detection.

In some embodiments, monitoring will be conducted by measuring tumor volume by mechanical means (caliper) and also by using in-vivo imaging systems (IVIS). In some embodiments, tumor burden can be quantified, and infiltrating T-cell populations can be analyzed by FACS.

C. Treatment Methods

The present invention provides methods for the treatment of cancers by employing the bicistronic iCAR/aCAR constructs or monocistronic aCAR and iCAR constructs for co-transduction as described herein. The methods of treatment for cancer as described herein can employ exploiting loss of heterozygosity, or other genetic loss or allelic imbalance phenotypes found in human tumors, including, without limitation, loss of function or expression, resulting from mutations affecting one or more nucleotides (for example, without limitation, in HLA-1 genes) by means of CAR-T therapy, or by modifying other cells of the immune system.

In yet another aspect, the present invention provides a method of selecting a personalized biomarker for a subject having a tumor characterized by loss of heterozygosity, or other genetic loss or allelic imbalance phenotypes found in human tumors, the method comprising (i) obtaining a tumor biopsy from the subject; (ii) obtaining a sample of normal tissue from the subject, e.g., PBMCs; (iii) identifying a single allelic variant of a polymorphic cell surface epitope that is not expressed by cells of the tumor due to loss of heterozygosity, or other genetic loss or allelic imbalance phenotypes found in human tumors, but that is expressed by the cells of the normal tissue, thereby identifying a personalized biomarker for the subject, and (iv) determining the appropriate bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction as described herein for use in treatment.

In a further aspect, the present invention provides a method for treating cancer in a patient having a tumor characterized by loss of heterozygosity, or other genetic loss or allelic imbalance phenotypes found in human tumors, comprising administering to the patient an effector immune cell as defined above, wherein the iCAR is directed to a single allelic variant encoding a polymorphic cell surface epitope absent from cells of the tumor due to loss of heterozygosity, or other genetic loss or allelic imbalance phenotypes found in human tumors but present at least on all cells of related mammalian normal tissue of the patient. In some embodiments, the effector immune cell comprises a bicistronic iCAR/aCAR construct as described herein.

In some embodiments, the treating results in reduced on-target, off-tumor reactivity, as compared with a treatment comprising administering to the cancer patient at least one population of immune effector cells expressing a bicistronic iCAR/aCAR construct as described herein.

In some embodiments, the safe effector immune cells used for treating cancer as defined above express on their surface an aCAR comprising an extracellular domain that specifically binds to a tumor-associated antigen or a non-polymorphic cell surface epitope of an antigen and an iCAR comprising an extracellular domain that specifically binds a single allelic variant of a polymorphic cell surface epitope of an antigen expressed at least in a tissue of origin of the tumor or of a housekeeping protein, which is a different antigen than that to which the extracellular domain of said aCAR binds. In some embodiments, the effector immune cell expresses the components of a bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction as described herein.

In some embodiments, the safe effector immune cells used for treating cancer as defined above express on their surface an aCAR comprising an extracellular domain that specifically binds to a tumor-associated antigen or a non-polymorphic cell surface epitope of an antigen and an iCAR comprising an extracellular domain that specifically binds a single allelic variant of a polymorphic cell surface epitope of an antigen expressed at least in a tissue of origin of the tumor or of a housekeeping protein, such as an HLA genes (including for example, HLA-A, HLA-B, HLA-C, HLA-G, HLA-E, HLA-F, HLA-K, HLA-L, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR) which is a different antigen than that to which the extracellular domain of said aCAR binds.

In some embodiments, the safe effector immune cells used for treating cancer as defined above express on their surface an aCAR comprising an extracellular domain that specifically binds to a tumor-associated antigen or a non-polymorphic cell surface epitope of an antigen and an iCAR comprising an extracellular domain that specifically binds a single allelic variant of a polymorphic cell surface epitope of an antigen expressed at least in a tissue of origin of the tumor, such as an HLA-A, which is a different antigen than that to which the extracellular domain of said aCAR binds.

In some embodiments, the safe effector immune cells used in the method of treating cancer are selected from T cells, natural killer cells or cytokine-induced killer cells. In some embodiments, the safe effector immune cell is autologous or universal (allogeneic) effector cells. In some embodiments, the iCAR used in any one of the methods of treating cancer defined above is directed to all tissues of the patient on which the target-antigen of the aCAR is present, wherein the target antigen of the aCAR is a non-polymorphic cell surface epitope of an antigen or a single allelic variant of a polymorphic cell surface epitope is present, and said epitope is a tumor-associated antigen or is shared at least by cells of related tumor and normal tissue.

In some embodiments, the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction for use in the treatment of cancer is any bicistronic iCAR/aCAR construct described herein. In some embodiments, the bicistronic iCAR/aCAR construct used to treat the cancer, such as any one of the cancer types recited above, is directed against or specifically binds to a single allelic variant of an HLA genes (including for example, HLA-A, HLA-B, HLA-C, HLA-G, HLA-E, HLA-F, HLA-K, HLA-L, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR, HLA-B gene or HLA-C gene or against a single allelic variant. In some embodiments, the treatment method employs administration of a safe effector cell comprising the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction. In some embodiments, the treatment method employs administration of a safe effector cell expressing the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction.

In some embodiments, the bicistronic iCAR/aCAR or monocistronic aCAR and iCAR constructs for co-transduction for use in the treatment of cancer comprises an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325. In some embodiments, the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction comprises an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:321, SEQ ID NO:323, and SEQ ID NO:325. In some embodiments, the treatment method employs administration of a safe effector cell comprising the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction. In some embodiments, the treatment method employs administration of a safe effector cell expressing the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction.

In some embodiments, the bicistronic iCAR/aCAR for use in the treatment of cancer comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:322, SEQ ID NO:324, and SEQ ID NO:326. In some embodiments, the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction comprises an amino acid sequence selected from the group consisting of SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:322, SEQ ID NO:324, and SEQ ID NO:326. In some embodiments, the treatment method employs administration of a safe effector cell comprising the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction. In some embodiments, the treatment method employs administration of a safe effector cell expressing the bicistronic iCAR/aCAR construct or monocistronic aCAR and iCAR constructs for co-transduction.

The compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added pharmaceutically acceptable carrier and/or preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

For purposes of clarity, and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values recited herein, should be interpreted as being preceded in all instances by the term “about.” Accordingly, the numerical parameters recited in the present specification are approximations that may vary depending on the desired outcome. For example, each numerical parameter may be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

EXAMPLES
Example 1. Development and Testing of Bicistronic Inhibitory Chimeric Antigen Receptor (iCAR)/Activating Chimeric Antigen Receptor (aCAR) Constructs
INTRODUCTION

This example provides the results related to development and testing of bicistronic inhibitory chimeric antigen receptor (iCAR)/activating chimeric antigen receptor (aCAR) constructs in order to develop cancer therapeutics for use in safely target tumors that have lost genomic segments encoding cell-membrane proteins with polymorphic protein coding changes). Data provide in the example and figures include T-REP identification of new iCAR leads, new human HLA-A2 scFv constructs, and bicistronic LV transduction—FaDu/MCF7-Luc immune killing assay—including development of novel iCAR leads.

Bicistronic iCAR/aCAR constructs have been developed and preliminary testing performed in order to prepare and examine these constructs for use as cancer therapeutics. See, FIGS. 1-59 as well as Tables 1-22 for illustrative design and evaluation of examples of iCAR and aCAR constructs as described herein, as well as sequences thereof.

Materials and Methods

mRNA Transcription In Vitro

Appropriate plasmids were linearized using SpeI or BamHI restriction enzymes. Linear plasmid was used to transcribe in-vitro mRNA using T7mScript Standard mRNA Production System (CELLSCRIPT, Madison, U.S.A.). The concentration and quality of the mRNA were assessed by spectrophotometry. Preparation was according to manufacturer's protocol.

PBMC Purification

Leukocyte enriched samples were acquired from The Sheba Medical Center blood bank, diluted with equal volumes of PBS and loaded on Ficoll-Paque PLUS (GE Healthcare) for density-based cell separation. Preparation was according to manufacturer's protocol. Mononuclear cells were collected from the plasma/Ficoll interface, washed several times and resuspended in Cryostor CS10 (Merck).

PBMC Culture and Transduction

PBMCs were thawed and seeded at a density of 1×10⁶cells/ml in LymphoOne medium (Takara-Bio, Kusatsu, Japan) supplemented with 100 U/ml IL2 (Miltenyi Biotech, Bergisch Gladbach, Germany). The next day concentrated lentiviruses were added at an MOI of 5, 10, or 20 (according to prior calibrations). After 3 days cells were transferred to 24-well G-Rex plates (Wilson Wolf, Saint Paul, MN) containing LymphoOne medium supplemented with 1% human serum (Access Biologicals, Vista, CA) and 100 U/ml IL2. On day 7 post-thaw 100 U/ml IL2 was added, and on day 8 the medium was replaced. Functional assays were typically performed.

mRNA Electroporation

On day 8 or 10 of PBMC's culture, 2×10⁶cells were washed twice with OptiMEM medium (GibcoBRL, Grand Island, NY). The cells were resuspended in 100 ul OptiMEM containing 1-10 ug mRNA and electroporated in 2 mm cuvette, using NepaGene21 electroporator (Nepa Gene Co., Ltd., Japan) at 200V, 2.5 ms, one pulse or using ECM830 electroporator (BTX.Ltd., US) at 300V, 2 ms, one pulse. The cells were resuspended in 5 ml growth medium and transferred into 6 well plates for further incubation.

IncuCyte Cytotoxicity Assay

Target cells expressing nuclear-GFP (nGFP) were seeded in black-walled 384-well plates with microclear bottom (Greiner Bio-One, Kremsmunster, Austria), 1.5×10⁴cells per well, in LymphoOne medium supplemented with 1% human serum. The next day, transduced or electroporated PBMCs were added to the wells at the desired E:T ratio. Annexin-V Red (Essen BioScience, Ann Arbor, MI) to detect apoptosis was added immediately before adding PBMCs). Plates were imaged for 3 days using the IncuCyte S3 (Essen BioScience) instrument at 37 C, 5% CO₂. Percent killing was calculated as nGFP+ Annexin-V-Red+ cell count divided by total nGFP+ cell count.

ELISA

Target cells expressing nuclear-GFP (nGFP) were seeded in 96 well plates (Thermo, NU-167008), 5×10³cells per well, in LymphoOne medium supplemented with 1% human serum. The next day, transduced or electroporated PBMCs were added to the wells at 5:1 E:T ratio. Cells are co-incubated for 15-18 hrs at 37 C, 5% CO₂. Following co-incubation, supernatant is harvested and transferred to non-binding 96-well plates (Greiner, #655901) at −200c. Supernatants are diluted 3 and 100-fold, ELISA performed as to manufactures instruction (Human IFN-gamma Quantikine, R&D, #SIF50) and quantified using Tecan plate reader.

Quantification of Antigen Expression by Flow Cytometry

The MESF/“Antibody Binding Capacity” (ABC) ratio of a particular antibody can be used to quantify the number of antigen sites per cell. To establish the MESF/ABC ratio of each antibody Lot, MFIs of stained SCQ beads were correlated to the MFIs of MESF standards. The slope of the curve constitutes the ratio of fluorochrome label in MESF units per antibody. The MESF/ABC of every antibody Lot was measured using mouse/human/rat Simple Cellular Quantum (SCQ) Beads and MESF standards purchased from Bangs laboratories. Each of the 4 populations of SCQ beads has a known Antibody Binding Capacity (ABC), typically in the range of several thousands to 500-800K, so by staining these beads with an antibody at near saturation, one can correlate the fluorescence measurement (MFI) on a flow cytometer to the amount of bound antibody (ABC). MESF standard beads are composed of 4-5 different bead populations labeled with a known amount of fluorochrome molecules. By running MESF beads on a flow cytometer, one can correlate an MFI measurement to MESF units and compare between data that was collected on multiple different occasions, PMT voltages and instruments. When using HLA-A2/NYESO1-PE tetramers to stain tag-less iCAR constructs, the MESF/ABC ratio was established by staining control Jurkat cell lines that express a tagged aCAR and iCAR at high and low levels, with both quantifiable Anti-Myc Tag antibody and HLA-A2/NYESO1-PE tetramers. For each staining 100-200K positive cells were washed twice with 100 ul of cold FACS buffer (2% FCS in PBS×1) by centrifugation, 300 g for 5 min at 4° C. For Flag tagged aCAR and Myc tagged iCAR quantification, the cells were stained with 50 ul of APC (130-119-584, Miltenyi) and FITC (130-116-485, Miltenyi) labeled antibodies diluted 1/25 with FACS buffer. For un-tagged trastuzumab aCAR and Anti-HLA-A2 iCAR quantification, primary human Anti-Trastuzumab scFv69 (Ab00618-10.0, Absolute Antibody), HLA-A2/NYESO1-PE tetramers (TB-M105-1, MBL) and secondary Anti-human Fc APC (BLG-409306, biolegend) were diluted in FACS buffer, 1/25, 1/5 and 1/10 respectively. For target cell line antigen quantification, Anti-EGFR PE (FAB9577P-100, R&D), Anti-HER2 APC (130-106-696, Miltenyi) and Anti-HLA-A2 APC (17-9876-42, ebioscience) were diluted with FACS buffer, 1/2.5, 1/10 and 1/5 respectively. The cells were incubated at 4° C. in the dark for 45-60 min and washed thrice with 100 ul cold FACS buffer as described previously. The cells were resuspended with 150 ul of FACS buffer or PBS×1 containing 0.5-1 ug/ml DAPI (MBD0015-1, Merck-Sigma). The cells were analyzed by flow cytometry (BD FACS Celesta or MACSQuant Analyzer 10) collecting 10K-50K double positive events from each sample. Next, without changing the PMT voltages on the instrument, 5-10K events of each population of relevant MESF standard beads (FITC 555P-5ML, APC 823-5ML, PE 827-5ML, Bangs), were collected. FlowJo software was used to gate and calculate MFIs (Geometric Mean Fluorescence) and MESF beads QuickCal files, provided by the manufacturer, were used to convert the MFIs in to MESF units. Next, the values were converted to ABC units Using the MESF/ABC curves of the specific antibody lots used.

Discussion

FaDu/MCF7-Luc Immune Killing Assay

Identification of novel iCAR using a nucGFP labeled target cells endpoint and bicistronic LV transduction.

The assay was useful regarding increasing the potency of iCAR inhibition (scFv avidity & activity) is necessary to decrease the iCAR/aCAR stoichiometry for efficient aCAR protection. Continued development and analysis related to dual differential expression in a lentiviral bicistronic format is ongoing and in progress.

Focused on HER2 (anti-Trastuzumab scFv) as an aCAR. Identified fully human or humanized scFv's to target HLA-A2. Identified novel iDomains (LIR1, KIR2DL1, KIR2DL2, and/or BTLA).

Lentiviral dualCAR Expression

Low transduction efficiency and variable differential expression.

iCAR constructs are identical, except for variations in the inhibitory domain.

aCAR constructs are also identical, except for variations in the scFv: Cetuximab or Panitumumab for EGFR and Trastuzumab or Pertuzumab for Her2.

All constructs are in iCAR/aCAR configuration with T2A cleavable linker.

IncuCyte Immune Cell Killing Assay: a cell imagining platform to monitor target killing and proliferation, and T-cell activation kinetics.

Quantum Bead Assay: a methodology to incorporate absolute aCAR & iCAR level, stoichiometry, and expression kinetics into screening and analysis.

IMPT001 GO: in vitro validation HLA-A2 scFv/PD-1 iCAR pairing with EGFR & HER2 scFv aCAR using mRNA co-electroporation of constructs into effector cells, using the Incucyte platform and FACS T-cell profiling IMPT001 go.

Lentiviral Technology: Design and evaluate the expression of mono- and dual lentiviral aCAR & iCAR to support IMPT001 and identify novel iCAR (64 constructs).

New Potent HLA-A2 scFv: Characterize a fully human HLA-A2 scFv alternative to murine BB7.2 as a lentiviral iCAR transduced into donor PBMC that appears to bind HLA-A tetramers more avidly.

FaDU/U87-Luc Immune Killing Assay: identification of novel iCAR using a Luciferase viability endpoint. LIR1 & KIR2DL1 iCARs identified.

Validation of an IncuCyte Immune Cell Killing Assay

Dependence of target cell killing & proliferation on E/T ratio.

Implemented an immune cell killing assay that simultaneously images the kinetics of target cell killing and proliferation, and T-cell activation.

The technology is applicable to diverse adherent cancer cell lines partially circumventing the time and cost associated with engineering isogenic cell lines.

The kinetic and endpoints are a quantitative metrics that will allow dual CAR ranking, i.e., directly proportional to E/T ratio and aCAR and iCAR level.

The sensitivity (E/T EC50) of target cancer cell lines to EGFR and HER2 aCAR killing varies >5-fold and does not correlate with EGFR expression level.

Cell-Surface Expression

Absolute iCAR/aCAR level and stoichiometry—Effector cells. Absolute iCAR/aCAR antigen level and stoichiometry—Target cells. See, for example, FIG. 14.

A highly reproducible FACS based method has been implemented to quantify absolute CAR and target antigen levels)

The level of aCAR and iCAR expression obtained with mRNA co-electroporation are linearly dependent on mRNA amount.

Stoichiometric expression by co-electroporation is heavily iCAR biased (e.g., iCAR/aCAR slope=6.0 on the Jurkat experiment (See, for example, FIGS. 12-13).

An EGFR x HLA-A2 Dual CAR

Validation with mRNA co-electroporation studies.

Pairing of Cetuximab aCAR with a BB7.2 PD-1 iCAR was assessed by mRNA co-electroporation in HLA-A2 NEG and HLA-A2 POS adherent cancer cell-lines.

Killing of FaDu A2 NEG cells by dual CAR T-cells was obtained at low E/T ratios without apparent loss of aCAR activity, U87 A2 POS (EGFR aCAR sensitive) cancer cells were fully protected.

All HLA-A2 POS cancer cell lines tested inhibited T-cell activation (CD107a, IFNg) at low E/T regardless of HLA-A2 level (˜10⁵to ˜10⁶per cell) and target cell killing efficiency.

CAR quantification has not yet been performed, however HLA-A2 dependent protection is associated with excess iCAR exposure (>10-fold Cmax). See, for example, FIG. 12A.

Pairing of Trastuzumab scFv aCAR with a BB7.2 scFv PD-1 iCAR was assessed by mRNA co-electroporation in HLA-A2 NEG and HLA-A2 POS adherent cancer cell-lines.

Killing of FaDu A2 NEG cells by dual CAR T-cells was obtained at E/T=10 without apparent loss of aCAR activity.

Protection of MDA-MB-231 A2 POS cancer cells appeared to depend on a 300-fold excess of iCAR over aCAR expression (Cmax).

In contrast, lower iCAR levels were sufficient to inhibit T-cell activation (CD107a, IFNg, TNFα) regardless of target cell killing efficiency.

Dual CAR Lentiviral Transduction

Absolute and stoichiometric expression in PBMCs.

iCAR constructs are identical, except for variations in the inhibitory domain.

aCAR constructs are also identical, except for variations in the scFv: Cetuximab or Panitumumab for EGFR and Trastuzumab or Pertuzumab for Her2.

All constructs are in iCAR/aCAR configuration with T2A cleavable linker.

The PBMC transduction efficiency (% gated double positive) of lentiviral bicistronic CARs was variable and most often too low (<20%) for IncuCyte co-culture assays.

iCAR expression (proximal gene) could exceed aCAR expression (distal gene) by 5-10 fold but exceptions and failures were not uncommon.

Identification of HUMAN Alternatives to BB7.2 HLA-A2 scFv

Mono-cistronic expression in PBMCs and HLA-A tetramer binding.

Binding to HLA-A2 tetramers was observed for BB7.2 (++), 3PF12 (+++), SN66E3 (+++), MBW1. Sequence Modifies (++). Binding to HLA-A2 tetramers was no observed for Ha5C2.A2 and murine BBM.1.

cMYC tag reports surface expression of iCARs.

HLA-A2 tetramer reports on HLA-A2 scFv binding.

Two fully human HLA-A2 scFv were identified as potential alternatives to BB7.2 (murine) that appear to bind HLA-A2 tetramer with higher avidity: 3PF12 and SN66E3.

FaDu/U87-Luc Immune Killing Assay

Identification of novel iCAR using a Luciferase viability endpoint.

FaDu/U87-Luc Assay was developed and internal controls were validated. EGFR aCARs show robust specific killing of FaDu and U87 cells. HLA-A2 aCAR shows specific killing in U87 HLA-A2 POS cells.

Achieves high E/T ratios without assay interference (E/T=64).

HLA-A2 dependent protection observed KIR2DL1 is consistent with T-REP (KIR2DL2) and Jurkat NFAT-Luc FA experiments (KIR2DL1+KIR2DL2).

FaDu/MCF7-Luc Immune Killing Assay

Identification of novel iCAR using a Luciferase viability endpoint.

FaDu/MCF7-Luc Assay was developed and internal controls were validated. HER2 aCARs show robust specific killing of FaDu and MCF7 cells. Achieves high E/T ratios without assay interference (E/T=20).

HLA-A2 dependent protection observed with KIR2DL1 is consistent with T-REP (KIR2DL2) and Jurkat NFAT-Luc FA experiments (KIR2DL1+KIR2DL2).

HLA-A2 dependent protection observed with LIR1 is consistent with Jurkat NFAT-Luc FA experiments.

SUMMARY

The data provide herein supports in vitro validation of a humanized BB7.2 iCAR scFv (see, for example, FIG. 59). This data confirmed that efficacy was observed for all constructs with a Hz BB7.2 version. This data also demonstrated that protection was observed for all constructs with a Hz BB7.2 version. VR428 and VR421 were identified as exemplary constructs.

Also provided by the data was in vivo validation of HzBB7.2 iCAR scFv (see, for example, FIG. 54 and FIG. 55). Both efficacy and protection were demonstrated in an in vivo study for low and high dose with VR428 administration. VR428 was identified as an exemplary construct.

Also provided by the data was in vitro validation of a fully human SN66E3.3 iCAR scFv (see, for example, FIG. 49). This data confirmed that efficacy was observed for all constructs with a fully human SN66E3 version. This data also demonstrated that protection was observed for all constructs with a fully human SN66E3 versions. VR447 and VR449 were identified as exemplary constructs.

All headings and section designations are used for clarity and reference purposes only and are not to be considered limiting in any way. For example, those of skill in the art will appreciate the usefulness of combining various aspects from different headings and sections as appropriate according to the spirit and scope of the invention described herein.

All references cited herein are hereby incorporated by reference herein in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled.

	Number	Date	Country
	63178452	Apr 2021	US
	63074812	Sep 2020	US

BICISTRONIC INHIBITORY CHIMERIC ANTIGEN RECEPTOR (ICAR)/ACTIVATING CHIMERIC ANTIGEN RECEPTOR (ACAR) CONSTRUCTS FOR USE IN CANCER THERAPIES

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