Chimeric receptors and methods of use thereof

Abstract

To provide acute myeloid leukemia antigen targets for chimeric receptors and methods of using same.SOLUTION: There is provided an isolated immunoresponsive cell comprising (a) a first chimeric receptor comprising an extracellular antigen-binding domain that binds to a first antigen, and (b) a second chimeric receptor comprising an extracellular antigen-binding domain that binds to a second antigen, wherein each antigen is selected from a group consisting of FLT3, CD33, CLEC12A, MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70, and wherein the first antigen is different from the second antigen.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 841,128, filed April 30, 2019, U.S. Provisional Application No. 62 / 854,151, filed May 29, 2019, and U.S. Provisional Application No. 62 / 893,106, filed August 28, 2019, each of which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created in XX month of 20XX, is named XXXXXUS_sequencelisting.txt and is X,XXX,XXX bytes in size. [Background technology]

[0003] background Chimeric antigen receptor (CAR)-based adoptive cell therapy, which is used to redirect the specificity and function of immune-responsive cells such as T cells, has shown efficacy in patients with lymphoid malignancies (Pule et al., Nat. Med. (14):1264-1270 (2008); Maude et al., N Engl J Med. (371):1507-17 (2014); Brentjens et al., Sci Transl Med. (5):177ra38 (2013) (Non-Patent Documents 1-3)). CAR T cells have been shown to induce complete remissions in patients with CD19-expressing malignancies where chemotherapy has led to drug resistance and tumor progression. The success of CD19 CAR therapy provides optimism for treating other hematologic malignancies, such as acute myeloid leukemia (AML), the most common acute leukemia in adults. AML is a cancer of the blood cells of the myeloid system, characterized by the rapid proliferation of abnormal cells that accumulate in the bone marrow and blood and interfere with normal blood cells. AML can sometimes spread to the brain, skin, or gums. Standard chemotherapy treatments for AML have remained virtually unchanged over the past 40 years (Pulte et al., 2008), and overall survival rates remain very low.

[0004] One challenge in developing CAR therapy for AML is the lack of suitable targets. The ability to identify appropriate CAR targets is critical for effectively targeting and treating tumors without damaging normal cells that express the same target antigen. Therefore, there remains a need for CAR-T cell-based AML therapies that target AML cells without targeting normal cells or tissues. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Pule et al., Nat.Med.(14):1264-1270(2008) [Non-patent document 2] Maude et al.,N Engl J Med.(371):1507-17(2014) [Non-patent document 3] Brentjens et al., Sci Transl Med.(5):177ra38(2013) Summary of the Invention

[0006] overview In one aspect, provided herein is an isolated immunoresponsive cell comprising: (a) a first chimeric receptor comprising an extracellular antigen-binding domain that binds a first antigen; and (b) a second chimeric receptor comprising an extracellular antigen-binding domain that binds a second antigen, wherein each antigen is selected from FLT3, CD33, CLEC12A, MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70; and wherein the first antigen is different from the second antigen.

[0007] In some embodiments, the first antigen is FLT3, and the extracellular antigen-binding domain of the first chimeric receptor comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 3, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 4, or a sequence at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 1, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 2, or a sequence at least 90% identical thereto; (c) a VH comprising the amino acid sequence of SEQ ID NO: 5, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 6, or a sequence at least 90% identical thereto; (d) a VH comprising the amino acid sequence of SEQ ID NO: 7, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 8, or a sequence at least 90% identical thereto. (e) a VH comprising the amino acid sequence of SEQ ID NO: 9 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 10 or a sequence at least 90% identical thereto, (f) a VH comprising the amino acid sequence of SEQ ID NO: 11 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 12 or a sequence at least 90% identical thereto, (g) a VH comprising the amino acid sequence of SEQ ID NO: 13 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 14 or a sequence at least 90% identical thereto, and (h) a VH comprising the amino acid sequence of SEQ ID NO: 15 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 16 or a sequence at least 90% identical thereto.

[0008] In some embodiments, the second antigen is CD33 and the extracellular antigen-binding domain of the second chimeric receptor comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from (a) a VH comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 18, or a sequence at least 90% identical thereto, and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20, or a sequence at least 90% identical thereto; or the second antigen is CLEC12A and (a) the sequences The heavy chain variable domain (VH) and light chain variable domain (VL) are selected from (a) a VH comprising the amino acid sequence of SEQ ID NO: 21 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 22 or a sequence at least 90% identical thereto, (b) a VH comprising the amino acid sequence of SEQ ID NO: 23 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 24 or a sequence at least 90% identical thereto, and (c) a VH comprising the amino acid sequence of SEQ ID NO: 25 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 26 or a sequence at least 90% identical thereto.

[0009] In some embodiments, the first antigen is CLEC12A, and the extracellular antigen-binding domain of the first chimeric receptor comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 21, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 22, or a sequence at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 23, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 24, or a sequence at least 90% identical thereto; and (c) a VH comprising the amino acid sequence of SEQ ID NO: 25, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 26, or a sequence at least 90% identical thereto. and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20 or a sequence at least 90% identical thereto.

[0010] In some embodiments, binding of a first chimeric receptor to a first antigen can activate an immunoresponsive cell, and / or binding of a second chimeric receptor to a second antigen can stimulate an immunoresponsive cell, and / or the immunoresponsive cell exhibits a greater degree of cytolytic activity against target cells positive for both the first and second antigens compared to cytolytic activity against target cells positive for only the first or second antigen, and / or the first chimeric receptor binds to the first antigen with a lower binding affinity than the binding affinity of the second chimeric receptor for the second antigen, and / or the first chimeric receptor binds to the first antigen with a lower avidity.

[0011] In some embodiments, the first chimeric receptor is a first CAR and the second chimeric receptor is a second CAR, each CAR comprising a CD3 zeta chain intracellular signaling domain, and optionally each CAR further comprising one or more additional intracellular signaling domains, the one or more additional intracellular signaling domains being selected from the group consisting of a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GI The antigen-binding domain is selected from a TR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, and a 2B4 intracellular signaling domain, and / or comprises a transmembrane domain, wherein the transmembrane domain is selected from a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 zeta chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, and a BTLA transmembrane domain, and / or comprises a spacer region between the antigen-binding domain and the transmembrane domain, wherein the spacer region has an amino acid sequence selected from SEQ ID NOs: 55 to 64.

[0012] In some embodiments, the cell further comprises an inhibitory chimeric receptor comprising the antigen binding domain, optionally, the inhibitory chimeric receptor inhibits one or more activities of the cell.

[0013] In some embodiments, the inhibitory chimeric receptor binds to an antigen expressed on a non-tumor cell, and optionally the antigen expressed on a non-tumor cell is derived from a tissue selected from brain, nervous tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0014] In some embodiments, the inhibitory chimeric receptor binds to an antigen selected from EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR2, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, and TMEM200A.

[0015] In some embodiments, the inhibitory chimeric receptor comprises an antigen-binding domain comprising a single-chain variable fragment (scFv), wherein the scFv is derived from an anti-EMCN antibody.

[0016] In some embodiments, the antigen-binding domain of the first chimeric receptor, the antigen-binding domain of the second chimeric receptor, and / or the antigen-binding domain of the inhibitory chimeric receptor comprises one or more single-chain variable fragments (scFvs), each of the one or more scFvs comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), optionally, the VH and VL are separated by a peptide linker, optionally, the peptide linker comprises the amino acid sequence of SEQ ID NO: 27.

[0017] In some embodiments, each of the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, where VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain.

[0018] In some embodiments, each of the one or more scFvs binds a distinct epitope on the same antigen.

[0019] In some embodiments, each of the one or more scFvs is separated by a peptide linker, optionally wherein the peptide linker comprises the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 27) or EAAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 74).

[0020] In some embodiments, the cells are T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, myeloid cells, macrophages, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs), and iPSC-derived cells; Optionally, the immunoresponsive cells are allogeneic.

[0021] In another aspect, provided herein is a pharmaceutical composition comprising an effective amount of an isolated immunoresponsive cell described in any of the embodiments herein, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

[0022] In another aspect, provided herein is a method of providing anti-tumor immunity in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of any of the isolated immunoresponsive cells described in any of the embodiments herein, or the pharmaceutical composition described in any of the embodiments herein.

[0023] In another aspect, provided herein is a method of treating or preventing a bone marrow disorder in a subject, comprising administering to the subject an effective amount of an isolated immunoresponsive cell described in any of the embodiments herein, or a pharmaceutical composition described in any of the embodiments herein, optionally wherein the bone marrow disorder is myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelomonocytic leukemia, acute myeloid leukemia (AML), acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, chronic myelogenous leukemia, and polycythemia vera.

[0024] In another aspect, provided herein is a kit for treating and / or preventing a bone marrow disorder, comprising an isolated immunoresponsive cell as described in any of the embodiments herein, or a pharmaceutical composition as described in any of the embodiments herein, optionally wherein the kit further comprises written instructions for using the cells to treat and / or prevent a bone marrow disorder in a subject.

[0025] In one aspect, provided herein is a chimeric receptor comprising an extracellular antigen-binding domain that binds to an antigen selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, and SPNS3.

[0026] In some embodiments, the antigen is MS4A3. In some embodiments, the antigen is VSTM1. In some embodiments, the antigen is LAT2. In some embodiments, the antigen is MLC1. In some embodiments, the antigen is CD131. In some embodiments, the antigen is GAPT. In some embodiments, the antigen is PRAM1. In some embodiments, the antigen is SLC22A16. In some embodiments, the antigen is SLC17A9. In some embodiments, the antigen is SPNS3.

[0027] In some embodiments, the chimeric receptor is a chimeric T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the chimeric receptor is a CAR.

[0028] In some embodiments, the CAR comprises one or more intracellular signaling domains selected from the group consisting of a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and a MyD88 intracellular signaling domain.

[0029] In some embodiments, the CAR comprises a transmembrane domain, wherein the transmembrane domain is selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 zeta chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, and a BTLA transmembrane domain.

[0030] In some embodiments, the CAR comprises a spacer region between the antigen binding domain and the transmembrane domain, wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-64.

[0031] In some embodiments, the antigen-binding domain comprises an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the VH and VL are separated by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.

[0032] In another aspect, provided herein is an isolated cell comprising the chimeric receptor of any one of the embodiments.

[0033] In some embodiments, the chimeric receptor is recombinantly expressed, hi some embodiments, the chimeric receptor is expressed from a selected locus from a vector or the genome of the cell.

[0034] In some embodiments, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell, a bone marrow cell, a macrophage, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, and an induced pluripotent stem cell (iPSC), and an iPSC-derived cell.

[0035] In some embodiments, the cells are autologous. In some embodiments, the cells are allogeneic.

[0036] In another aspect, provided herein is an isolated cell comprising: (a) a first chimeric receptor comprising an extracellular antigen-binding domain that binds a first antigen; and (b) a second chimeric receptor comprising an extracellular antigen-binding domain that binds a second antigen, wherein each antigen is selected from the group consisting of the antigens listed in Table 1; or wherein the first and second antigens are selected from the group consisting of antigen pairs listed in Table 3, and the first antigen is different from the second.

[0037] In another aspect, provided herein is an isolated cell comprising: (a) a first chimeric receptor comprising an extracellular antigen-binding domain that binds a first antigen; and

[0038] and (b) a second chimeric receptor comprising an extracellular antigen-binding domain that binds to a second antigen, wherein each antigen is selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70, and wherein the first antigen is different from the second antigen.

[0039] In some embodiments, the first antigen is MS4A3 and the second antigen is selected from the group consisting of VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0040] In some embodiments, the first antigen is VSTM1 and the second antigen is selected from the group consisting of MS4A3, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0041] In some embodiments, the first antigen is LAT2 and the second antigen is selected from the group consisting of MS4A3, VSTM1, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0042] In some embodiments, the first antigen is MLC1 and the second antigen is selected from the group consisting of MS4A3, VSTM1, LAT2, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0043] In some embodiments, the first antigen is CD131 and the second antigen is selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0044] In some embodiments, the first antigen is GAPT and the second antigen is selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0045] In some embodiments, the first antigen is PRAM1 and the second antigen is selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0046] In some embodiments, the first antigen is SLC22A16 and the second antigen is selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0047] In some embodiments, the first antigen is SLC17A9 and the second antigen is selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0048] In some embodiments, the first antigen is SPNS3 and the second antigen is selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0049] In some embodiments, the first antigen is FLT3. In some embodiments, the antigen-binding domain that binds to the first antigen is selected from the group consisting of (a) a VH comprising the amino acid sequence of SEQ ID NO: 1, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 2, or a sequence at least 90% identical thereto, (b) a VH comprising the amino acid sequence of SEQ ID NO: 3, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 4, or a sequence at least 90% identical thereto, (c) a VH comprising the amino acid sequence of SEQ ID NO: 5, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 6, or a sequence at least 90% identical thereto, (d) a VH comprising the amino acid sequence of SEQ ID NO: 7, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 8, or a sequence at least 90% identical thereto, (e) an VH comprising the amino acid sequence of SEQ ID NO: 9, or a sequence at least 90% identical thereto, (f) a VH comprising the amino acid sequence of SEQ ID NO: 11 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 12 or a sequence at least 90% identical thereto; (g) a VH comprising the amino acid sequence of SEQ ID NO: 13 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 14 or a sequence at least 90% identical thereto; and (h) a VH comprising the amino acid sequence of SEQ ID NO: 15 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 16 or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds to the first antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 1, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 2, or a sequence at least 90% identical thereto.In some embodiments, the antigen-binding domain that binds to the first antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 3, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 4, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds to the first antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 5, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 6, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds to the first antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 7, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 8, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds to the first antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 9, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 10, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds to the first antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 11, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 12, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds to the first antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 13, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds to the first antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 16, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the second antigen is CD33.In some embodiments that may be combined with any of the preceding embodiments, the antigen-binding domain that binds to the second antigen comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 18, or a sequence at least 90% identical thereto, and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the antigen-binding domain that binds to the second antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 18, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the antigen-binding domain that binds to the second antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 19, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 20, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the second antigen is CLECL12A. In some embodiments that may be combined with any of the preceding embodiments, the antigen-binding domain that binds to the second antigen comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 21, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 22, or a sequence at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 23, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 24, or a sequence at least 90% identical thereto; and (c) a VH comprising the amino acid sequence of SEQ ID NO: 25, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 26, or a sequence at least 90% identical thereto.In some embodiments that may be combined with any of the preceding embodiments, the antigen-binding domain that binds to the second antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 21, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 22, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the antigen-binding domain that binds to the second antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 23, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 24, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the antigen-binding domain that binds the second antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 25, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 26, or a sequence at least 90% identical thereto.

[0050] In some embodiments, the first antigen is CLECL12A. In some embodiments, the antigen-binding domain that binds to the first antigen comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 21, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 22, or a sequence at least 90% identical thereto, (b) a VH comprising the amino acid sequence of SEQ ID NO: 23, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 24, or a sequence at least 90% identical thereto, and (c) a VH comprising the amino acid sequence of SEQ ID NO: 25, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 26, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds to the first antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 21, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 22, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds to the first antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 23, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 24, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the second antigen is CD33.In some embodiments that may be combined with any of the preceding embodiments, the antigen-binding domain that binds to the second antigen comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 18, or a sequence at least 90% identical thereto, and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the antigen-binding domain that binds to the second antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 18, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the antigen-binding domain that binds to the second antigen comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 19, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 20, or a sequence at least 90% identical thereto.

[0051] In some embodiments, the cell is an immunoresponsive cell. In some embodiments, binding of a first chimeric receptor to a first antigen can activate the immunoresponsive cell. In some embodiments, binding of a second chimeric receptor to a second antigen can stimulate the immunoresponsive cell.

[0052] In some embodiments, binding of a first chimeric receptor to a first antigen and binding of a second chimeric receptor to a second antigen are required to activate an immunoresponsive cell.

[0053] In some embodiments, the immunoresponsive cells exhibit a greater degree of cytolytic activity against cells that are positive for both the first antigen and the second antigen compared to cells that are positive for the first antigen alone.

[0054] In some embodiments, binding of a first chimeric receptor to a first antigen or binding of a second chimeric receptor to a second antigen can activate an immunoresponsive cell.

[0055] In some embodiments, the first chimeric receptor binds to the first antigen with low binding affinity. In some embodiments, the first chimeric receptor binds to the first antigen with a binding affinity lower than the binding affinity with which the second chimeric receptor binds to the second antigen. In some embodiments, the first chimeric receptor binds to the first antigen with low avidity.

[0056] In some embodiments, the first chimeric receptor and / or the second chimeric receptor is a chimeric T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the first chimeric receptor and / or the second chimeric receptor is a CAR. In some embodiments, the first chimeric receptor is a first CAR and the second chimeric receptor is a second CAR.

[0057] In some embodiments, the CAR comprises one or more intracellular signaling domains selected from the group consisting of a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and a MyD88 intracellular signaling domain.

[0058] In some embodiments, one or more intracellular signaling domains of a first CAR are different from one or more intracellular signaling domains of a second CAR.

[0059] In some embodiments, the first CAR and the second CAR each comprise a CD3 zeta chain intracellular signaling domain.

[0060] In some embodiments, the first CAR and second CAR each further comprise an additional intracellular signaling domain selected from the group consisting of a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and a MyD88 intracellular signaling domain.

[0061] In some embodiments, the additional intracellular signaling domain of the first CAR is different from the additional intracellular signaling domain of the second CAR.

[0062] In some embodiments, each CAR comprises a transmembrane domain selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 zeta chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, and a BTLA transmembrane domain.

[0063] In some embodiments, the transmembrane domain of the first CAR is different from the transmembrane domain of the second CAR.

[0064] In some embodiments, each CAR comprises a spacer region between the antigen binding domain and the transmembrane domain, wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-64.

[0065] In some embodiments, the antigen-binding domain of the first chimeric receptor and / or the second chimeric receptor comprises an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the VH and VL are separated by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.

[0066] In some embodiments, the first chimeric receptor is recombinantly expressed.

[0067] In some embodiments, the first chimeric receptor is expressed from a selected locus from a vector or the genome of the cell.

[0068] In some embodiments, the second chimeric receptor is recombinantly expressed.

[0069] In some embodiments, the second chimeric receptor is expressed from a selected locus from the vector or the genome of the cell.

[0070] In some embodiments, the cells further comprise an inhibitory chimeric receptor comprising an antigen binding domain.

[0071] In some embodiments, the inhibitory chimeric receptor inhibits one or more activities of a cell.

[0072] In some embodiments, the inhibitory chimeric receptor binds to an antigen that is not expressed on tumor cells.

[0073] In some embodiments, the inhibitory chimeric receptor binds to an antigen expressed on a non-tumor cell.

[0074] In some embodiments, the inhibitory chimeric receptor binds to an antigen expressed on a non-tumor cell derived from a tissue selected from the group consisting of brain, nervous tissue, endocrine, bone, bone marrow, immune system, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0075] In some embodiments, the tumor cells are acute myeloid leukemia (AML) cells.

[0076] In some embodiments, the inhibitory chimeric receptor comprises an enzyme inhibitory domain.

[0077] In some embodiments, the enzyme inhibitory domain inhibits activation of an immunoreceptor when in proximity to the immunoreceptor.

[0078] In some embodiments, the enzyme inhibitory domain comprises an enzyme catalytic domain.

[0079] In some embodiments, the enzyme catalytic domain is derived from an enzyme selected from the group consisting of CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, and RasGAP.

[0080] In some embodiments, the inhibitory chimeric receptor further comprises one or more intracellular inhibitory co-signaling domains.

[0081] In some embodiments, the one or more intracellular inhibitory co-signaling domains are selected from the group consisting of PD-1, CTLA4, TIGIT, LAIR1, GRB-2, Dok-1, Dok-2, SLAP, LAG3, HAVR, BTLA, GITR, and PD-L1.

[0082] In some embodiments, the inhibitory chimeric receptor binds to an antigen selected from the group consisting of EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, and FFAR2.

[0083] In some embodiments, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell, a bone marrow cell, a macrophage, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, and an induced pluripotent stem cell (iPSC), and an iPSC-derived cell.

[0084] In some embodiments, the cells are autologous.

[0085] In some embodiments, the cells are allogeneic.

[0086] In another aspect, provided herein is a chimeric receptor comprising two or more antigen-binding domains, wherein each antigen-binding domain binds to an antigen selected from the group consisting of the antigens listed in Table 1, or wherein the two or more antigen-binding domains bind to an antigen pair selected from the group consisting of the antigen pairs listed in Table 3, and each antigen-binding domain binds to a different antigen.

[0087] In another aspect, provided herein is a chimeric receptor comprising two or more antigen-binding domains, wherein each antigen-binding domain binds to an antigen selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70, and wherein each antigen-binding domain binds to a different antigen.

[0088] In some embodiments, one antigen-binding domain binds to MS4A3 and the second antigen-binding domain binds to an antigen selected from the group consisting of VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0089] In some embodiments, one antigen-binding domain binds to VSTM1 and the second antigen-binding domain binds to an antigen selected from the group consisting of MS4A3, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0090] In some embodiments, one antigen-binding domain binds to LAT2 and the second antigen-binding domain binds to an antigen selected from the group consisting of MS4A3, VSTM1, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0091] In some embodiments, one antigen-binding domain binds to MLC1 and the second antigen-binding domain binds to an antigen selected from the group consisting of MS4A3, VSTM1, LAT2, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0092] In some embodiments, one antigen-binding domain binds to CD131 and the second antigen-binding domain binds to an antigen selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0093] In some embodiments, one antigen-binding domain binds to GAPT and the second antigen-binding domain binds to an antigen selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0094] In some embodiments, one antigen binding domain binds to PRAM1 and the second antigen binding domain binds to an antigen selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0095] In some embodiments, one antigen-binding domain binds to SLC22A16 and the second antigen-binding domain binds to an antigen selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0096] In some embodiments, one antigen-binding domain binds to SLC17A9 and the second antigen-binding domain binds to an antigen selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0097] In some embodiments, one antigen-binding domain binds to SPNS3 and the second antigen-binding domain binds to an antigen selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0098] In some embodiments, one antigen-binding domain binds to FLT3. In some embodiments, the antigen-binding domain that binds to FLT3 is selected from the group consisting of (a) a VH comprising the amino acid sequence of SEQ ID NO: 1, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 2, or a sequence at least 90% identical thereto, (b) a VH comprising the amino acid sequence of SEQ ID NO: 3, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 4, or a sequence at least 90% identical thereto, (c) a VH comprising the amino acid sequence of SEQ ID NO: 5, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 6, or a sequence at least 90% identical thereto, (d) a VH comprising the amino acid sequence of SEQ ID NO: 7, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 8, or a sequence at least 90% identical thereto, (e) a VH comprising the amino acid sequence of SEQ ID NO: 9, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 10, or a sequence at least 90% identical thereto, (f) a VH comprising the amino acid sequence of SEQ ID NO: 11, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 12, or a sequence at least 90% identical thereto, (g) a VH comprising the amino acid sequence of SEQ ID NO: 13, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto, (h) a VH comprising the amino acid sequence of SEQ ID NO: 1 (f) a VH comprising the amino acid sequence of SEQ ID NO: 11 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 12 or a sequence at least 90% identical thereto; (g) a VH comprising the amino acid sequence of SEQ ID NO: 13 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 14 or a sequence at least 90% identical thereto; and (h) a VH comprising the amino acid sequence of SEQ ID NO: 15 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 16 or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds to FLT3 comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 1, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 2, or a sequence at least 90% identical thereto.In some embodiments, the antigen-binding domain that binds to FLT3 comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 3, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 4, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds to FLT3 comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 5, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 6, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds to FLT3 comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 7, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 8, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds FLT3 comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 9, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 10, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds FLT3 comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 11, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 12, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds FLT3 comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 13, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds FLT3 comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 16, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the second antigen-binding domain binds CD33.In some embodiments that may be combined with any of the preceding embodiments, the second antigen-binding domain that binds CD33 comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 18, or a sequence at least 90% identical thereto, and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the second antigen-binding domain that binds CD33 comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 18, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the second antigen-binding domain that binds CD33 comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 19, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 20, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the second antigen-binding domain binds CLEC12A. In some embodiments that may be combined with any of the preceding embodiments, the second antigen-binding domain that binds to CLEC12A comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 21, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 22, or a sequence at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 23, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 24, or a sequence at least 90% identical thereto; and (c) a VH comprising the amino acid sequence of SEQ ID NO: 25, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 26, or a sequence at least 90% identical thereto.In some embodiments that may be combined with any of the preceding embodiments, the second antigen-binding domain that binds CLEC12A comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 21, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 22, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the second antigen-binding domain that binds CLEC12A comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 23, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 24, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the second antigen-binding domain that binds CLEC12A comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 25, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 26, or a sequence at least 90% identical thereto.

[0099] In some embodiments, one antigen-binding domain binds to CLEC12A. In some embodiments, the antigen-binding domain that binds to CLEC12A comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 21, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 22, or a sequence at least 90% identical thereto, (b) a VH comprising the amino acid sequence of SEQ ID NO: 23, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 24, or a sequence at least 90% identical thereto, and (c) a VH comprising the amino acid sequence of SEQ ID NO: 25, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 26, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds to CLEC12A comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 21, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 22, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain that binds to CLEC12A comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 23, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 24, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the second antigen-binding domain binds CD33.In some embodiments that may be combined with any of the preceding embodiments, the second antigen-binding domain that binds CD33 comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 18, or a sequence at least 90% identical thereto, and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the second antigen-binding domain that binds CD33 comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 18, or a sequence at least 90% identical thereto. In some embodiments that may be combined with any of the preceding embodiments, the second antigen-binding domain that binds CD33 comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 19, or a sequence at least 90% identical thereto, and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 20, or a sequence at least 90% identical thereto.

[0100] In some embodiments, the chimeric receptor is a chimeric T cell receptor or a chimeric antigen receptor (CAR).

[0101] In some embodiments, the chimeric receptor is a CAR.

[0102] In some embodiments, the CAR is a bispecific CAR.

[0103] In some embodiments, the CAR comprises one or more intracellular signaling domains selected from the group consisting of a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and a MyD88 intracellular signaling domain.

[0104] In some embodiments, the CAR comprises a transmembrane domain, wherein the transmembrane domain is selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 zeta chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, and a BTLA transmembrane domain.

[0105] In some embodiments, the CAR comprises a spacer region between the antigen binding domain and the transmembrane domain, wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-64.

[0106] In some embodiments, each antigen-binding domain comprises an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the VH and VL are separated by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.

[0107] In another aspect, provided herein is an isolated cell comprising the chimeric receptor of any one of the embodiments.

[0108] In some embodiments, the cell further comprises an additional chimeric receptor comprising an antigen binding domain.

[0109] In some embodiments, the additional chimeric receptor binds to an antigen selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0110] In some embodiments, each of the two chimeric receptors binds a different antigen.

[0111] In some embodiments, the cell is an immunoresponsive cell.

[0112] In some embodiments, binding of the chimeric receptor to either of the two antigens can activate immunoresponsive cells.

[0113] In some embodiments, binding of the additional chimeric receptor to its cognate antigen can stimulate immunoresponsive cells.

[0114] In some embodiments, binding of a chimeric receptor to one of two antigens and binding of an additional chimeric receptor to its cognate antigen is required to activate an immunoresponsive cell.

[0115] In some embodiments, immunoresponsive cells exhibit a greater degree of cytolytic activity against cells that are positive for either of the two antigens bound by the chimeric receptor and positive for the antigen bound by the additional chimeric receptor, compared to cells that are positive for only a single antigen.

[0116] In some embodiments, the additional chimeric receptor is a chimeric T cell receptor or a chimeric antigen receptor (CAR).

[0117] In some embodiments, the additional chimeric receptor is a CAR.

[0118] In some embodiments, the chimeric receptor is a first CAR and the additional chimeric receptor is a second CAR.

[0119] In some embodiments, the second CAR comprises one or more intracellular signaling domains selected from the group consisting of a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and a MyD88 intracellular signaling domain.

[0120] In some embodiments, one or more intracellular signaling domains of a first CAR are different from one or more intracellular signaling domains of a second CAR.

[0121] In some embodiments, the first CAR and the second CAR each comprise a CD3 zeta chain intracellular signaling domain.

[0122] In some embodiments, the first CAR and second CAR each further comprise an additional intracellular signaling domain selected from the group consisting of a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and a MyD88 intracellular signaling domain.

[0123] In some embodiments, the additional intracellular signaling domain of the first CAR is different from the additional intracellular signaling domain of the second CAR.

[0124] In some embodiments, the second CAR comprises a transmembrane domain, wherein the transmembrane domain is selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 zeta chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, and a BTLA transmembrane domain.

[0125] In some embodiments, the transmembrane domain of the first CAR is different from the transmembrane domain of the second CAR.

[0126] In some embodiments, the second CAR comprises a spacer region between the antigen-binding domain and the transmembrane domain, wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-64.

[0127] In some embodiments, the cells further comprise an inhibitory chimeric receptor comprising an antigen binding domain.

[0128] In some embodiments, the additional chimeric receptor is an inhibitory chimeric receptor that comprises an antigen-binding domain.

[0129] In some embodiments, the inhibitory chimeric receptor inhibits one or more activities of a cell.

[0130] In some embodiments, the inhibitory chimeric receptor binds to an antigen that is not expressed on tumor cells.

[0131] In some embodiments, the inhibitory chimeric receptor binds to an antigen expressed on a non-tumor cell.

[0132] In some embodiments, the inhibitory chimeric receptor binds to an antigen expressed on a non-tumor cell derived from a tissue selected from the group consisting of brain, nervous tissue, endocrine, bone, bone marrow, immune system, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0133] In some embodiments, the tumor cells are acute myeloid leukemia (AML) cells.

[0134] In some embodiments, the inhibitory chimeric receptor comprises an enzyme inhibitory domain.

[0135] In some embodiments, the enzyme inhibitory domain inhibits activation of an immunoreceptor when in proximity to the immunoreceptor.

[0136] In some embodiments, the enzyme inhibitory domain comprises an enzyme catalytic domain.

[0137] In some embodiments, the enzyme catalytic domain is derived from an enzyme selected from the group consisting of CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, and RasGAP.

[0138] In some embodiments, the inhibitory chimeric receptor further comprises one or more intracellular inhibitory co-signaling domains.

[0139] In some embodiments, the one or more intracellular inhibitory co-signaling domains are selected from the group consisting of PD-1, CTLA4, TIGIT, LAIR1, GRB-2, Dok-1, Dok-2, SLAP, LAG3, HAVR, BTLA, GITR, and PD-L1.

[0140] In some embodiments, the inhibitory chimeric receptor binds to an antigen selected from the group consisting of EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, and FFAR2.

[0141] In some embodiments, the antigen-binding domain of the additional chimeric receptor and / or the inhibitory chimeric receptor comprises an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the VH and VL are separated by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.

[0142] In some embodiments, each chimeric receptor is recombinantly expressed.

[0143] In some embodiments, each chimeric receptor is expressed from a selected locus from the vector or the genome of the cell.

[0144] In some embodiments, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell, a bone marrow cell, a macrophage, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, and an induced pluripotent stem cell (iPSC), and an iPSC-derived cell.

[0145] In some embodiments, the cells are autologous.

[0146] In some embodiments, the cells are allogeneic.

[0147] In another aspect, provided herein is a chimeric inhibitory receptor comprising an extracellular antigen-binding domain that binds to an antigen selected from the group consisting of EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, and FFAR2.

[0148] In some embodiments, the antigen is EMCN.

[0149] In some embodiments, the antigen is JAM2.

[0150] In some embodiments, the antigen is MS4A15.

[0151] In some embodiments, the antigen is C4BPA.

[0152] In some embodiments, the antigen is TRPM1.

[0153] In some embodiments, the antigen is SCTR.

[0154] In some embodiments, the antigen is SLC2A2.

[0155] In some embodiments, the antigen is KCNQ2.

[0156] In some embodiments, the antigen is PERP.

[0157] In some embodiments, when expressed on a cell, the inhibitory chimeric receptor inhibits one or more activities of the cell.

[0158] In some embodiments, the antigen is not expressed on tumor cells.

[0159] In some embodiments, the antigen is expressed on a non-tumor cell.

[0160] In some embodiments, the antigen is expressed on a non-tumor cell derived from a tissue selected from the group consisting of brain, nervous tissue, endocrine, bone, bone marrow, immune system, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0161] In some embodiments, the inhibitory chimeric receptor comprises an enzyme inhibitory domain.

[0162] In some embodiments, the enzyme inhibitory domain inhibits activation of an immunoreceptor when in proximity to the immunoreceptor.

[0163] In some embodiments, the enzyme inhibitory domain comprises an enzyme catalytic domain.

[0164] In some embodiments, the enzyme catalytic domain is derived from an enzyme selected from the group consisting of CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, and RasGAP.

[0165] In some embodiments, the inhibitory chimeric receptor further comprises one or more intracellular inhibitory co-signaling domains.

[0166] In some embodiments, the one or more intracellular inhibitory co-signaling domains are selected from the group consisting of PD-1, CTLA4, TIGIT, LAIR1, GRB-2, Dok-1, Dok-2, SLAP, LAG3, HAVR, BTLA, GITR, and PD-L1.

[0167] In some embodiments, the antigen-binding domain comprises an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the VH and VL are separated by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.

[0168] In another aspect, provided herein is an isolated cell comprising the chimeric inhibitory receptor of any one of the embodiments.

[0169] In some embodiments, the chimeric inhibitory receptor is recombinantly expressed.

[0170] In some embodiments, the chimeric inhibitory receptor is expressed from a selected locus from a vector or the genome of the cell.

[0171] In some embodiments, the cells further comprise a chimeric receptor comprising an extracellular antigen-binding domain that binds to an antigen selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0172] In another aspect, provided herein is an isolated cell comprising: (a) a chimeric inhibitory receptor comprising an extracellular antigen-binding domain that binds a first antigen, wherein the first antigen is selected from the group consisting of EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, and FFAR2; and (b) a chimeric receptor comprising one or more extracellular antigen-binding domains, wherein each antigen-binding domain binds to an antigen selected from the group consisting of the antigens listed in Table 1.

[0173] In another aspect, provided herein is an isolated cell comprising: (a) a chimeric inhibitory receptor comprising an extracellular antigen-binding domain that binds a first antigen, wherein the first antigen is selected from the group consisting of EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, and FFAR2; and (b) a chimeric receptor comprising one or more extracellular antigen-binding domains, wherein each antigen-binding domain binds to a chimeric inhibitory receptor selected from the group consisting of EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, and FFAR2. and a chimeric receptor that binds to an antigen selected from the group consisting of 4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70.

[0174] In some embodiments, the chimeric receptor is a chimeric T cell receptor or a chimeric antigen receptor (CAR).

[0175] In some embodiments, the chimeric receptor is a CAR.

[0176] In some embodiments, the CAR comprises one or more intracellular signaling domains selected from the group consisting of a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and a MyD88 intracellular signaling domain.

[0177] In some embodiments, the CAR comprises a transmembrane domain, wherein the transmembrane domain is selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 zeta chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, and a BTLA transmembrane domain.

[0178] In some embodiments, the CAR comprises a spacer region between the antigen binding domain and the transmembrane domain, wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-64.

[0179] In some embodiments, the antigen-binding domain of the chimeric inhibitory receptor and / or chimeric receptor comprises an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the VH and VL are separated by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.

[0180] In some embodiments, the cell is an immunoresponsive cell.

[0181] In some embodiments, binding of the chimeric inhibitory receptor to the first antigen can inhibit an immunoresponsive cell.

[0182] In some embodiments, binding of the chimeric receptor to a second antigen can activate an immunoresponsive cell.

[0183] In some embodiments, the chimeric receptor binds the second antigen with low binding affinity.

[0184] In some embodiments, the chimeric receptor binds to the second antigen with a lower binding affinity than the binding affinity with which the chimeric inhibitory receptor binds to the first antigen.

[0185] In some embodiments, the chimeric receptor binds the first antigen with low avidity.

[0186] In some embodiments, the chimeric receptor is recombinantly expressed.

[0187] In some embodiments, the chimeric receptor is expressed from a selected locus from a vector or the genome of the cell.

[0188] In some embodiments, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell, a bone marrow cell, a macrophage, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, and an induced pluripotent stem cell (iPSC), and an iPSC-derived cell.

[0189] In some embodiments, the cells are autologous.

[0190] In some embodiments, the cells are allogeneic.

[0191] In another aspect, provided herein is an isolated nucleic acid encoding the chimeric receptor of any one of the embodiments.

[0192] In another aspect, provided herein is an isolated nucleic acid encoding the chimeric receptor of any one of the embodiments.

[0193] In another aspect, provided herein is an isolated nucleic acid encoding the chimeric inhibitory receptor of any one of the embodiments.

[0194] In another aspect, provided herein is a vector comprising the nucleic acid of claim 175.

[0195] In some embodiments, the vector further comprises a nucleic acid of the embodiments.

[0196] In another aspect, provided herein is a vector comprising a nucleic acid of the embodiments.

[0197] In another aspect, provided herein are genetically modified cells comprising the nucleic acids of the embodiments.

[0198] In some embodiments, the cells further comprise a nucleic acid of the embodiments.

[0199] In another aspect, provided herein are genetically modified cells comprising the nucleic acids of the embodiments.

[0200] In another aspect, provided herein is a genetically engineered cell comprising the vector of any one of the embodiments.

[0201] In another aspect, provided herein is a method of reducing tumor burden in a subject, comprising administering to the subject an effective amount of the isolated cells described in any one of the embodiments.

[0202] In some embodiments, the method reduces the number of tumor cells.

[0203] In some embodiments, the method reduces tumor size.

[0204] In some embodiments, the method eradicates the tumor in the subject.

[0205] In another aspect, provided herein is a method of treating or preventing a bone marrow disorder in a subject, comprising administering to the subject an effective amount of the isolated cells described in any one of the embodiments.

[0206] In some embodiments, the bone marrow disorder is myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelomonocytic leukemia, acute myeloid leukemia (AML), acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, chronic myelogenous leukemia, and polycythemia vera.

[0207] In some embodiments, the bone marrow disorder is acute myeloid leukemia (AML).

[0208] In some embodiments, the method reduces or eradicates tumor burden in the subject.

[0209] In another aspect, provided herein is a pharmaceutical composition comprising an effective amount of the isolated cell of any one of the embodiments and a pharmaceutically acceptable excipient.

[0210] In some embodiments, the pharmaceutical composition is for treating and / or preventing a bone marrow disorder.

[0211] In another aspect, provided herein is a kit for treating and / or preventing a bone marrow disorder, comprising the isolated cells of any one of the embodiments.

[0212] In some embodiments, the kit further comprises written instructions for using the cells to treat and / or prevent a bone marrow disorder in a subject.

[0213] In another aspect, provided herein is a kit for treating and / or preventing a bone marrow disorder, comprising an isolated nucleic acid according to any one of the embodiments.

[0214] In some embodiments, the kit further comprises written instructions for using the nucleic acid to produce one or more antigen-specific cells for treating and / or preventing a bone marrow disorder in a subject.

[0215] In another aspect, provided herein is a kit for treating and / or preventing a bone marrow disorder, comprising a vector according to any one of the embodiments.

[0216] In some embodiments, the kit further comprises written instructions for using the vector to produce one or more antigen-specific cells for treating and / or preventing a bone marrow disorder in a subject.

[0217] In another aspect, provided herein is a method for treating and / or preventing bone marrow disorders, comprising administering an effective amount of at least one antibody that binds to an antigen, wherein the antigen is selected from the group consisting of MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, and SPNS3.

[0218] In some embodiments, the bone marrow disorder is selected from the group consisting of myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelomonocytic leukemia, or acute myeloid leukemia (AML), acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, chronic myelogenous leukemia, and polycythemia vera.

[0219] In some embodiments, the bone marrow disorder is acute myeloid leukemia (AML).

[0220] In some embodiments, the method reduces or eradicates tumor burden in the subject.

[0221] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0222] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description and accompanying drawings. [The present invention 1001] 1. An isolated immunoresponsive cell, comprising: (a) a first chimeric receptor comprising an extracellular antigen-binding domain that binds to a first antigen; (b) a second chimeric receptor comprising an extracellular antigen-binding domain that binds a second antigen; and Including, each antigen is selected from the group consisting of FLT3, CD33, CLEC12A, MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70; the first antigen is different from the second antigen; The isolated immunoresponsive cell. [The present invention 1002] the first antigen is FLT3, and the extracellular antigen-binding domain of the first chimeric receptor is (a) a VH comprising the amino acid sequence of SEQ ID NO: 3 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 4 or a sequence at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 90% identical thereto; (c) a VH comprising the amino acid sequence of SEQ ID NO: 5 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 6 or a sequence at least 90% identical thereto; (d) a VH comprising the amino acid sequence of SEQ ID NO: 7 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 8 or a sequence at least 90% identical thereto; (e) a VH comprising the amino acid sequence of SEQ ID NO: 9 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 10 or a sequence at least 90% identical thereto; (f) a VH comprising the amino acid sequence of SEQ ID NO: 11 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 12 or a sequence at least 90% identical thereto; (g) a VH comprising the amino acid sequence of SEQ ID NO: 13 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 14 or a sequence at least 90% identical thereto; and (h) a VH comprising the amino acid sequence of SEQ ID NO: 15 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 16 or a sequence at least 90% identical thereto; 1001. An isolated immunoresponsive cell of the present invention, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: [The present invention 1003] i) the second antigen is CD33 and the extracellular antigen-binding domain of the second chimeric receptor comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 17 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 18 or a sequence at least 90% identical thereto; and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20 or a sequence at least 90% identical thereto; or comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: ii) the second antigen is CLEC12A; (a) a VH comprising the amino acid sequence of SEQ ID NO: 21 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 22 or a sequence at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 23 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 24 or a sequence at least 90% identical thereto; and (c) a VH comprising the amino acid sequence of SEQ ID NO: 25 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 26 or a sequence at least 90% identical thereto; comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: 1002. An isolated immunoresponsive cell of the present invention. [The present invention 1004] the first antigen is CLEC12A, and the extracellular antigen-binding domain of the first chimeric receptor is (a) a VH comprising the amino acid sequence of SEQ ID NO: 21 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 22 or a sequence at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 23 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 24 or a sequence at least 90% identical thereto; and (c) a VH comprising the amino acid sequence of SEQ ID NO: 25 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 26 or a sequence at least 90% identical thereto; a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: Optionally, the second antigen is CD33 and the extracellular antigen-binding domain of the second chimeric receptor is (a) a VH comprising the amino acid sequence of SEQ ID NO: 17 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 18 or a sequence at least 90% identical thereto; and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20 or a sequence at least 90% identical thereto; comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: 1001. An isolated immunoresponsive cell of the present invention. [The present invention 1005] i) binding of the first chimeric receptor to the first antigen is capable of activating the immunoresponsive cell, and / or binding of the second chimeric receptor to the second antigen is capable of stimulating the immunoresponsive cell, and / or ii) the immunoresponsive cells exhibit a greater degree of cytolytic activity against target cells that are positive for both the first antigen and the second antigen compared to cytolytic activity against target cells that are positive for only the first antigen or only the second antigen; and / or iii) the first chimeric receptor binds to the first antigen with a binding affinity that is lower than the binding affinity of the second chimeric receptor for the second antigen; and / or iv) the first chimeric receptor binds the first antigen with low avidity; An isolated immunoresponsive cell according to any one of 1001 to 1004 of the present invention. [The present invention 1006] the first chimeric receptor is a first CAR; the second chimeric receptor is a second CAR; Each car, i) comprises a CD3 zeta chain intracellular signaling domain, and optionally each CAR further comprises one or more additional intracellular signaling domains selected from the group consisting of a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, and a 2B4 intracellular signaling domain; and / or ii) comprises a transmembrane domain, wherein the transmembrane domain is selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 zeta chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, and a BTLA transmembrane domain; and / or iii) comprising a spacer region between the antigen-binding domain and the transmembrane domain, wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 55 to 64. An isolated immunoresponsive cell according to any one of 1001 to 1005 of the present invention. [The present invention 1007] The isolated immunoresponsive cell of any of claims 1001 to 1006, wherein the cell further comprises an inhibitory chimeric receptor comprising an antigen-binding domain, and optionally, the inhibitory chimeric receptor inhibits one or more activities of the cell. [The present invention 1008] The isolated immunoresponsive cell of the present invention 1007, wherein the inhibitory chimeric receptor binds to an antigen expressed on a non-tumor cell, and optionally the antigen expressed on the non-tumor cell is derived from a tissue selected from the group consisting of brain, nervous tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin. [The present invention 1009] 1007. The isolated immunoresponsive cell of claim 1008, wherein the chimeric inhibitory receptor binds to an antigen selected from the group consisting of EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR2, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, and TMEM200A. [The present invention 1010] 1009. The isolated immunoresponsive cell of any one of claims 1007 to 1009, wherein the inhibitory chimeric receptor comprises an antigen-binding domain comprising a single-chain variable fragment (scFv), and the scFv is derived from an anti-EMCN antibody. [The present invention 1011] The isolated immunoresponsive cell of any of claims 1007 to 1010, wherein the antigen-binding domain of the first chimeric receptor, the antigen-binding domain of the second chimeric receptor, and / or the antigen-binding domain of the inhibitory chimeric receptor comprises one or more single-chain variable fragments (scFv), each of the one or more scFvs comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), optionally wherein the VH and VL are separated by a peptide linker, and optionally wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 27. [The present invention 1012] 1011. The isolated immunoresponsive cell of the present invention, wherein each of the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, where VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain. [The present invention 1013] The isolated immunoresponsive cell of invention 1011 or invention 1012, wherein each of the one or more scFvs binds to a distinct epitope on the same antigen. [The present invention 1014] The isolated immunoresponsive cell of any of claims 1011 to 1013, wherein each of the one or more scFvs is separated by a peptide linker, and optionally, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 27) or EAAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 74). [The present invention 1015] the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell, a bone marrow cell, a macrophage, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, and an induced pluripotent stem cell (iPSC), and an iPSC-derived cell; Optionally, the immunoresponsive cells are allogeneic. An isolated immunoresponsive cell according to any one of claims 1001 to 1014 of the present invention. [The present invention 1016] A pharmaceutical composition comprising an effective amount of any of the isolated immunoresponsive cells of the present invention 1001 to 1015, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. [The present invention 1017] 1. A method of providing anti-tumor immunity in a subject, comprising: Administering to a subject in need thereof a therapeutically effective amount of any of the isolated immunoresponsive cells of the present inventions 1001 to 1015 or the pharmaceutical composition of the present invention 1016. The method. [The present invention 1018] 1. A method of treating or preventing bone marrow disorders in a subject, comprising: Administering to the subject an effective amount of any of the isolated immunoresponsive cells of the present inventions 1001 to 1015 or the pharmaceutical composition of the present invention 1016, Optionally, the bone marrow disorder is myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelomonocytic leukemia, acute myeloid leukemia (AML), acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, chronic myelogenous leukemia, and polycythemia vera. The method. [The present invention 1019] A kit for treating and / or preventing bone marrow disorders, comprising the isolated immunoresponsive cell of any one of the present inventions 1001 to 1015 or the pharmaceutical composition of the present invention 1016, Optionally, the kit further comprises written instructions for using the cells to treat and / or prevent a bone marrow disorder in a subject. [Brief explanation of the drawings]

[0223] [Figure 1] Microarray and RNA-Seq data for FLT3 expression in the indicated tissues or cell types are provided. [Figure 2] Microarray and RNA-Seq data for MS4A3 expression in the indicated tissues or cell types are provided. [Figure 3]Microarray and RNA-Seq data for CD33 expression in the indicated tissues or cell types are provided. [Figure 4] Microarray and RNA-Seq data for CLEC12A expression in the indicated tissues or cell types are provided. [Figure 5] Microarray and RNA-Seq data for CD312 / ADGRE2 expression in the indicated tissues or cell types are provided. [Figure 6] Microarray and RNA-Seq data for SLC22A16 expression in the indicated tissues or cell types are provided. [Figure 7] Microarray and RNA-Seq data for CD123 / ILR3RA expression in the indicated tissues or cell types are provided. [Figure 8] Microarray and RNA-Seq data for LAT2 expression in the indicated tissues or cell types are provided. [Figure 9] Microarray and RNA-Seq data for PIEZO1 / FAM38A expression in the indicated tissues or cell types are provided. [Figure 10] Microarray and RNA-Seq data for CD38 expression in the indicated tissues or cell types are provided. [Figure 11] Microarray and RNA-Seq data for EMB expression in the indicated tissues or cell types are provided. [Figure 12] Microarray and RNA-Seq data for CD131 / CSF2RB expression in the indicated tissues or cell types are provided. [Figure 13] Microarray and RNA-Seq data for P2RY8 expression in the indicated tissues or cell types are provided. [Figure 14] Microarray and RNA-Seq data for LILRA2 / CD85H expression in the indicated tissues or cell types are provided. [Figure 15]Microarray and RNA-Seq data for SLC17A9 expression in the indicated tissues or cell types are provided. [Figure 16] Microarray and RNA-Seq data for MYADM expression in the indicated tissues or cell types are provided. [Figure 17] Microarray and RNA-Seq data for CD300LF expression in the indicated tissues or cell types are provided. [Figure 18] Microarray and RNA-Seq data for CD244 / SLAMF4 expression in the indicated tissues or cell types are provided. [Figure 19] Microarray and RNA-Seq data for PLAUR expression in the indicated tissues or cell types are provided. [Figure 20] Microarray and RNA-Seq data for CD93 expression in the indicated tissues or cell types are provided. [Figure 21] Microarray and RNA-Seq data for SPNS3 expression in the indicated tissues or cell types are provided. [Figure 22] Microarray and RNA-Seq data for GAPT expression in the indicated tissues or cell types are provided. [Figure 23] Microarray and RNA-Seq data for RASGRP4 expression in the indicated tissues or cell types are provided. [Figure 24] Microarray and RNA-Seq data for CD117 / c-Kit expression in the indicated tissues or cell types are provided. [Figure 25] Microarray and RNA-Seq data for CD123 / ILR3RA expression in the indicated tissues or cell types are provided. [Figure 26] Microarray and RNA-Seq data for EMCN (Endomucin) expression in the indicated tissues or cell types are provided. [Figure 27]Microarray and RNA-Seq data for JAM2 expression in the indicated tissues or cell types are provided. [Figure 28] Microarray and RNA-Seq data for MS4A15 expression in the indicated tissues or cell types are provided. [Figure 29] Microarray and RNA-Seq data for SLC34A2 expression in the indicated tissues or cell types are provided. [Figure 30] Microarray and RNA-Seq data for SLC2A2 expression in the indicated tissues or cell types are provided. [Figure 31] Microarray and RNA-Seq data for TRPM1 expression in the indicated tissues or cell types are provided. [Figure 32] Microarray and RNA-Seq data for SCTR expression in the indicated tissues or cell types are provided. [Figure 33] Microarray and RNA-Seq data for KCNQ2 expression in the indicated tissues or cell types are provided. [Figure 34] Microarray and RNA-Seq data for PERP expression in the indicated tissues or cell types are provided. [Figure 35] A shows the % of cells expressing YFP and FLT3 CAR from donor 1 for each of the six FLT3 scFvs tested. B shows the mean fluorescence intensity of YFP-positive cells from donor 1 for each of the six FLT3 scFvs tested. C shows the % of cells expressing YFP and FLT3 CAR from donor 2 for each of the six FLT3 scFvs tested. D shows the mean fluorescence intensity of YFP-positive cells from donor 2 for each of the six FLT3 scFvs tested. [Figure 36]A shows that FLT3 CAR T cells from donor 1 induced the killing of SEM cells. B shows that FLT3 CAR T cells from donor 1 induced the killing of MOLM13 cells. C shows that FLT3 CAR T cells from donor 2 induced the killing of SEM cells. D shows that FLT3 CAR T cells from donor 2 induced the killing of MOLM13 cells. [Figure 37] A shows TNFα secretion by FLT3 CAR T cells from donor 1 after culture with SEM cells. B shows IFNγ secretion by FLT3 CAR T cells from donor 1 after culture with SEM cells. C shows IL-2 secretion by FLT3 CAR T cells from donor 1 after culture with SEM cells. D shows TNFα secretion by FLT3 CAR T cells from donor 2 after culture with SEM cells. E shows IFNγ secretion by FLT3 CAR T cells from donor 2 after culture with SEM cells. F shows IL-2 secretion by FLT3 CAR T cells from donor 1 after culture with SEM cells. [Figure 38] A shows TNFα secretion by FLT3 CAR T cells from donor 1 after culture with MOLM-13 cells. B shows IFNγ secretion by FLT3 CAR T cells from donor 1 after culture with MOLM-13 cells. C shows IL-2 secretion by FLT3 CAR T cells from donor 1 after culture with MOLM-13 cells. D shows TNFα secretion by FLT3 CAR T cells from donor 2 after culture with MOLM-13 cells. E shows IFNγ secretion by FLT3 CAR T cells from donor 2 after culture with MOLM-13 cells. F shows IL-2 secretion by FLT3 CAR T cells from donor 1 after culture with MOLM-13 cells. [Figure 39] 1 shows a heat mat of FLT3, CD33, and CLEC12A microarray expression data in various tissues and / or cell lines. [Figure 40]A shows flow cytometry histogram plots depicting FLT3 protein expression (x-axis) in the AML cell lines MOLM-13, THP-1, and the ALL cell line SEM. B shows that FLT3 CAR T cells killed MOLM-13, THP-1, and SEM human leukemia cells. C shows dose-dependent FLT3 CAR T cell killing of MOLM-13 AML cells. [Figure 41] A shows that FLT3 CAR T cells induced TNF-α, IFN-γ, and IL-2 production after culture with MOLM-14 cells. B shows that FLT3 CAR T cells induced TNF-α, IFN-γ, and IL-2 production after culture with MOLM-13 cells. C shows that FLT3 CAR T cells induced TNF-α, IFN-γ, and IL-2 production after culture with EOL-1 cells. [Figure 42] (A) Flow cytometry histogram plot showing expression of CD33 protein expression (x-axis) in AML cell lines MOLM-13, MV4-1, and THP-1. (B) CD33 CAR T cells killed MOLM-13, MV-11, and THP-1 human leukemia cells. (C) Dose-dependent CD33 CAR T cell killing of MOLM-13 AML cells by both CD33 CAR T cells. [Figure 43] A shows that CD33 CAR T cells induced TNF-α, IFN-γ, and IL-2 production after culture with MOLM-13 cells. B shows that CD33 CAR T cells induced TNF-α, IFN-γ, and IL-2 production after culture with MV4-11 cells. C shows that CD33 CAR T cells induced TNF-α, IFN-γ, and IL-2 production in THP-1 cells. [Figure 44] Flow cytometry plots of CLEC12A expression in U937, THP-1, HL-60, MV-14, MOLM-14, MOLM-13, Nalm6, Raji, K562, and SEM cell lines are shown. [Figure 45](A) Flow cytometry histogram plot showing the expression of CLEC12A protein expression in four AML cell lines. (B) In vitro coculture cytotoxicity assay showing that CLEC12A CAR T cells kill human AML cells (E:T ratio 1:1) with percent killing activity on the y-axis. (C) CLEC12A CAR T cells have potent cytokine secretion (IL-2) when cocultured with human acute myeloid leukemia (AML) cell lines, as detected by Luminex assay. CAR#1 = SB01261, CLEC12A(357) CAR T cells; CAR#2 = SB01262, CLEC12A(378) CAR T cells; CAR#3 = SB01263, CLEC12A(161) CAR T cells. [Figure 46] A shows the cytotoxic activity and cytokine production of CLEC12A CAR T cells against MOLM-13 (low CLEC12A expression). B shows the cytotoxic activity and cytokine production of CLEC12A CAR T cells against MV4-11 (low CLEC12A expression). C shows the cytotoxic activity and cytokine production of CLEC12A CAR T cells against MOLM-14 (high CLEC12A expression). D shows the cytotoxic activity and cytokine production of CLEC12A CAR T cells against U937. E shows the cytotoxic activity and cytokine production of CLEC12A CAR T cells against THP-1. F shows the cytotoxic activity and cytokine production of CLEC12A CAR T cells against HL-60. Each CLEC12A CAR contained a CD8 hinge and transmembrane domain and 4-1 BB and CD3ζ costimulatory domains. The FLT3(NC7) CAR contained a 4-1BB costimulatory domain. The CD33(hu195)CAR had a CD28 costimulatory domain. [Figure 47] A schematic diagram of the synthesized CLEC12A CAR is provided. [Figure 48] A shows transduction of the indicated CLEC12A CARs in T cells. B shows mean fluorescence intensity (MFI) of the indicated CLEC12A CARs in T cells. [Figure 49]Shows the cytotoxicity of the indicated CLEC12A CART cells against HL-60, MOLM14, MV4-11, and MOLM-13 cells, as indicated by the killing rate of the cell lines after culture with CAR T cells. [Figure 50] Shows IL-2 production of the indicated CLEC12A CAR T cells after culture with HL-60, MOLM14, MV4-11, MOLM-13, and K562 cells. [Figure 51] Shown is IFN-γ production of the indicated CLEC12A CAR T cells after culture with HL-60, MOLM14, MV4-11, MOLM-13, and K562 cells. [Figure 52] Shown is TNF-α production of the indicated CLEC12A CAR T cells after culture with HL-60, MOLM14, MV4-11, MOLM-13, and K562 cells. [Figure 53-1]A shows fLuc bioluminescence in non-tumor mice treated with CD33 CAR T cells. B shows fLuc bioluminescence in non-tumor mice treated with FLT3 CAR T cells. C shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with control unmanipulated T cells. The endpoint criterion was reached on day 14, and the mice were sacrificed. D shows fLuc bioluminescence in non-tumor mice treated with control unmanipulated T cells. E shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with FLT3 CAR T cells once on day 5. F shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with FLT3 CAR T cells once on day 8. The endpoint criterion was reached on day 14, and the mice were sacrificed. G shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with FLT3 CAR T cells twice on days 5 and 12. H shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with CD33 CAR T cells once on day 5. I shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with CD33 CAR T cells once on day 8. The endpoint criteria were reached on day 14, and mice were sacrificed. J shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with CD33 CAR T cells twice on days 5 and 12. K provides images of in vivo fLuc bioluminescence in each treatment group. [Figure 53-2] See description of Figure 53-1. [Figure 54-1](A) shows in vivo imaging results of a T cell dose-escalation assay using FLT3 CAR T cells and CD33 CAR T cells alone or in combination. (B) shows fLuc bioluminescence in tumor-free mice treated with unengineered T cells. (C) shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with unengineered T cells. (D) shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with 9×10 FLT3 T cells. (E) shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with 18×10 FLT3 T cells. (F) shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with 0.625×10 CD33 T cells. (G) shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with 1.25×10 CD33 T cells. H shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with 2.5×10 CD33 T cells. I shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with 5×10 CD33 T cells. J shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with 10×10 CD33 T cells. K shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with 4.5×10 FLT3 CAR T cells and 2.5×10 CD33 T cells. L shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with 9×10 FLT3 CAR T cells and 5×10 CD33 T cells. M shows fLuc bioluminescence in MOLM-13 tumor-bearing mice treated with 18×10 6 FLT3 CAR T cells and 10×10 6 CD33 T cells. [Figure 54-2] See description of Figure 54-1. [Figure 55-1] A shows the fold change in relative bioluminescence of in vivo tumor MOLM-14 cells after treatment with the indicated CARs at day 7. B shows the fold change in relative bioluminescence of in vivo tumor MOLM-14 cells after treatment with the indicated CARs at day 11. C shows the bioluminescence of in vivo tumor MOLM-14 cells after treatment with the indicated CARs. [Figure 55-2] See description of Figure 55-1. [Figure 56] A provides a schematic of the indicated CARs with alternative hinge sequences. B shows the cytotoxicity and IL-2 production of the indicated CARs after culture with MOLM-13 cells. C shows the cytotoxicity and IL-2 production of the indicated CARs after culture with MOLM-14 cells. D shows the cytotoxicity and IL-2 production of the indicated CARs after culture with MV4-11 cells. E shows the cytotoxicity and IL-2 production of the indicated CARs after culture with SEM cells. [Figure 57] A shows the normalized killing rate relative to untransduced CAR T cells by the indicated CAR T cells after culture with MOLM-13 cells. B shows IL-2 production by the indicated CARs after culture with MOLM-13 cells. C shows IFN-γ production by the indicated CAR T cells after culture with MOLM-13 cells. D shows TNF-α production by the indicated CAR T cells after culture with MOLM-13 cells. [Figure 58A] A shows in vivo survival after treatment with the indicated CAR T cells in the MOLM-13 model. [Figure 58B] B shows bioluminescence of tumor MOLM-13 cells in vivo after treatment with the indicated CARs. [Figure 59] A shows the expression of two bicistronic FLT3 and CD33 CARs in T cells, SB01266 and SB01659. B shows the cytotoxicity and IL-2 production of the indicated CARs after culture with MOLM-13 cells. C shows the cytotoxicity and IL-2 production of the indicated CARs after culture with MV4-11 cells. D shows the cytotoxicity and IL-2 production of the indicated CARs after culture with SEM cells. [Figure 60] A shows FLT3 and CD33 staining in unengineered K562 cells, B shows FLT3 and CD33 staining in K562 cells engineered to express FLT3, and C shows FLT3 and CD33 staining in K562 cells engineered to express CD33. [Figure 61]Cytotoxicity, IL-2 production, IFN-γ production, and TNF-α production by monovalent FLT3 CAR T cells, monovalent CD33 CAR T cells, and bicistronic FLT3 and CD33 CAR T cells cultured with K562 cells expressing FLT3 (top panel), CD33 (middle panel), or a 1:1 mixture of cells expressing FLT3 and CD33 (bottom panel) are shown. [Figure 62] Cytotoxicity, IL-2 production, IFN-γ production, and TNF-α production by monovalent FLT3 CAR T cells, monovalent CD33 CAR T cells, and bicistronic FLT3 and CD33 CAR T cells cultured with MOLM-13 cells (top panel), MV4-11 cells (middle panel), or SEM cells (bottom panel) are shown. [Figure 63] A shows a schematic diagram of a loop FLT3 bivalent CAR. B shows a schematic diagram of a tandem FLT3 bivalent CAR. C shows the cytotoxicity of monovalent FLT3 CAR T cells, loop bivalent FLT3 CAR T cells, and tandem FLT3 CAR T cells after culture with MV4-11 cells (top) or SEM cells (bottom). [Figure 64] A shows the cytotoxicity and IL-2, IFN-γ, and TNF-α secretion of the indicated CAR constructs after culture with MOLM-13 cells. B shows the cytotoxicity and IL-2, IFN-γ, and TNF-α secretion of the indicated CAR constructs after culture with MV4-11 cells. C shows the cytotoxicity and IL-2, IFN-γ, and TNF-α secretion of the indicated CAR constructs after culture with CD33-expressing K562 cells. [Figure 65] A shows EMCN expression in the indicated cell types. B shows EMCN expression in HSCs and LSCs. C shows a summary of EMCN expression in HSCs and LSCs from multiple donors. [Figure 66]A shows FLT3, CD33, and FLT3 / CD33 CAR NK cell killing of SEM cells. B shows IFN-γ secretion by FLT3 CAR NK cells after culture with SEM cells. C shows TNF-α secretion by FLT3 CAR NK cells after culture with SEM cells. D shows FLT3, CD33, and FLT3 / CD33 CAR NK cell killing of PL-21 cells. DETAILED DESCRIPTION OF THE INVENTION

[0224] Detailed Description The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of molecular biology, chemistry, biochemistry, virology, and immunology within the skill of the art. Such techniques are fully described in the literature, e.g., Hepatitis C Viruses: Genomes and Molecular Biology (SLTan ed., Taylor & Francis, 2006), Fundamental Virology, 3 rd Edition, vol. I & II (BNFields and DMKnipe, eds.), Handbook of Experimental Immunology, Vols. I-IV (DMWeir and CC Blackwell eds., Blackwell Scientific Publications), ALLehninger, Biochemistry (Worth Publishers, Inc., current addition), Sambrook, et al., Molecular Cloning: A Laboratory Manual (3 rd Edition, 2001), Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

[0225] definition Unless otherwise defined, all terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by those skilled in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a difference to that commonly understood in the art. The techniques and procedures described or referenced herein are generally well known and commonly used by those skilled in the art using conventional methodologies, such as the widely used molecular cloning methodology described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and conditions unless otherwise specified.

[0226] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Terms such as "including," "e.g.," and the like are intended to convey inclusion without limitation unless otherwise indicated.

[0227] As used herein, the term "comprising" also specifically includes embodiments "consisting of" and "consisting essentially of" the listed elements, unless otherwise indicated.

[0228] The term "about" denotes and includes the indicated value and a range above and below that value. In certain embodiments, the term "about" denotes ±10%, ±5%, or ±1% of the specified value. In certain embodiments, where applicable, the term "about" denotes the specified value(s) ± one standard deviation around that value(s).

[0229] As used herein, the term "activating an immunoresponsive cell" refers to the induction of signal transduction or changes in protein expression in a cell that result in the initiation of an immune response. For example, clustering of CD3 chains in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs) generates a signaling cascade. In certain embodiments, binding of an endogenous TCR or an exogenous CAR to an antigen results in the formation of an immunological synapse, involving the clustering of many molecules in the vicinity of the bound receptor (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.). This clustering of membrane-bound signaling molecules allows the ITAM motifs contained within the CD3 chains to become phosphorylated. This phosphorylation then initiates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors increase IL-2 production for proliferation, induce the expression of key regulatory T cell proteins, and induce general gene expression in T cells to initiate a T cell-mediated immune response.

[0230] As used herein, the term "stimulating immunoresponsive cells" refers to signals that result in a robust and sustained immune response. In various embodiments, this is mediated following immune cell (e.g., T cell) activation or simultaneously through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, and ICOS. Without being bound by theory, receiving multiple stimulatory signals is important for initiating a robust and long-lasting T cell-mediated immune response. Without receiving these stimulatory signals, T cells are quickly inhibited and become unresponsive to antigens. While the effects of these costimulatory signals are varied and remain partially understood, they generally increase gene expression to generate long-lived, proliferative, and anti-apoptotic T cells that robustly respond to antigens for complete and sustained eradication.

[0231] As used herein, the term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that comprises a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, the domains within a CAR polypeptide construct are within the same polypeptide chain, e.g., comprising a chimeric fusion protein. In some embodiments, the domains within a CAR polypeptide construct are not adjacent to each other, but rather are within different polypeptide chains, e.g., as provided in the RCARs described herein. In one aspect, the stimulatory molecule is a zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., the primary signaling domain of CD3-zeta). In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one aspect, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, ICOS, and / or CD28. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule.In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, where the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

[0232] As used herein, the term "intracellular signaling domain" refers to a functional portion of a protein that acts by transmitting information intracellularly to regulate cellular activity through a defined signaling pathway, either by generating second messengers or by functioning as an effector by responding to such messengers. In some embodiments, the signaling domain of a chimeric receptor of the present disclosure is derived from a stimulatory molecule or costimulatory molecule described herein, or is a synthetic or engineered signaling domain.

[0233] The term "antibody," as used herein, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. The term "antibody," as used herein, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. An antibody may be a tetramer of immunoglobulin molecules.

[0234] As used herein, the term "antibody fragment" refers to at least a portion of an intact antibody, or a recombinant variant thereof, including an antigen-binding domain, such as an antigen-determining variable region of the intact antibody, sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAbs (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments such as two Fab fragments linked by a disulfide bridge at the hinge region, and bivalent fragments comprising isolated CDRs or other epitope-binding fragments of an antibody. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0235] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are linked via a short flexible polypeptide linker and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. As used herein, unless specified, an scFv can have the VL and VH variable regions in either order, e.g., with respect to the N- and C-termini of the polypeptide, and can comprise a VL-linker-VH or a VH-linker-VL.

[0236] As used herein, the term "complementarity-determining region" or "CDR" refers to a sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, there are typically three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3), and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any of several well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al. (1997) JMB 273, 927-948 ("Chothia" numbering scheme), or a combination thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both.For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of a VH, e.g., a mammalian VH, e.g., a human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of a VL, e.g., a mammalian VL, e.g., a human VL.

[0237] The portion of the chimeric receptor of the present disclosure comprising an antibody or antibody fragment thereof can exist in a variety of forms in which the antigen-binding domain is expressed as part of a contiguous polypeptide chain, including, for example, scFv antibody fragments, linear antibodies, single-domain antibodies such as sdAbs (either VL or VH), camelid VHH domains, humanized antibodies, bispecific antibodies, and antibody complexes (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen-binding domain of a chimeric receptor of the present disclosure comprises an antibody fragment. In a further embodiment, the chimeric receptor comprises an antibody fragment comprising an scFv.

[0238] As used herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations and which usually determine the class to which the antibody belongs.

[0239] As used herein, the term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0240] As used herein, the term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, where the DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, where the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

[0241] As used herein, the term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve either antibody production, or activation of cells with specific immunological capabilities, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can function as an antigen.

[0242] As used herein, the term "anti-tumor effect" or "anti-tumor activity" refers to a biological effect that can be manifested by various means, including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in lifespan, a reduction in tumor cell proliferation, a reduction in tumor cell viability, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present disclosure to prevent tumors from developing in the first place.

[0243] As used herein, the term "autologous" refers to any material derived from the same individual that is subsequently reintroduced into the individual.

[0244] As used herein, the term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to each other if the genes at one or more loci are not identical. In some embodiments, allogeneic materials derived from individuals of the same species may be sufficiently different to interact antigenically.

[0245] As used herein, the term "affinity" refers to a measure of binding strength. Without being bound by theory, affinity depends on the closeness of the stereochemical fit between the antibody binding site and the antigenic determinant, the size of the contact area between them, and the distribution of charged and hydrophobic groups. Affinity also includes the term "avidity," which refers to the strength of antigen-antibody binding after the formation of a reversible complex. Methods for calculating the affinity of an antibody to an antigen are known in the art, including the use of binding experiments to calculate affinity. Antibody activity in functional assays (e.g., flow cytometry assays) also reflects antibody affinity. Antibodies and affinities can be phenotypically characterized and compared using functional assays (e.g., flow cytometry assays).

[0246] As used herein, the term "immunosuppressive activity" refers to the induction of signaling or alteration of protein expression in cells, such as activated immune response cells, that results in a decrease in the immune response. Non-limiting examples of polypeptides known to suppress or decrease immune responses through their binding include CD47, PD-1, CTLA-4, and their corresponding ligands, including SIRPa, PD-L1, PD-L2, B7-1, and B7-2. Such polypeptides may be present in the tumor microenvironment and may inhibit the immune response against tumor cells. In various embodiments, inhibiting, blocking, or antagonizing the interaction of immunosuppressive polypeptides and / or their ligands may enhance the immune response of immunoresponsive cells.

[0247] As used herein, the term "enzyme inhibitory domain" refers to a protein domain that inhibits an intracellular signaling cascade, e.g., a natural T cell activation cascade. In some embodiments, the enzyme inhibitory domain of a chimeric inhibitory receptor of the present disclosure comprises at least a portion of the extracellular domain, the transmembrane domain, and / or the intracellular domain. In some embodiments, the enzyme inhibitory domain comprises at least a portion of an enzyme. In some embodiments, the enzyme is selected from CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, and RasGAP (see, e.g., Stanford et al., Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 Sep;137(1):1-19). In some embodiments, the portion of the enzyme comprises an enzyme domain(s), an enzyme fragment(s), or a variant(s) thereof. In some embodiments, the portion of the enzyme is the catalytic domain of the enzyme. In some embodiments, the enzyme domain(s), enzyme fragment(s), or variant(s) thereof are selected to maximize efficacy and minimize basal inhibition.

[0248] As used herein, the term "immunostimulatory activity" refers to the induction of signaling or changes in protein expression in cells, such as activated immunoresponsive cells, that result in an increased immune response. Immunostimulatory activity can include proinflammatory activity. Non-limiting examples of polypeptides known to stimulate or increase immune responses through their binding include CD28, OX-40, 4-1BB, and their corresponding ligands, including B7-1, B7-2, OX-40L, and 4-1BBL. Such polypeptides can be present in the tumor microenvironment and can activate an immune response against tumor cells. In various embodiments, promoting, stimulating, or receptor-stimulating proinflammatory polypeptides and / or their ligands can enhance the immune response of immunoresponsive cells.

[0249] Isolated nucleic acid molecules of the present disclosure include any nucleic acid molecule encoding a polypeptide of the present disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% homologous or identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A nucleic acid having "substantial identity" or "substantial homology" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. As used herein, "hybridization" refers to the pairing to form a double-stranded molecule between a complementary polynucleotide sequence (e.g., a gene described herein) or a portion thereof under various stringency conditions. For example, stringent salt concentrations can typically be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. While low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, high stringency hybridization can be achieved in the presence of at least about 35% formamide or at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30°C, at least about 37°C, or at least about 42°C. Various additional parameters, such as hybridization time, detergent concentration, such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed.

[0250] "Substantially identical" or "substantially homologous" means that a polypeptide or nucleic acid molecule exhibits at least 50% homology or identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least about 60%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% homologous or identical at the amino acid level or at the nucleic acid level to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following group: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine. An exemplary method for measuring the degree of identity may use the BLAST program, where a probability score of e-3 to e-100 indicates closely related sequences.

[0251] As used herein, the term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the 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, or a biological property resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA. Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may include introns, to the extent that a nucleotide sequence encoding a protein may, in some versions, contain intron(s).

[0252] As used herein, the term "ligand" refers to a molecule that binds to a receptor. Specifically, a ligand binds to a receptor on another cell, allowing for intercellular recognition and / or interaction.

[0253] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, substance, or composition described herein that is effective to achieve a particular biological result. In some embodiments, an "effective amount" or "therapeutically effective amount" is an amount sufficient to prevent, ameliorate, or inhibit the continued proliferation, growth, or metastasis of a disease or disorder of interest, e.g., a bone marrow disorder.

[0254] As used herein, the term "immunoresponsive cell" refers to a cell that functions in an immune response (e.g., an immune effector response) or a precursor, or progeny thereof. Examples of immune effector cells include, but are not limited to, alpha / beta T cells, gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0255] As used herein, the term "immune effector response" or "immune effector function" refers to a function or response, e.g., of an immunocompetent cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response can refer to a property of a T cell or NK cell that promotes the killing or inhibition of growth or proliferation of a target cell. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.

[0256] As used herein, the term "flexible polypeptide linker" or "linker" refers to a peptide linker consisting of amino acids such as glycine and / or serine residues, used alone or in combination, to link the variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker, having the amino acid sequence (Gly-Gly-Gly-Ser) n wherein n is a positive integer greater than 1 (SEQ ID NO: 224). For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, n=10. In some embodiments, the flexible polypeptide linker includes, but is not limited to, (Gly4Ser)4 (SEQ ID NO: 225) or (Gly4Ser)3 (SEQ ID NO: 226). In other embodiments, the linker includes multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 229). Also included within the scope of the present disclosure are linkers described, for example, in WO2012 / 138475.

[0257] As used herein, the term "specifically binds" refers to a polypeptide or fragment thereof that recognizes and binds to a biological molecule (e.g., a polypeptide) of interest, but does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample that naturally contains the polypeptide of the present disclosure. In certain embodiments, "specifically binds" refers to the binding of an antibody to an epitope, antigen, or antigenic determinant, for example, in such a way that binding can be displaced or competed with by a second preparation of the same or similar epitope, antigen, or antigenic determinant.

[0258] As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or alleviation of the progression, severity, and / or duration of a proliferative disorder, or the alleviation of one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more therapies (e.g., one or more therapeutic agents, such as a CAR of the present disclosure). In some embodiments, the terms "treat," "treatment," and "treating" refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, that is not necessarily discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to an inhibition of the progression of a proliferative disorder, either physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physical parameter, or both. In some embodiments, the terms "treat," "treatment," and "treating" refer to a reduction or stabilization of tumor size or cancerous cell number.

[0259] As used herein, the term "subject" is intended to include living organisms in which an immune response can be elicited (eg, mammals, humans).

[0260] Other aspects of the disclosure are described in the following sections and are within the scope of the claims.

[0261] Other Rules of Interpretation Ranges recited herein are understood to be shorthand for all of the values ​​within the range, inclusive of the recited endpoints. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subrange from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0262] Unless otherwise indicated, reference to a compound having one or more stereocenters contemplates each stereoisomer and all stereoisomeric combinations thereof.

[0263] bone marrow antigens Certain aspects of the present disclosure relate to chimeric receptors and cells, such as immunoresponsive cells, genetically modified to express one or more of such chimeric receptors that bind to an antigen of interest, and methods of using such receptors and cells to treat and / or prevent myeloid malignancies, such as AML, and other conditions in which an antigen-specific immune response is desired. Malignant cells have developed a series of mechanisms to protect themselves from immune recognition and elimination. The present disclosure provides immunogenicity within the tumor microenvironment for treating such malignant cells.

[0264] Certain aspects of the present disclosure relate to chimeric receptors that specifically bind to one or more antigens expressed on myeloid cells, and immunoresponsive cells genetically modified to express such chimeric receptors, useful for treating myeloid malignancies. Myeloid malignancies are clonal diseases caused by dysfunction of hematopoietic stem or progenitor cells, resulting from genetic and epigenetic alterations that disrupt key processes such as cell proliferation and differentiation. Myeloid malignancies can be chronic or acute. Chronic diseases include myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDSs), and chronic myelomonocytic leukemia (CMML). Acute diseases include acute myeloid leukemia (AML).

[0265] AML is characterized by the rapid proliferation of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. Symptoms of AML include fatigue, shortness of breath, increased susceptibility to infection, and easy bruising and bleeding. The majority of AML cases are de novo, although some cases may be secondary to chronic illness. There are eight distinct subtypes of AML based on the cell type from which the leukemia originates and its degree of maturity. AML subtypes include myeloblastic (M0), minimally mature myeloblastic (M1), fully mature myeloblastic (M2), promyelocytic (M3), myelomonocytic (M4), monocytic (M5), erythroleukemic (M6), and megakaryocytic (M7).

[0266] In certain embodiments, the present disclosure relates to AML antigens and combinations of AML antigens suitable for use in chimeric receptors (e.g., chimeric TCRs or CARs) to increase efficacy and reduce extratumoral toxicity in the treatment of AML.

[0267] Table 1 provides AML antigens suitable for use in the chimeric receptors described in the methods and compositions presented herein.

[0268] (Table 1) AML antigen TIFF2025143407000002.tif229163TIFF2025143407000003.tif241163TIFF2025143407000004.tif246163TIFF2025143407000005.tif235163TIFF2025143407000006.tif164163

[0269] In some embodiments, the AML antigen is a FLT3 antigen. In some embodiments, the AML antigen is an MS4A3 antigen. In some embodiments, the AML antigen is a CD33 antigen. In some embodiments, the AML antigen is a CLEC12A antigen. In some embodiments, the AML antigen is a CD312 / ADGRE2 antigen. In some embodiments, the AML antigen is a SLC22A16 antigen. In some embodiments, the AML antigen is a CD123 / ILR3RA antigen. In some embodiments, the AML antigen is a LAT2 antigen. In some embodiments, the AML antigen is a PIEZO1 / FAM38A antigen. In some embodiments, the AML antigen is a CD38 antigen. In some embodiments, the AML antigen is an EMB antigen. In some embodiments, the AML antigen is a CD131 / CSF2RB antigen. In some embodiments, the AML antigen is a P2RY8 antigen. In some embodiments, the AML antigen is a LILRA2 / CD85H antigen. In some embodiments, the AML antigen is a SLC17A9 antigen. In some embodiments, the AML antigen is a MYADM antigen. In some embodiments, the AML antigen is a CD300LF antigen. In some embodiments, the AML antigen is a CD244 / SLAMF4 antigen. In some embodiments, the AML antigen is a PLAUR antigen. In some embodiments, the AML antigen is a CD93 antigen. In some embodiments, the AML antigen is a SPNS3 antigen. In some embodiments, the AML antigen is a GAPT antigen. In some embodiments, the AML antigen is a RASGRP4 antigen. In some embodiments, the AML antigen is a CD117 / c-Kit antigen. In some embodiments, the AML antigen is a CD123 / ILR3RA antigen. In some embodiments, the AML antigen is a SLC34A2 antigen. In some embodiments, the AML antigen is a VSTM1 antigen. In some embodiments, the AML antigen is an MLC1 antigen. In some embodiments, the AML antigen is a PRAM1 antigen. In some embodiments, the AML antigen is an HCK antigen. In some embodiments, the AML antigen is an ICAM3 antigen.In some embodiments, the AML antigen is an LRRC37A2 antigen. In some embodiments, the AML antigen is an ITGAM antigen. In some embodiments, the AML antigen is an ITGB2 antigen. In some embodiments, the AML antigen is an LILRA1 antigen. In some embodiments, the AML antigen is a PRTN3 antigen. In some embodiments, the AML antigen is a CARD9 antigen. In some embodiments, the AML antigen is a SIGLEC5 antigen. In some embodiments, the AML antigen is a SELL antigen. In some embodiments, the AML antigen is an MLKL antigen. In some embodiments, the AML antigen is an INPP5D antigen. In some embodiments, the AML antigen is an APBB1IP antigen. In some embodiments, the AML antigen is an ITGA4 antigen. In some embodiments, the AML antigen is a C3AR1 antigen. In some embodiments, the AML antigen is an ITGA5 antigen. In some embodiments, the AML antigen is an FMNL1 antigen. In some embodiments, the AML antigen is an IL1RAP antigen. In some embodiments, the AML antigen is a CCR1 / CD191 antigen. In some embodiments, the AML antigen is a LILRB2 antigen. In some embodiments, the AML antigen is a CD70 antigen.

[0270] Chimeric receptors Certain aspects of the present disclosure relate to chimeric receptors that bind to antigens of interest and nucleic acids encoding such chimeric receptors.

[0271] Antibodies and antigen-binding fragments In some embodiments, the chimeric receptor comprises one or more of the amino acid sequences listed in Table A1 or Table A2. Table A1 provides the variable domains of antibody heavy or light chains. The CDRs were determined using the Kabat method and are underlined in Table A1 and shown in Table A2 for each variable heavy or variable light chain. In some embodiments, a nucleic acid encoding any of the chimeric receptors of the present disclosure comprises one or more of the nucleic acid sequences listed in Table B.

[0272] (Table A1) TIFF2025143407000007.tif193163TIFF2025143407000008.tif180163

[0273] (Table B) TIFF2025143407000009.tif234163TIFF2025143407000010.tif246163TIFF20251434070 00011.tif217163TIFF2025143407000012.tif248163TIFF2025143407000013.tif213163

[0274] (Table A2) TIFF2025143407000014.tif208170

[0275] Certain aspects of the present disclosure relate to chimeric receptors (e.g., CARs or chimeric TCRs) that comprise an extracellular antigen-binding domain that binds to one or more antigens of the present disclosure. In some embodiments, the antigen-binding domain is derived from an antibody, or an antigen-binding fragment thereof.

[0276] In some embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 1, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 2, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 3, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 4, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 5, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 8, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 9, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 10, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 11, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 12, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 13, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 16, or a sequence at least 90% identical thereto.In some embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 18, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 19, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 20, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 21, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 22, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 23, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 24, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 25, or a sequence at least 90% identical thereto. In some embodiments, the antigen-binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 26, or a sequence at least 90% identical thereto.

[0277] In some embodiments, the antigen binding domain comprises the CDR-H1 sequence set forth in SEQ ID NO: 75, the CDR-H2 sequence set forth in SEQ ID NO: 76, and the CDR-H3 sequence set forth in SEQ ID NO: 77. In some embodiments, the antigen binding domain comprises the CDR-L1 sequence set forth in SEQ ID NO: 78, the CDR-L2 sequence set forth in SEQ ID NO: 79, and the CDR-L3 sequence set forth in SEQ ID NO: 80. In some embodiments, the antigen binding domain comprises the CDR-H1 sequence set forth in SEQ ID NO: 81, the CDR-H2 sequence set forth in SEQ ID NO: 82, and the CDR-H3 sequence set forth in SEQ ID NO: 83. In some embodiments, the antigen binding domain comprises the CDR-L1 sequence set forth in SEQ ID NO: 84, the CDR-L2 sequence set forth in SEQ ID NO: 85, and the CDR-L3 sequence set forth in SEQ ID NO: 86. In some embodiments, the antigen binding domain comprises the CDR-H1 sequence set forth in SEQ ID NO: 87, the CDR-H2 sequence set forth in SEQ ID NO: 88, and the CDR-H3 sequence set forth in SEQ ID NO: 89. In some embodiments, the antigen binding domain comprises the CDR-L1 sequence set forth in SEQ ID NO: 90, the CDR-L2 sequence set forth in SEQ ID NO: 91, and the CDR-L3 sequence set forth in SEQ ID NO: 92. In some embodiments, the antigen binding domain comprises the CDR-H1 sequence set forth in SEQ ID NO: 93, the CDR-H2 sequence set forth in SEQ ID NO: 94, and the CDR-H3 sequence set forth in SEQ ID NO: 95. In some embodiments, the antigen binding domain comprises the CDR-L1 sequence set forth in SEQ ID NO: 96, the CDR-L2 sequence set forth in SEQ ID NO: 97, and the CDR-L3 sequence set forth in SEQ ID NO: 98. In some embodiments, the antigen binding domain comprises the CDR-H1 sequence set forth in SEQ ID NO: 99, the CDR-H2 sequence set forth in SEQ ID NO: 100, and the CDR-H3 sequence set forth in SEQ ID NO: 101. In some embodiments, the antigen binding domain comprises the CDR-L1 sequence set forth in SEQ ID NO: 102, the CDR-L2 sequence set forth in SEQ ID NO: 103, and the CDR-L3 sequence set forth in SEQ ID NO: 104. In some embodiments, the antigen binding domain comprises the CDR-H1 sequence set forth in SEQ ID NO: 105, the CDR-H2 sequence set forth in SEQ ID NO: 106, and the CDR-H3 sequence set forth in SEQ ID NO: 107.In some embodiments, the antigen binding domain comprises the CDR-L1 sequence set forth in SEQ ID NO: 108, the CDR-L2 sequence set forth in SEQ ID NO: 109, and the CDR-L3 sequence set forth in SEQ ID NO: 110. In some embodiments, the antigen binding domain comprises the CDR-H1 sequence set forth in SEQ ID NO: 111, the CDR-H2 sequence set forth in SEQ ID NO: 112, and the CDR-H3 sequence set forth in SEQ ID NO: 113. In some embodiments, the antigen binding domain comprises the CDR-L1 sequence set forth in SEQ ID NO: 114, the CDR-L2 sequence set forth in SEQ ID NO: 115, and the CDR-L3 sequence set forth in SEQ ID NO: 116. In some embodiments, the antigen binding domain comprises the CDR-H1 sequence set forth in SEQ ID NO: 117, the CDR-H2 sequence set forth in SEQ ID NO: 118, and the CDR-H3 sequence set forth in SEQ ID NO: 119. In some embodiments, the antigen binding domain comprises the CDR-L1 sequence set forth in SEQ ID NO: 120, the CDR-L2 sequence set forth in SEQ ID NO: 121, and the CDR-L3 sequence set forth in SEQ ID NO: 122. In some embodiments, the antigen binding domain comprises the CDR-H1 sequence set forth in SEQ ID NO: 123, the CDR-H2 sequence set forth in SEQ ID NO: 124, and the CDR-H3 sequence set forth in SEQ ID NO: 125. In some embodiments, the antigen binding domain comprises the CDR-L1 sequence set forth in SEQ ID NO: 126, the CDR-L2 sequence set forth in SEQ ID NO: 127, and the CDR-L3 sequence set forth in SEQ ID NO: 128. In some embodiments, the antigen binding domain comprises the CDR-H1 sequence set forth in SEQ ID NO: 129, the CDR-H2 sequence set forth in SEQ ID NO: 130, and the CDR-H3 sequence set forth in SEQ ID NO: 131. In some embodiments, the antigen binding domain comprises the CDR-L1 sequence set forth in SEQ ID NO: 132, the CDR-L2 sequence set forth in SEQ ID NO: 133, and the CDR-L3 sequence set forth in SEQ ID NO: 134. In some embodiments, the antigen binding domain comprises the CDR-H1 sequence set forth in SEQ ID NO: 135, the CDR-H2 sequence set forth in SEQ ID NO: 136, and the CDR-H3 sequence set forth in SEQ ID NO: 137. In some embodiments, the antigen binding domain comprises the CDR-L1 sequence set forth in SEQ ID NO: 138, the CDR-L2 sequence set forth in SEQ ID NO: 139, and the CDR-L3 sequence set forth in SEQ ID NO: 140.In some embodiments, the antigen binding domain comprises the CDR-H1 sequence set forth in SEQ ID NO: 141, the CDR-H2 sequence set forth in SEQ ID NO: 142, and the CDR-H3 sequence set forth in SEQ ID NO: 143. In some embodiments, the antigen binding domain comprises the CDR-L1 sequence set forth in SEQ ID NO: 144, the CDR-L2 sequence set forth in SEQ ID NO: 145, and the CDR-L3 sequence set forth in SEQ ID NO: 146. In some embodiments, the antigen binding domain comprises the CDR-H1 sequence set forth in SEQ ID NO: 147, the CDR-H2 sequence set forth in SEQ ID NO: 148, and the CDR-H3 sequence set forth in SEQ ID NO: 149. In some embodiments, the antigen binding domain comprises the CDR-L1 sequence set forth in SEQ ID NO: 150, the CDR-L2 sequence set forth in SEQ ID NO: 151, and the CDR-L3 sequence set forth in SEQ ID NO: 152.

[0278] Suitable antibodies of the present disclosure include antibodies or fragments thereof, whether natural or synthetic, either full-length, monoclonal or polyclonal, that bind sufficiently strongly and specifically to myeloid (e.g., AML) antigens. In some embodiments, the antibodies have a binding affinity of up to about 10 -6 M, maximum approx. 10 -7 M, maximum approx. 10 -8 M, maximum approx. 10 -9 M, maximum approx. 10 -10 M, maximum approx. 10 -11 M, or up to about 10 -12 K of M d may have:

[0279] In some embodiments, antibodies and derivatives thereof that may be used include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, primatized (CDR-grafted) antibodies, veneered antibodies, single-chain antibodies, phage-produced antibodies (e.g., from a phage display library), and functional binding fragments of antibodies. For example, antibody fragments, or portions thereof, capable of binding to myeloid (e.g., AML) antigens include, but are not limited to, Fv, Fab, Fab', and F(ab')2 fragments. Such fragments can be produced by enzymatic cleavage or recombinant techniques. For example, but not limited to, papain or pepsin cleavage can generate Fab or F(ab')2 fragments, respectively. Other proteases with the required substrate specificity can also be used to generate Fab or F(ab')2 fragments. Antibodies can also be produced in various truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain, and hinge region of the heavy chain.

[0280] Methods for raising antibodies that target specific antigens are generally known in the art. Synthetic and engineered antibodies are described, for example, in US4816567, EP0125023B1, US4816397, EP0120694B1, WO86 / 01533, EP0194276B1, US5225539, EP0239400B1, EP0451216B1, EP0519596A1, and US4946778.

[0281] In some embodiments, commercially available antibodies can be used to bind to myeloid (e.g., AML) antigens. The CDRs of commercially available antibodies can be easily accessed by those skilled in the art using conventional sequencing techniques. Furthermore, those skilled in the art can construct nucleic acids encoding scFvs and chimeric receptors (e.g., CARs and TCRs) based on the CDRs of such commercially available antibodies.

[0282] In some embodiments, the chimeric receptor comprises an antigen-binding domain that specifically binds to FLT3. In some embodiments, the FLT3-specific antigen-binding domain is derived from an anti-FLT3 antibody, such as the D4-3, NC7, or EB10 antibodies described in U.S. Patent No. 8,071,099. In some embodiments, the FLT3-specific antigen-binding domain is derived from an anti-FLT3 antibody, such as the 4G8 or BV10 antibodies described in U.S. Patent No. 9,023,996. In some embodiments, the FLT3-specific antigen-binding domain is derived from an anti-FLT3 antibody, such as the FL_16 or FL_39 antibodies described in U.S. Patent Publication No. 2017 / 0037149, published February 9, 2017. In some embodiments, the FLT3-specific antigen-binding domain is derived from an anti-FLT3 antibody, such as the ml0006 antibody described in International Patent Publication WO2018 / 119279, published June 28, 2018. The antigen-binding domain can be an scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, a chimeric receptor can have a multispecific antigen-binding domain. For example, a chimeric receptor can have a FLT ’ 3 and may be specific for one or more additional antigens, for example CD33 and / or CLEC12A.

[0283] In some embodiments, the chimeric receptor comprises an antigen-binding domain that specifically binds to CD33. In some embodiments, the CD33-specific antigen-binding domain is derived from an anti-CD33 antibody, such as lintuzumab, described in U.S. Patent Publication No. 2018 / 0002397, published January 4, 2018. In some embodiments, the CD33-specific antigen-binding domain is derived from an anti-CD33 antibody, such as gemtuzumab, described in U.S. Patent No. 5,739,116. The antigen-binding domain may be an scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor may have a multispecific antigen-binding domain. For example, the chimeric receptor may be specific for CD33 and one or more additional antigens, such as FLT3 and / or CLEC12A.

[0284] In some embodiments, the chimeric receptor comprises an antigen-binding domain that specifically binds to CLEC12A. In some embodiments, the CLEC12A-specific antigen-binding domain is derived from an anti-CLEC12A antibody, such as the SC02-357, SC02-378, or SC02-161 antibodies described in U.S. Patent No. 7,741,443. The antigen-binding domain may be an scFv comprising a light chain variable domain (VL) and a heavy chain variable domain (VH). In some embodiments, the chimeric receptor may have a multispecific antigen-binding domain. For example, the chimeric receptor may be specific for CLEC12A and one or more additional antigens, such as FLT3 and / or CD33.

[0285] T cell receptor (TCR) Certain aspects of the present disclosure relate to chimeric receptors that specifically bind to antigens expressed on myeloid cells, such as AML cells. In some embodiments, the chimeric receptor is a chimeric T cell receptor (TCR). The TCRs of the present disclosure are disulfide-linked heterodimeric proteins comprising two variable chains expressed as part of a complex with an invariant CD3 chain molecule. TCRs are found on the surface of T cells and are responsible for recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, the TCRs of the present disclosure comprise an alpha chain encoded by TRA and a beta chain encoded by TRB. In certain embodiments, the TCRs comprise a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0286] Each chain of the TCR consists of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is proximal to the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. Each variable region has three complementarity-determining regions (CDRs).

[0287] In certain embodiments, the TCR can form a receptor complex with three dimeric signaling modules, CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or CD247ζ / η. Upon complexation of the TCR complex with its antigen and MHC (peptide / MHC), a T cell expressing the TCR complex is activated.

[0288] In some embodiments, the TCR of the present disclosure is a recombinant TCR. In certain embodiments, the TCR is a non-naturally occurring TCR. In certain embodiments, the TCR differs from a naturally occurring TCR by at least one amino acid residue. In some embodiments, the TCR differs from a naturally occurring TCR by at least two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid residues, at least six amino acid residues, at least seven amino acid residues, at least eight amino acid residues, at least nine amino acid residues, at least ten amino acid residues, at least 11 amino acid residues, at least 12 amino acid residues, at least 13 amino acid residues, at least 14 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, or more amino acid residues. In certain embodiments, the TCR is a naturally occurring TCR modified by at least one amino acid residue. In some embodiments, the TCR is a naturally occurring TCR modified by at least two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid residues, at least six amino acid residues, at least seven amino acid residues, at least eight amino acid residues, at least nine amino acid residues, at least ten amino acid residues, at least eleven amino acid residues, at least 12 amino acid residues, at least 13 amino acid residues, at least 14 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, or more amino acid residues.

[0289] Chimeric TCR In some embodiments, the TCRs of the present disclosure comprise one or more antigen-binding domains that can be grafted onto one or more constant domains of a TCR chain, e.g., a TCR alpha chain or a TCR beta chain, to create a chimeric TCR that specifically binds to a target antigen of the present disclosure (e.g., an AML antigen). Without wishing to be bound by theory, it is believed that the chimeric TCR can signal through the TCR complex upon antigen binding. For example, an antibody or antibody fragment (e.g., an scFv) can be grafted onto the constant domains of a TCR chain, such as the TCR alpha chain and / or the TCR beta chain, e.g., at least a portion of the extracellular constant domain, transmembrane domain, and cytoplasmic domain. As another example, the CDRs of an antibody or antibody fragment can be grafted onto the TCR alpha chain and / or beta chain to create a chimeric TCR that specifically binds to an antigen of the present disclosure (e.g., an AML antigen). Such chimeric TCRs can be produced by methods known in the art (see, for example, Willemsen RA et al., Gene Therapy 2000;7:1369-1377, Zhang T et al., Cancer Gene Ther 2004 11:487-496, and Aggen et al., Gene Ther. 2012 Apr;19(4):365-74).

[0290] Chimeric antigen receptor (CAR) Certain aspects of the present disclosure relate to chimeric receptors that specifically bind to antigens expressed on myeloid cells, such as AML cells. In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR).

[0291] In some embodiments, CARs are engineered receptors that transfer or confer desired specificity to immune effector cells. In certain embodiments, CARs can be used to transfer antibody specificity to immune responsive cells, such as T cells. In some embodiments, the CARs of the present disclosure comprise an extracellular antigen-binding domain (e.g., scFv) fused to a transmembrane domain, which is fused to one or more intracellular signaling domains.

[0292] In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce activation of an immunoresponsive cell. In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce stimulation of an immunoresponsive cell. In some embodiments, activation of an immunoresponsive cell results in killing of a target cell. In some embodiments, activation of an immunoresponsive cell results in cytokine or chemokine expression and / or secretion by the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell results in cytokine or chemokine expression and / or secretion by the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell induces differentiation of the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell induces proliferation of the immunoresponsive cell.

[0293] The CAR of the present disclosure can be a first-, second-, or third-generation CAR. A "first-generation" CAR generally contains a single intracellular signaling domain derived from a T cell receptor chain. A "first-generation" CAR generally has an intracellular signaling domain from the CD3-zeta (CD3ζ) chain, which is the primary transmitter of signals from endogenous TCRs. A "first-generation" CAR provides novel antigen recognition independent of HLA-mediated antigen presentation and transduces CD4 ζ via the CD3ζ chain signaling domain within a single fusion molecule. + and CD8 +"Second-generation" CARs can induce both activation of T cells and activation of T cells. "Second-generation" CARs add a second intracellular signaling domain from one of various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide an additional signal to T cells. "Second-generation" CARs provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). Preclinical studies have shown that "second-generation" CARs can improve the anti-tumor activity of immunoresponsive cells such as T cells. "Third-generation" CARs have multiple intracellular costimulatory signaling domains (e.g., CD28 and 4-1BB) and an intracellular activation signaling domain (CD3ζ).

[0294] In some embodiments, the extracellular antigen binding domain of a CAR of the present disclosure binds to one or more antigens expressed on myeloid cells, such as AML cells, at a concentration of about 2×10 -7 M or less, approximately 1×10 -7 M or less, approximately 9 x 10 -8 M or less, approximately 1×10 -8 M or less, approximately 9 x 10 -9 M or less, about 5 x 10 -9 M or less, approximately 4 x 10 -9 M or less, about 3 x 10 -9 M or less, approximately 2×10 -9 M or less, or approximately 1 x 10 -9 M or less. In some embodiments, the Kd is about 2×10 -7 M ~ approx. 1×10 -9 It is in the range of M.

[0295] The binding of the extracellular antigen-binding domain of the CAR of the present disclosure can be determined, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a specific protein-antibody complex of interest by using a labeled reagent (e.g., antibody or scFv) specific to the complex of interest. For example, scFv can be radiolabeled and used in an RIA assay. Radioisotopes can be detected by means such as the use of a gamma counter or scintillation counter, or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet).

[0296] In some embodiments, a CAR of the present disclosure comprises an extracellular antigen-binding domain that binds to one or more antigens expressed on myeloid (e.g., AML) cells, a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the extracellular antigen-binding domain comprises a Fab fragment, which can be cross-linked. In certain embodiments, the extracellular binding domain is a F(ab)2 fragment.

[0297] Extracellular antigen-binding domain In some embodiments, the extracellular antigen-binding domain of the CAR of the present disclosure specifically binds to one or more antigens expressed on myeloid cells, such as AML cells. In certain embodiments, the extracellular antigen-binding domain binds to one or more antigens expressed on AML cells (AML antigens). In some embodiments, the one or more AML antigens are human polypeptides.

[0298] Antigen-binding domains of the present disclosure can include monoclonal antibodies, polyclonal antibodies, recombinant antibodies, bispecific antibodies, conjugated antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including, but not limited to, single-domain antibodies (sdAbs), such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid-derived nanobodies (VHH), as well as any domain associated with an alternative scaffold known in the art to function as an antigen-binding domain, such as a recombinant fibronectin domain, a T cell receptor (TCR), an affinity-improved recombinant TCR, or a fragment thereof, e.g., a single-chain TCR. In some cases, it is beneficial for the antigen-binding domain to be derived from the same species in which the CAR will ultimately be used. For example, for human use, it may be beneficial for the antigen-binding domain of the CAR to include human or humanized residues for the antigen-binding domain of an antibody or antibody fragment.

[0299] In some embodiments, the extracellular antigen-binding domain comprises an antibody. In certain embodiments, the antibody is a human antibody. In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody is a chimeric antibody. In some embodiments, the extracellular antigen-binding domain comprises an antigen-binding fragment of an antibody.

[0300] In some embodiments, the extracellular antigen-binding domain comprises an F(ab) fragment. In certain embodiments, the extracellular antigen-binding domain comprises an F(ab') fragment.

[0301] In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the extracellular antigen-binding domain comprises two single-chain variable fragments (scFvs). In some embodiments, each of the two scFvs binds to a distinct epitope on the same antigen. In some embodiments, the extracellular antigen-binding domain comprises a first scFv and a second scFv. In some embodiments, the first scFv and the second scFv bind to a distinct epitope on the same antigen. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a chimeric scFv. In certain embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In certain embodiments, the VH and VL are separated by a peptide linker. In certain embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 74. In certain embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 218 or 219. In certain embodiments, the peptide linker is encoded by a nucleic acid comprising the sequence of SEQ ID NO: 54, 220, 221, or 223. In certain embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.

[0302] In some embodiments, each of the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is a peptide linker, and VL is the light chain variable domain. When two or more scFvs are linked together, each scFv can be linked to the next scFv via a peptide linker. In some embodiments, each of the one or more scFvs is separated by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence GGGGSGGGSGGGGS (SEQ ID NO: 27) or EAAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 74).

[0303] In some embodiments, the cell comprises a first chimeric receptor and a second chimeric receptor. The antigen-binding domain of the first chimeric receptor and the antigen-binding domain of the second chimeric receptor can be any suitable antigen-binding domain described herein or known in the art. For example, the first or second antigen-binding domain can be one or more antibodies, antigen-binding fragments of antibodies, F(ab) fragments, F(ab') fragments, single-chain variable fragments (scFv), or single-domain antibodies (sdAbs). In some embodiments, the antigen-binding domain of the first chimeric receptor and / or the second chimeric receptor comprises two single-chain variable fragments (scFv). In some embodiments, each of the two scFvs binds to a distinct epitope on the same antigen.

[0304] In some embodiments, the extracellular antigen-binding domain comprises a single domain antibody (sdAb). In certain embodiments, the sdAb is a humanized sdAb. In certain embodiments, the sdAb is a chimeric sdAb.

[0305] In some embodiments, a CAR of the present disclosure may comprise two or more antigen-binding domains, three or more antigen-binding domains, four or more antigen-binding domains, five or more antigen-binding domains, six or more antigen-binding domains, seven or more antigen-binding domains, eight or more antigen-binding domains, nine or more antigen-binding domains, or ten or more antigen-binding domains. In some embodiments, each of the two or more antigen-binding domains binds to the same antigen. In some embodiments, each of the two or more antigen-binding domains binds to a different epitope of the same antigen. In some embodiments, each of the two or more antigen-binding domains binds to a different antigen. In some embodiments, the two or more antigen-binding domains provide the CAR with logical gating, such as OR logical gating.

[0306] In some embodiments, the CAR comprises two antigen-binding domains. In some embodiments, the two antigen-binding domains are linked to each other via a flexible linker. In some embodiments, each of the two antigen-binding domains can be independently selected from an antibody, an antigen-binding fragment of an antibody, an scFv, an sdAb, a recombinant fibronectin domain, a T cell receptor (TCR), an affinity-enhanced recombinant TCR, and a single-chain TCR. In some embodiments, the CAR comprising two antigen-binding domains is a bispecific CAR or a tandem CAR (tanCAR).

[0307] In certain embodiments, a bispecific CAR or tanCAR comprises an antigen-binding domain comprising a bispecific antibody or antibody fragment (e.g., scFv). In some embodiments, within each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, the VH can be upstream or downstream of the VL. In some embodiments, an upstream antibody or antibody fragment (e.g., scFv) is positioned with its VH (VH1) upstream of its VL (VL1), and a downstream antibody or antibody fragment (e.g., scFv) is positioned with its VL (VL2) upstream of its VH (VH2), such that the overall bispecific antibody molecule has the configuration VH1-VL1-VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL1) upstream of its VH (VH1), and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH2) upstream of its VL (VL2), with the overall bispecific antibody molecule having the configuration VL1VH1-VH2-VL2. In some embodiments, a linker is placed between the two antibodies or antibody fragments (e.g., scFvs), for example, between VL1 and VL2 when the construct is arranged as VH1-VL1-VL2-VH2, or between VH1 and VH2 when the construct is arranged as VL1-VH1-VH2-VL2. The linker can be a linker described herein, e.g., a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6 (SEQ ID NO: 227). Generally, the linker between two scFvs must be long enough to avoid mismatching between the domains of the two scFvs. In some embodiments, the linker is placed between the VL and VH of the first scFv. In some embodiments, the linker is placed between the VL and VH of the second scFv. In constructs with multiple linkers, any two or more linkers may be the same or different. Thus, in some embodiments, a bispecific CAR or tanCAR comprises a VL, a VH, and may further comprise one or more linkers in the arrangements described herein.

[0308] In some embodiments, the chimeric receptor comprises a bivalent CAR. In some embodiments, the bivalent CAR is a FLT3 bivalent CAR. In some embodiments, the bivalent FLT3 CAR comprises an NC7 scFv and a D4-3 scFv. In some embodiments, the bivalent CAR is a CD33 bivalent CAR. In some embodiments, the bivalent CAR is a CLEC12A bivalent CAR.

[0309] In some embodiments, the chimeric receptor comprises a bivalent chimeric antigen receptor. In some embodiments, the bivalent chimeric receptor comprises a FLT3 CAR and a CD33 CAR. In some embodiments, the bivalent chimeric receptor comprises a FLT3 CAR and a CLEC12A CAR. In some embodiments, the bivalent chimeric receptor comprises a CLEC12A CAR and a CD33 CAR. In some embodiments, the bivalent chimeric receptor comprises an EMCN CAR. In some embodiments, the bivalent chimeric receptor comprises a CAR having an antigen binding domain that targets any of the antigens provided in Table 1. In some embodiments, the bivalent chimeric receptor comprises a CAR having an antigen binding domain that targets any of the antigens provided in Table 2. In some embodiments, the bivalent chimeric receptor comprises a CAR having two or more antigen binding domains that target any of the antigen pairs provided in Table 3. In some embodiments, the bivalent chimeric antigen receptor comprises a CAR having any combination of two or more antigen binding domains described herein.

[0310] In some embodiments, the chimeric receptor comprises a bicistronic chimeric antigen receptor. In some embodiments, the bicistronic chimeric antigen receptor comprises a FLT3 CAR and a CD33 CAR. In some embodiments, the bicistronic chimeric antigen receptor comprises a FLT3 CAR and a CLEC12A CAR. In some embodiments, the bicistronic chimeric antigen receptor comprises a CLEC12A CAR and a CD33 CAR. In some embodiments, the bicistronic chimeric antigen receptor comprises an EMCN CAR. In some embodiments, the bicistronic chimeric antigen receptor comprises a CAR having an antigen binding domain that targets any of the antigens provided in Table 1. In some embodiments, the bicistronic chimeric antigen receptor comprises a CAR having an antigen binding domain that targets any of the antigens provided in Table 2. In some embodiments, the bicistronic chimeric antigen receptor comprises a CAR having two or more antigen binding domains that target any of the antigen pairs provided in Table 3. In some embodiments, the bicistronic chimeric antigen receptor comprises a CAR having any combination of two or more antigen binding domains described herein.

[0311] Transmembrane domain In some embodiments, the transmembrane domain of the CAR of the present disclosure comprises a hydrophobic alpha helix spanning at least a portion of the cell membrane. It has been shown that different transmembrane domains can result in different receptor stabilities. After antigen recognition, the receptors cluster and a signal is transmitted to the cell. In some embodiments, the transmembrane domain of the CAR of the present disclosure can comprise the transmembrane domain of a CD8 polypeptide, a CD28 polypeptide, a CD3-zeta polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a LIR-1 (LILRB1) polypeptide, or can be a synthetic peptide, or any combination thereof.

[0312] In some embodiments, the transmembrane domain is derived from a CD8 polypeptide. Any suitable CD8 polypeptide may be used. Exemplary CD8 polypeptides include, but are not limited to, NCBI reference numbers NP_001139345 and AAA92533.1. In some embodiments, the transmembrane domain is derived from a CD28 polypeptide. Any suitable CD28 polypeptide may be used. Exemplary CD28 polypeptides include, but are not limited to, NCBI reference numbers NP_006130.1 and NP_031668.3. In some embodiments, the transmembrane domain is derived from a CD3-zeta polypeptide. Any suitable CD3-zeta polypeptide may be used. Exemplary CD3-zeta polypeptides include, but are not limited to, NCBI reference numbers NP_932170.1 and NP_001106862.1. In some embodiments, the transmembrane domain is derived from a CD4 polypeptide. Any suitable CD4 polypeptide may be used. Exemplary CD4 polypeptides include, but are not limited to, NCBI reference numbers NP_000607.1 and NP_038516.1. In some embodiments, the transmembrane domain is derived from a 4-1BB polypeptide. Any suitable 4-1BB polypeptide may be used. Exemplary 4-1BB polypeptides include, but are not limited to, NCBI reference numbers NP_001552.2 and NP_001070977.1. In some embodiments, the transmembrane domain is derived from an OX40 polypeptide. Any suitable OX40 polypeptide may be used. Exemplary OX40 polypeptides include, but are not limited to, NCBI reference numbers NP_003318.1 and NP_035789.1. In some embodiments, the transmembrane domain is derived from an ICOS polypeptide. Any suitable ICOS polypeptide may be used. Exemplary ICOS polypeptides include, but are not limited to, NCBI reference numbers NP_036224 and NP_059508. In some embodiments, the transmembrane domain is derived from a CTLA-4 polypeptide. Any suitable CTLA-4 polypeptide can be used.Exemplary CTLA-4 polypeptides include, but are not limited to, NCBI reference numbers NP_005205.2 and NP_033973.2. In some embodiments, the transmembrane domain is derived from a PD-1 polypeptide. Any suitable PD-1 polypeptide may be used. Exemplary PD-1 polypeptides include, but are not limited to, NCBI reference numbers NP_005009 and NP_032824. In some embodiments, the transmembrane domain is derived from a LAG-3 polypeptide. Any suitable LAG-3 polypeptide may be used. Exemplary LAG-3 polypeptides include, but are not limited to, NCBI reference numbers NP_002277.4 and NP_032505.1. In some embodiments, the transmembrane domain is derived from a 2B4 polypeptide. Any suitable 2B4 polypeptide may be used. Exemplary 2B4 polypeptides include, but are not limited to, NCBI reference numbers NP_057466.1 and NP_061199.2. In some embodiments, the transmembrane domain is derived from a BTLA polypeptide. Any suitable BTLA polypeptide may be used. Exemplary BTLA polypeptides include, but are not limited to, NCBI reference numbers NP_861445.4 and NP_001032808.2. Any suitable LIR-1 (LILRB1) polypeptide may be used. Exemplary LIR-1 (LILRB1) polypeptides include, but are not limited to, NCBI reference numbers NP_001075106.2 and NP_001751072.

[0313] In some embodiments, the transmembrane domain is selected from the group consisting of NP_001139345, AAA92533.1, NP_006130.1, NP_031668.3, NP_932170.1, NP_001106862.1, NP_000607.1, NP_038516.1, NP_001552.2, NP_001070977.1, NP_003318.1, NP_035789.1, NP_036224, NP_059508, NP_005205.2, NP_033973.2, NP_005009 , NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2, or a fragment thereof. In some embodiments, homology can be determined using standard software such as BLAST or FASTA. In some embodiments, the polypeptide can contain one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions.In some embodiments, the polypeptide is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, or at least 240 amino acids in length, see NCBI Reference Nos. NP_001139345, AAA92533.1, NP_006130.1, NP_006130.1, NP_006130.2, NP_006130.3, NP_006130.4, NP_006130.5, NP_006130.6, NP_006130.7, NP_006130.8, NP_006130.9, NP_006130.1 ... NP_005009, NP_032824, NP_002277.4, NP_032505.1, NP_057466.1, NP_061199.2, NP_861445.4, or NP_001032808.2.

[0314] Further examples of suitable polypeptides from which the transmembrane domain may be derived include T cell receptor, CD27, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, CD2, CD27, LFA-1 (CD11a, CD18), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, These include, but are not limited to, the transmembrane region(s) of the alpha, beta, or zeta chain of ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (antennary), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, and NG2C.

[0315] In some embodiments, the transmembrane domain comprises the sequence set forth in SEQ ID NO: 209. In some embodiments, the transmembrane domain comprises the sequence set forth in SEQ ID NO: 210. In some embodiments, the transmembrane domain comprises the sequence set forth in SEQ ID NO: 211.

[0316] Spacer region In some embodiments, the CAR of the present disclosure can also include a spacer region linking the extracellular antigen-binding domain to the transmembrane domain. The spacer region can be sufficiently flexible to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. In some embodiments, the spacer region can be a hinge derived from a human protein. For example, the hinge can be a human Ig (immunoglobulin) hinge, including, but not limited to, an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge. In some embodiments, the spacer region can include an IgG4 hinge, an IgG2 hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, an LNGFR hinge, or a PDGFR-beta extracellular linker. In some aspects, the spacer region is located between the antigen-binding domain and the transmembrane domain. In some embodiments, the spacer region may comprise any of the amino acid sequences listed in Table C, or any of the amino acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the amino acid sequences listed in Table C. In some embodiments, a nucleic acid encoding any of the spacer regions of the present disclosure may comprise any of the nucleic acid sequences listed in Table D, or any of the nucleic acid sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the nucleic acid sequences listed in Table D.

[0317] (Table C) TIFF2025143407000015.tif123163

[0318] (Table D) TIFF2025143407000016.tif190161

[0319] In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 55. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 56. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 57. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 58. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 59. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 60. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 61. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 62. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 63. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 64. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 206. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 207. In some embodiments, the spacer region comprises the sequence set forth in SEQ ID NO: 208.

[0320] In some embodiments, a CAR of the present disclosure may further comprise a short oligopeptide or polypeptide linker between 2 and 10 amino acid residues in length that can form a bond between the transmembrane domain and the cytoplasmic region of the CAR. A non-limiting example of a suitable linker is a glycine-serine duplex. In some embodiments, the linker comprises the amino acid sequence GGCKJSGGCKJS (SEQ ID NO: 228).

[0321] Intracellular signaling domains In some embodiments, a CAR of the present disclosure comprises one or more cytoplasmic domains or regions. The cytoplasmic domain or region of a CAR may comprise an intracellular signaling domain. The intracellular signaling domain is typically involved in activating one or more effector functions of an immune cell (e.g., a T cell or NK cell) engineered to express a CAR of the present disclosure. For example, the effector function of a T cell can be cytolytic activity or helper activity, such as cytokine secretion. Thus, in some embodiments, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be used, although it is often not necessary to use the entire chain. In embodiments in which a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the corresponding intact chain, so long as the truncated portion transmits the effector function signal.

[0322] Examples of suitable intracellular signaling domains that may be used in the CARs of the present disclosure include, but are not limited to, the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act cooperatively to initiate signal transduction after antigen receptor engagement, as well as any derivatives or variants of these sequences, and any recombinant sequences that have the same functional capabilities.

[0323] Without wishing to be bound by theory, it is believed that signals generated through the TCR alone are insufficient for full activation of T cells, and therefore secondary and / or costimulatory signals are also required for full activation. Thus, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains).

[0324] In some embodiments, the primary signaling domain controls the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of suitable ITAM-containing primary intracellular signaling domains that can be used in the CARs of the present disclosure include, but are not limited to, those of CD3-zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, DAP10, DAP12, and CD66d.

[0325] In some embodiments, a CAR of the present disclosure comprises an intracellular signaling domain, such as the primary signaling domain of a CD3-zeta polypeptide. The CD3-zeta polypeptide of the present disclosure may have an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homologous to the sequence of NCBI reference number NP_932170 or NP_001106864.2. In some embodiments, the CD3-zeta polypeptide may contain one conservative amino acid substitution, up to two conservative amino acid substitutions, or up to three conservative amino acid substitutions. In some embodiments, the polypeptide can have an amino acid sequence that is a contiguous portion of NCBI Reference No. NP_932170 or NP_001106864.2 that is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, or at least 160, at least 170, or at least 180 amino acids in length.

[0326] In other embodiments, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain, that has altered (e.g., increased or decreased) activity compared to the native ITAM domain. In one embodiment, the primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In one embodiment, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

[0327] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure can comprise a CD3-zeta signaling domain by itself, or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a CAR of the present disclosure. For example, the intracellular signaling domain of a CAR can comprise a portion of a CD3-zeta chain and a costimulatory signaling domain. A costimulatory signaling domain can refer to a portion of a CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule of the present disclosure is a cell surface molecule other than an antigen receptor or its ligand that may be required for an efficient response of lymphocytes to an antigen.Examples of suitable costimulatory molecules include CD97, CD2, ICOS, CD27, CD154, CD8, OX40, 4-1BB, CD28, ZAP40, CD30, GITR, HVEM, DAP10, DAP12, MyD88, 2B4, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, an MHC class I molecule, a TNF receptor protein, an immunoglobulin G receptor protein, and the like. Cholesterol-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, CDS, ICAM-1, (CD11a / CD18), BAFFR, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, IL2R beta, IL2R gamma, IL7R alpha, ITGA4 , VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGA X, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, C These include, but are not limited to, D96 (antennary), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, etc.

[0328] In some embodiments, the intracellular signaling sequences within the cytoplasmic portion of the CAR of the present disclosure can be linked to each other in a random or specific order. In some embodiments, a short oligopeptide or polypeptide linker, for example, 2 to 10 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), can form a linkage between the intracellular signaling sequences. In one embodiment, a glycine-serine duplex can be used as a suitable linker. In one embodiment, a single amino acid, for example, alanine or glycine, can be used as a suitable linker.

[0329] In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains, e.g., two costimulatory signaling domains, three costimulatory signaling domains, four costimulatory signaling domains, five costimulatory signaling domains, six costimulatory signaling domains, seven costimulatory signaling domains, eight costimulatory signaling domains, nine costimulatory signaling domains, ten costimulatory signaling domains, or more costimulatory signaling domains. In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the two or more costimulatory signaling domains are separated by a linker of the present disclosure. In one embodiment, the linker is a glycine residue. In another embodiment, the linker is an alanine residue.

[0330] In some embodiments, a cell of the present disclosure expresses a CAR that comprises an antigen binding domain that binds to a target antigen of the present disclosure, a transmembrane domain of the present disclosure, a primary signaling domain, and one or more costimulatory signaling domains.

[0331] In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 153, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 155, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 157, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 159, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 161, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 163, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 165, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 167, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 169, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 171, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 173, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 175, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 177, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 179, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 181, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 183, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 185, or a sequence at least 90% identical thereto.In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 187, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 189, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 191, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 19 ...5, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 197, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 199, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 201, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 203, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 205, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 212, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 214, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 216, or a sequence at least 90% identical thereto.

[0332] In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 154, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 156, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 158, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 160, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 162, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 164, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 166, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 168, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 170, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 172, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 174, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 176, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 178, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 180, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 182, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 184, or a sequence at least 90% identical thereto.In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 186, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 188, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 190, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 192, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 194, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 196, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 198, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 200, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 202, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 204, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 213, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 215, or a sequence at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 217, or a sequence at least 90% identical thereto.

[0333] Natural killer cell receptor (NKR) CAR In some embodiments, a CAR of the present disclosure comprises one or more components of natural killer cell receptors (NKRs), thereby forming an NKR-CAR. The NKR components include KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3S1. The CAR may be a transmembrane domain, hinge domain, or cytoplasmic domain from any suitable natural killer cell receptor, including, but not limited to, killer cell immunoglobulin-like receptors (KIRs) such as DPI; natural cytotoxicity receptors (NCRs) such as NKp30, NKp44, and NKp46; the signaling lymphocyte activation molecule (SLAM) family of immune cell receptors such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10; Fc receptors (FcRs) such as CD16 and CD64; and Ly49 receptors such as LY49A and LY49C. In some embodiments, the NKR-CAR may interact with an adapter molecule or an intracellular signaling domain such as DAP12. Exemplary configurations and sequences of CARs containing NKR components are described in International Patent Publication WO2014 / 145252, published September 18, 2014.

[0334] Chimeric inhibitory receptors Certain aspects of the present disclosure relate to chimeric inhibitory receptors. Chimeric inhibitory receptors are not useful as logic gates for controlling cellular activity, such as immune cell activity. In some embodiments, the chimeric inhibitory receptors of the present disclosure specifically bind to one or more antigens that are expressed on normal cells but not on tumor cells.

[0335] In some embodiments, a chimeric inhibitory receptor comprises an antigen-binding domain, a transmembrane domain of the present disclosure (e.g., any suitable transmembrane domain for use in conjunction with a chimeric receptor of the present disclosure), and an intracellular domain. In some embodiments, a chimeric inhibitory receptor can inhibit one or more activities of a cell, such as an immunoresponsive cell.

[0336] In some embodiments, the chimeric inhibitory receptor may contain an enzyme inhibitory domain. When the chimeric inhibitory receptor is located proximal to a receptor, such as an immune receptor, in the cell membrane, binding of the cognate antigen to the antigen-binding domain activates the enzyme inhibitory domain, inhibiting receptor activation. As used herein, the term "enzyme inhibitory domain" refers to a protein domain that inhibits an intracellular signaling cascade, such as the natural T cell activation cascade. Thus, the disclosed chimeric inhibitory receptors can be engineered to contain an appropriate antigen-binding domain that reduces an immune response, for example, in the presence of a cognate antigen. Uses of the chimeric inhibitory receptors of the present disclosure include, but are not limited to, reducing immune responses, controlling T cell activation, and controlling CAR-T responses.

[0337] In some embodiments, the enzyme inhibitory domain of a chimeric inhibitory receptor of the present disclosure comprises at least a portion of an extracellular domain, a transmembrane domain, and / or an intracellular domain. In some embodiments, the enzyme inhibitory domain comprises at least a portion of an enzyme. In some embodiments, the enzyme is selected from CSK, SHP-1, PTEN, CD45, CD148, PTP-MEG1, PTP-PEST, c-CBL, CBL-b, PTPN22, LAR, PTPH1, SHIP-1, and RasGAP (see, e.g., Stanford et al., Regulation of TCR signaling by tyrosine phosphatases: from immune homeostasis to autoimmunity, Immunology, 2012 Sep;137(1):1-19). In some embodiments, the portion of the enzyme comprises an enzyme domain(s), enzyme fragment(s), or variant(s) thereof. In some embodiments, the portion of the enzyme is the catalytic domain of the enzyme. In some embodiments, the enzyme domain(s), enzyme fragment(s), or variant(s) thereof are selected to maximize efficacy and minimize basal inhibition.

[0338] In some embodiments, the enzyme inhibitory domain comprises one or more modifications that modulate basal inhibition. Examples of modifications include, but are not limited to, truncation mutation(s), amino acid substitution(s), introduction of sites for post-translational modifications (examples of which are known to those of skill in the art), and addition of new functional groups. In some embodiments, the enzyme domain(s), enzyme fragment(s), or variant(s) thereof are selected to maximize efficacy and minimize basal inhibition. In some embodiments, the one or more modifications reduce basal inhibition. In other embodiments, the one or more modifications increase basal inhibition.

[0339] In some embodiments, the enzyme inhibitory domain inhibits immunoreceptor activation, for example, upon recruitment of a chimeric inhibitory receptor of the present disclosure into proximity with the immunoreceptor. In some embodiments, the immunoreceptor is a naturally occurring immunoreceptor. In some embodiments, the immunoreceptor is a naturally occurring antigen receptor. In some embodiments, the immunoreceptor is selected from a T cell receptor, a pattern recognition receptor (PRR), a NOD-like receptor (NLR), a Toll-like receptor (TLR), a killer activating receptor (KAR), a killer inhibitor receptor (KIR), a complement receptor, an Fc receptor, a B cell receptor, and a cytokine receptor. In some embodiments, the immunoreceptor is a T cell receptor. In some embodiments, the immunoreceptor is a chimeric immunoreceptor. In some embodiments, the chimeric immunoreceptor is a chimeric TCR or a CAR.

[0340] In some embodiments, a chimeric inhibitory receptor of the present disclosure may also comprise one or more intracellular inhibitory co-signaling domains. In some embodiments, the intracellular inhibitory co-signaling domain comprises an inhibitory domain. In some embodiments, the one or more intracellular inhibitory co-signaling domains comprise one or more ITIM-containing proteins, or fragments thereof. ITIMs are conserved amino acid sequences found in the cytoplasmic tails of many inhibitory immunoreceptors. In some embodiments, the one or more ITIM-containing proteins, or fragments thereof, are selected from PD-1, CTLA4, TIGIT, and LAIR1. In some embodiments, the one or more intracellular inhibitory co-signaling domains comprise one or more non-ITIM scaffolding proteins, or fragments thereof. In some embodiments, the one or more non-ITIM scaffolding proteins, or fragments thereof, are selected from GRB-2, Dok-1, Dok-2, SLAP, LAG3, HAVR, BTLA, GITR, and PD-L1. Further examples of suitable intracellular inhibitory co-signaling domains include, but are not limited to, PD-L1, TIM3, VISTA, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF-beta.

[0341] In some embodiments, the chimeric inhibitory receptor binds to an antigen expressed on a non-tumor cell. Exemplary antigens for use in chimeric inhibitory receptors are listed in Table 2.

[0342] (Table 2) TIFF2025143407000017.tif239161TIFF2025143407000018.tif228161TIFF2025143407000019.tif240161TIFF2025143407000020.tif152161

[0343] In some embodiments, the chimeric inhibitory receptor binds to the EMCN antigen. In some embodiments, the chimeric inhibitory receptor binds to the JAM2 antigen. In some embodiments, the chimeric inhibitory receptor binds to the MS4A15 antigen. In some embodiments, the chimeric inhibitory receptor binds to the C4BPA antigen. In some embodiments, the chimeric inhibitory receptor binds to the TRPM1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the SCTR antigen. In some embodiments, the chimeric inhibitory receptor binds to the SLC2A2 antigen. In some embodiments, the chimeric inhibitory receptor binds to the KCNQ2 antigen. In some embodiments, the chimeric inhibitory receptor binds to the PERP antigen. In some embodiments, the chimeric inhibitory receptor binds to the PERP antigen. In some embodiments, the chimeric inhibitory receptor binds to the FFAR2 antigen.

[0344] In some embodiments, the chimeric inhibitory receptor is a multispecific receptor that includes two or more antigen-binding domains, such that the chimeric inhibitory receptor can bind to two or more antigens. Alternatively, cells can be edited to express two or more chimeric inhibitory receptors that bind different antigens.

[0345] In some embodiments, the chimeric inhibitory receptor binds to the PTPRB antigen. In some embodiments, the chimeric inhibitory receptor binds to the NCKAP1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the MPZL2 antigen. In some embodiments, the chimeric inhibitory receptor binds to the PLSCR4 antigen. In some embodiments, the chimeric inhibitory receptor binds to the TMEM47 antigen. In some embodiments, the chimeric inhibitory receptor binds to the ADGRL4 antigen. In some embodiments, the chimeric inhibitory receptor binds to the MET antigen. In some embodiments, the chimeric inhibitory receptor binds to the BACE2 antigen. In some embodiments, the chimeric inhibitory receptor binds to the ATP8B1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the LIFR antigen. In some embodiments, the chimeric inhibitory receptor binds to the ART4 antigen. In some embodiments, the chimeric inhibitory receptor binds to the CALCRL antigen. In some embodiments, the chimeric inhibitory receptor binds to the CNTNAP3 antigen. In some embodiments, the chimeric inhibitory receptor binds to the PCDH9 antigen. In some embodiments, the chimeric inhibitory receptor binds to the IL18R1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the SLC8A3 antigen. In some embodiments, the chimeric inhibitory receptor binds to the CDH26 antigen. In some embodiments, the chimeric inhibitory receptor binds to the TMEM163 antigen. In some embodiments, the chimeric inhibitory receptor binds to the ABCA13 antigen. In some embodiments, the chimeric inhibitory receptor binds to the CACHD1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the CYYR1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the ABCB1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the ADGRG6 antigen. In some embodiments, the chimeric inhibitory receptor binds to the ATP9A antigen. In some embodiments, the chimeric inhibitory receptor binds to the CALN1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the CALN1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the CDCP1 antigen.In some embodiments, the chimeric inhibitory receptor binds to the IL12RB2 antigen. In some embodiments, the chimeric inhibitory receptor binds to the SLC16A14 antigen. In some embodiments, the chimeric inhibitory receptor binds to the TMEM136 antigen. In some embodiments, the chimeric inhibitory receptor binds to the TMEM200A antigen.

[0346] immunoresponsive cells Certain aspects of the present disclosure relate to cells, e.g., immunoresponsive cells, genetically engineered to contain one or more chimeric receptors of the present disclosure or one or more nucleic acids encoding such chimeric receptors, and methods of using such cells to treat myeloid malignancies (e.g., AML).

[0347] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a primary cell. In some embodiments, the mammalian cell is a cell line. In some embodiments, the mammalian cell is a bone marrow cell, a blood cell, a skin cell, a bone cell, a muscle cell, a neuron, an adipocyte, a liver cell, or a cardiac cell. In some embodiments, the cell is a stem cell. Exemplary stem cells include, but are not limited to, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), adult stem cells, and tissue-specific stem cells such as hematopoietic stem cells (blood stem cells), mesenchymal stem cells (MSCs), neural stem cells, epithelial stem cells, or skin stem cells. In some embodiments, the cell is derived from or differentiated from a stem cell of the present disclosure. In some embodiments, the cell is an immune cell. The immune cells of the present disclosure can be isolated or differentiated from the stem cells of the present disclosure (e.g., from ESCs or iPSCs). Exemplary immune cells include, but are not limited to, T cells (e.g., helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, alpha beta T cells, and gamma delta T cells), B cells, natural killer (NK) cells, dendritic cells, myeloid cells, macrophages, and monocytes. In some embodiments, the cell is a neuronal cell. Neuronal cells of the present disclosure can be isolated or differentiated from stem cells of the present disclosure (e.g., from ESCs or iPSCs). Exemplary neuronal cells include, but are not limited to, neural progenitor cells, neurons (e.g., sensory neurons, motor neurons, cholinergic neurons, GABAergic neurons, glutamatergic neurons, dopaminergic neurons, or serotonergic neurons), astrocytes, oligodendrocytes, and microglia.

[0348] In some embodiments, the cell is an immunoresponsive cell. The immunoresponsive cells of the present disclosure can be isolated or differentiated from the stem cells of the present disclosure (e.g., from ESCs or iPSCs). Exemplary immunoresponsive cells of the present disclosure include, but are not limited to, cells of the lymphoid lineage. The lymphoid lineage, including B cells, T cells, and natural killer (NK) cells, produces antibodies, regulates the cellular immune system, detects foreign substances in the blood, detects cells foreign to the host, and the like. Examples of immunoresponsive cells of the lymphoid lineage include, but are not limited to, T cells, natural killer (NK) cells, embryonic stem cells, pluripotent stem cells, and induced pluripotent stem cells (e.g., those derived from or capable of differentiating into lymphocytes). T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. In some embodiments, the T cells of the present disclosure include T helper cells, cytotoxic T cells, memory T cells (central memory T cells, stem cell-like memory T cells (or stem-like memory T cells)), and two types of effector memory T cells, e.g., T EM Cells and T EMRA The T cells can be any type of T cell, including, but not limited to, T cells, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. The patient's own T cells can be genetically modified to target specific antigens through the introduction of one or more chimeric receptors, such as chimeric TCRs or CARs.

[0349] Natural killer (NK) cells are lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation to exert their cytotoxic effect on target cells.

[0350] In some embodiments, the immunoresponsive cells of the present disclosure are T cells. The T cells of the present disclosure can be derived in vitro from autologous, allogeneic, or engineered progenitor or stem cells.

[0351] In some embodiments, the immunoresponsive cells of the present disclosure are universal T cells with defective TCR-αβ. Methods for developing universal T cells are described in the art, for example, Valton et al., Molecular Therapy (2015); 23 9, 1507-1518, and Torikai et al., Blood 2012 119:5697-5705.

[0352] In some embodiments, the immunoresponsive cells of the present disclosure are isolated immunoresponsive cells comprising one or more chimeric receptors of the present disclosure, hi some embodiments, the immunoresponsive cells comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more chimeric receptors of the present disclosure.

[0353] In some embodiments, the immunoresponsive cells are T cells. In some embodiments, the immunoresponsive cells are natural killer (NK) cells.

[0354] Cells expressing multiple chimeric receptors In some embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises two or more chimeric receptors of the present disclosure. In some embodiments, the cell comprises two or more chimeric receptors, and one of the two or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises three or more chimeric receptors, and at least one of the three or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises four or more chimeric receptors, and at least one of the four or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises five or more chimeric receptors, and at least one of the five or more chimeric receptors is a chimeric inhibitory receptor.

[0355] In some embodiments, each of the two or more chimeric receptors comprises a different antigen-binding domain, e.g., antigen-binding domains that bind to the same antigen or different antigens. In some embodiments, each antigen bound by the two or more chimeric receptors is expressed on the same myeloid cell type (e.g., the same AML cell type). In one embodiment, a cell comprises a first chimeric receptor that targets a first antigen and comprises an intracellular signaling domain with a costimulatory signaling domain but not a primary signaling domain, and a second chimeric receptor that targets a second, different antigen and comprises an intracellular signaling domain with a primary signaling domain but not a costimulatory signaling domain. Without wishing to be bound by theory, it is believed that placing a costimulatory signaling domain (e.g., 4-1BB, CD28, or OX-40) on the first chimeric receptor and a primary signaling domain (e.g., CD3-zeta chain) on the second chimeric receptor may restrict chimeric receptor activity to cells in which both targets are expressed. Thus, in some embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises (a) a first chimeric receptor comprising an antigen binding domain that binds a first antigen, a transmembrane domain, and a costimulatory signaling domain, and (b) a second chimeric receptor comprising an antigen binding domain that binds a second antigen, a transmembrane domain, and a primary signaling domain. In some embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises (a) a first chimeric receptor comprising an antigen binding domain that binds a first antigen, a transmembrane domain, and a primary signaling domain, and (b) a second chimeric receptor comprising an antigen binding domain that binds a second antigen, a transmembrane domain, and a costimulatory signaling domain. In some embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises (a) a first chimeric receptor comprising an antigen binding domain that binds a first antigen, a transmembrane domain, a primary signaling domain, and a costimulatory domain, and (b) a second chimeric receptor comprising an antigen binding domain that binds a second antigen, a transmembrane domain, a primary signaling domain, and a costimulatory domain.In embodiments in which both the first chimeric receptor and the second chimeric receptor each comprise a costimulatory signaling domain, the costimulatory signaling domain of the first chimeric receptor and the costimulatory signaling domain of the second chimeric receptor can be derived from the same protein, e.g., 4-1BB, CD28, or OX40. Alternatively, the costimulatory signaling domain of the first chimeric receptor can be derived from a different protein than the costimulatory signaling domain of the second chimeric receptor.

[0356] In embodiments in which a cell (e.g., an immunoresponsive cell) of the present disclosure expresses two or more distinct chimeric receptors, the antigen-binding domains of each of the different chimeric receptors can be designed so that the antigen-binding domains do not interact with each other. For example, a cell (e.g., an immunoresponsive cell) of the present disclosure that expresses a first chimeric receptor and a second chimeric receptor can include a first chimeric receptor that comprises an antigen-binding domain that does not form an association with the antigen-binding domain of the second chimeric receptor. For example, the antigen-binding domain of the first chimeric receptor can comprise an antibody fragment such as an scFv, while the antigen-binding domain of the second chimeric receptor can comprise a VHH.

[0357] Without wishing to be bound by theory, it is believed that in cells bearing multiple chimeric membrane-embedded receptors, each comprising an antigen-binding domain, interactions between the antigen-binding domains of each receptor may be undesirable, as such interactions may inhibit the ability of one or more of the antigen-binding domains to bind to their cognate antigen. Thus, in embodiments in which a cell (e.g., an immunoresponsive cell) of the present disclosure expresses two or more chimeric receptors, the chimeric receptors comprise antigen-binding domains that minimize such inhibitory interactions. In one embodiment, the antigen-binding domain of one chimeric receptor comprises an scFv, and the antigen-binding domain of a second chimeric receptor comprises a single VH domain, e.g., a camel, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence.

[0358] In some embodiments, when present on the surface of a cell, binding of the antigen-binding domain of a first chimeric receptor to its cognate antigen is not substantially reduced by the presence of a second chimeric receptor. In some embodiments, binding of the antigen-binding domain of a first chimeric receptor to its cognate antigen in the presence of the second chimeric receptor is 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the binding of the antigen-binding domain of the first chimeric receptor to its cognate antigen in the absence of the second chimeric receptor. In some embodiments, when present on the surface of a cell, the antigen-binding domains of the first chimeric receptor and the second chimeric receptor associate with each other less than when both are scFv antigen-binding domains. In some embodiments, the antigen-binding domains of the first chimeric receptor and the second chimeric receptor associate with each other 85%, 90%, 95%, 96%, 97%, 98%, or 99% less than when both are scFv antigen-binding domains.

[0359] In embodiments in which a cell (e.g., an immunoresponsive cell) of the present disclosure comprises two or more distinct chimeric receptors of the present disclosure that bind different antigens, the two or more chimeric receptors provide logical gating to the cell, such as OR logical gating, AND logical gating, NOT logical gating, or any combination of such logical gating. Thus, in certain embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises two or more chimeric receptors, and binding of a first chimeric receptor to a first antigen can activate the cell. In some embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises two or more chimeric receptors, and binding of a second chimeric receptor to a second antigen can stimulate the cell. In some embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises two or more chimeric receptors, and binding of a first chimeric receptor to a first antigen and binding of a second chimeric receptor to a second antigen are required to activate the cell. In some embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises two or more chimeric receptors, and binding of a first chimeric receptor to a first antigen and binding of a second chimeric receptor to a second antigen are required to stimulate the cell. In some embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises two or more chimeric receptors, and the cell exhibits a greater degree of cytolytic activity against cells positive for both the first and second antigens compared to cytolytic activity against cells positive for only the first antigen or only the second antigen. In some embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises two or more chimeric receptors, and binding of a first chimeric receptor to a first antigen or binding of a second chimeric receptor to a second antigen can activate the immunoresponsive cell.

[0360] In some embodiments, cells (e.g., immunoresponsive cells) of the present disclosure comprise a split-chimeric receptor system, such as a split-CAR system. Exemplary split-chimeric receptor systems are described in WO2014 / 055442 and WO2014 / 055657. In some embodiments, the split-chimeric receptor system comprises a cell expressing a first chimeric receptor having a first antigen-binding domain and a costimulatory domain (e.g., 4-1BB) and a second chimeric receptor having a second antigen-binding domain and an intracellular signaling domain (e.g., CD3-zeta). In such embodiments, when the cell encounters the first antigen, the costimulatory domain is activated, causing the cell to proliferate. Additionally, when the cell encounters the second antigen, the intracellular signaling domain is activated, inducing cell-killing activity. Thus, in some embodiments, cells (e.g., immunoresponsive cells) of the present disclosure are only fully activated in the presence of both antigens.

[0361] In certain embodiments, cells (e.g., immunoresponsive cells) of the present disclosure exhibit a greater degree of cytolytic activity against cells positive for both the first and second antigens compared to cells positive for the first antigen alone. In certain embodiments, the first chimeric receptor binds to the first antigen with low binding affinity or low avidity. In certain embodiments, the first chimeric receptor binds to the first antigen at a less accessible epitope. In certain embodiments, the first chimeric receptor binds to the first antigen with a lower binding affinity compared to the binding affinity with which the second chimeric receptor binds to the second antigen. In some embodiments, the first chimeric receptor binds to the first antigen with a binding affinity that is at least 5-fold lower compared to the binding affinity with which the second chimeric receptor binds to the second antigen. In some embodiments, the first chimeric receptor binds to the first antigen with a binding affinity that is at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 5000-fold, 1000-fold, 5000-fold, or 10000-fold lower than the binding affinity with which the second chimeric receptor binds to the second antigen.

[0362] In some embodiments, pairing selection should favor redundant expression of two target antigens in tumors to minimize the risk of antigen escape. Thus, in some embodiments, the cells (e.g., immunocompetent cells) of the present disclosure comprise (i) a first chimeric receptor that binds to a first antigen and (ii) a second chimeric receptor that binds to a second antigen, and the combination of both chimeric receptors that bind to the target antigens produces a therapeutic effect. In embodiments, binding to only one target antigen does not achieve a therapeutic effect.

[0363] In some embodiments, the chimeric receptor binds to the FLT3 antigen. In some embodiments, the chimeric receptor binds to the MS4A3 antigen. In some embodiments, the chimeric receptor binds to the CD33 antigen. In some embodiments, the chimeric receptor binds to the CLEC12A antigen. In some embodiments, the chimeric receptor binds to the CD312 / ADGRE2 antigen. In some embodiments, the chimeric receptor binds to the SLC22A16 antigen. In some embodiments, the chimeric receptor binds to the CD123 / ILR3RA antigen. In some embodiments, the chimeric receptor binds to the LAT2 antigen. In some embodiments, the chimeric receptor binds to the PIEZO1 / FAM38A antigen. In some embodiments, the chimeric receptor binds to the CD38 antigen. In some embodiments, the chimeric receptor binds to the EMB antigen. In some embodiments, the chimeric receptor binds to the CD131 / CSF2RB antigen. In some embodiments, the chimeric receptor binds to the P2RY8 antigen. In some embodiments, the chimeric receptor binds to the LILRA2 / CD85H antigen. In some embodiments, the chimeric receptor binds to the SLC17A9 antigen. In some embodiments, the chimeric receptor binds to the MYADM antigen. In some embodiments, the chimeric receptor binds to the CD300LF antigen. In some embodiments, the chimeric receptor binds to the CD244 / SLAMF4 antigen. In some embodiments, the chimeric receptor binds to the PLAUR antigen. In some embodiments, the chimeric receptor binds to the CD93 antigen. In some embodiments, the chimeric receptor binds to the SPNS3 antigen. In some embodiments, the chimeric receptor binds to the GAPT antigen. In some embodiments, the chimeric receptor binds to the RASGRP4 antigen. In some embodiments, the chimeric receptor binds to the CD117 / c-Kit antigen. In some embodiments, the chimeric receptor binds to the CD123 / ILR3RA antigen.

[0364] In some embodiments, the chimeric receptor binds to the VSTM1 antigen. In some embodiments, the chimeric receptor binds to the MLC1 antigen. In some embodiments, the chimeric receptor binds to the PRAM1 antigen. In some embodiments, the chimeric receptor binds to the HCK antigen. In some embodiments, the chimeric receptor binds to the ICAM3 antigen. In some embodiments, the chimeric receptor binds to the LRRC37A2 antigen. In some embodiments, the chimeric receptor binds to the ITGAM antigen. In some embodiments, the chimeric receptor binds to the ITGB2 antigen. In some embodiments, the chimeric receptor binds to the LILRA1 antigen. In some embodiments, the chimeric receptor binds to the PRTN3 antigen. In some embodiments, the chimeric receptor binds to the CARD9 antigen. In some embodiments, the chimeric receptor binds to the SIGLEC5 antigen. In some embodiments, the chimeric receptor binds to the SELL antigen. In some embodiments, the chimeric receptor binds to the MLKL antigen. In some embodiments, the chimeric receptor binds to the INPP5D antigen. In some embodiments, the chimeric receptor binds to the APBB1IP antigen. In some embodiments, the chimeric receptor binds to the ITGA4 antigen. In some embodiments, the chimeric receptor binds to the C3AR1 antigen. In some embodiments, the chimeric receptor binds to the ITGA5 antigen. In some embodiments, the chimeric receptor binds to the FMNL1 antigen. In some embodiments, the chimeric receptor binds to the IL1RAP antigen. In some embodiments, the chimeric receptor binds to the CCR1 / CD191 antigen. In some embodiments, the chimeric receptor binds to the LILRB2 antigen. In some embodiments, the chimeric receptor binds to the CD70 antigen. In some embodiments, the chimeric receptor is a multispecific receptor comprising two or more antigen binding domains such that the chimeric receptor can bind to two or more antigens.

[0365] In some embodiments, immunoresponsive cells may contain one or more tumor-targeting chimeric receptors and one or more inhibitory chimeric receptors that target antigens not expressed on tumors. The combination of tumor-targeting chimeric receptors and inhibitory chimeric receptors in the same immunoresponsive cells can be used to reduce extratumoral toxicity on the target. For example, if healthy cells express both the antigen recognized by the tumor-targeting chimeric receptor and the antigen recognized by the inhibitory chimeric receptor, the immunoresponsive cells expressing the tumor antigen may bind to the healthy cells. In such cases, the inhibitory chimeric antigen also binds to its cognate ligand on the healthy cells, and the inhibitory function of the inhibitory chimeric receptor reduces, reduces, prevents, or inhibits the activation of immunoresponsive cells via the tumor-targeting chimeric receptor.

[0366] In some embodiments, the inhibitory chimeric receptor binds to the EMCN (endomucin) antigen. In some embodiments, the inhibitory chimeric receptor binds to the JAM2 antigen. In some embodiments, the inhibitory chimeric receptor binds to the MS4A15 antigen. In some embodiments, the inhibitory chimeric receptor binds to the SLC34A2 antigen. In some embodiments, the inhibitory chimeric receptor binds to the SLC2A2 antigen. In some embodiments, the inhibitory chimeric receptor binds to the TRPM1 antigen. In some embodiments, the inhibitory chimeric receptor binds to the SCTR antigen. In some embodiments, the inhibitory chimeric receptor binds to the KCNQ2 antigen. In some embodiments, the inhibitory chimeric receptor binds to the PERP antigen. In some embodiments, the inhibitory chimeric receptor binds to the WLS antigen. In some embodiments, the inhibitory chimeric receptor binds to the FFAR2 antigen. In some embodiments, the inhibitory chimeric receptor binds to the PTPRB antigen. In some embodiments, the inhibitory chimeric receptor binds to the NCKAP1 antigen. In some embodiments, the inhibitory chimeric receptor binds to the MPZL2 antigen. In some embodiments, the inhibitory chimeric receptor binds to the PLSCR4 antigen. In some embodiments, the inhibitory chimeric receptor binds to the TMEM47 antigen. In some embodiments, the inhibitory chimeric receptor binds to the ADGRL4 antigen. In some embodiments, the inhibitory chimeric receptor binds to the MET antigen. In some embodiments, the inhibitory chimeric receptor binds to the BACE2 antigen. In some embodiments, the inhibitory chimeric receptor binds to the ATP8B1 antigen. In some embodiments, the inhibitory chimeric receptor binds to the LIFR antigen. In some embodiments, the inhibitory chimeric receptor binds to the ART4 antigen. In some embodiments, the inhibitory chimeric receptor binds to the CALCRL antigen. In some embodiments, the inhibitory chimeric receptor binds to the CNTNAP3 antigen. In some embodiments, the inhibitory chimeric receptor binds to the PCDH9 antigen. In some embodiments, the inhibitory chimeric receptor binds to the IL18R1 antigen. In some embodiments, the inhibitory chimeric receptor binds to the SLC8A3 antigen.In some embodiments, the inhibitory chimeric receptor binds to the CDH26 antigen. In some embodiments, the inhibitory chimeric receptor binds to the SLC8A3 antigen. In some embodiments, the inhibitory chimeric receptor binds to the TMEM163 antigen. In some embodiments, the inhibitory chimeric receptor binds to the ABCA13 antigen. In some embodiments, the inhibitory chimeric receptor binds to the CACHD1 antigen. In some embodiments, the inhibitory chimeric receptor binds to the CYYR1 antigen. In some embodiments, the inhibitory chimeric receptor binds to the ABCB1 antigen. In some embodiments, the inhibitory chimeric receptor binds to the ADGRG6 antigen. In some embodiments, the inhibitory chimeric receptor binds to the ATP9A antigen. In some embodiments, the inhibitory chimeric receptor binds to the CALN1 antigen. In some embodiments, the inhibitory chimeric receptor binds to the CDCP1 antigen. In some embodiments, the inhibitory chimeric receptor binds to the IL12RB2 antigen. In some embodiments, the inhibitory chimeric receptor binds to the SLC16A14 antigen. In some embodiments, the inhibitory chimeric receptor binds to the TMEM136 antigen. In some embodiments, the inhibitory chimeric receptor binds to the TMEM200A antigen.

[0367] Alternatively, the cells express two or more chimeric receptors that bind different antigens. Exemplary pairs of antigens are shown in Table 3.

[0368] (Table 3) TIFF2025143407000021.tif144128TIFF2025143407000022.tif135128

[0369] In some embodiments, the two or more antigens are ITGA4 and SLC17A9. In some embodiments, the two or more antigens are ITGA4 and LRRC37A2. In some embodiments, the two or more antigens are ITGA4 and EMB. In some embodiments, the two or more antigens are ITGA4 and MLKL. In some embodiments, the two or more antigens are ITGA4 and MYADM. In some embodiments, the two or more antigens are ITGA4 and ADGRE2.

[0370] In some embodiments, the two or more antigens are ITGA5 and ITGAM. In some embodiments, the two or more antigens are ITGA5 and LRRC37A2. In some embodiments, the two or more antigens are ITGA5 and PRTN3. In some embodiments, the two or more antigens are ITGA5 and MLC1. In some embodiments, the two or more antigens are ITGA5 and ITGB2. In some embodiments, the two or more antigens are ITGA5 and LAT2. In some embodiments, the two or more antigens are ITGA5 and MS4A3. In some embodiments, the two or more antigens are ITGA5 and PIEZO1.

[0371] In some embodiments, the two or more antigens are ITGAM and PIEZO1. In some embodiments, the two or more antigens are ITGAM and LRRC37A2. In some embodiments, the two or more antigens are ITGAM and PRTN3. In some embodiments, the two or more antigens are ITGAM and MLC1. In some embodiments, the two or more antigens are ITGAM and LAT2. In some embodiments, the two or more antigens are ITGAM and MS4A3.

[0372] In some embodiments, the two or more antigens are CSF2RB and SLC17A9. In some embodiments, the two or more antigens are CSF2RB and LRRC37A2. In some embodiments, the two or more antigens are CSF2RB and EMB. In some embodiments, the two or more antigens are CSF2RB and MLKL. In some embodiments, the two or more antigens are CSF2RB and ADGRE2.

[0373] In some embodiments, the two or more antigens are SLC17A9 and CMTM7. In some embodiments, the two or more antigens are SLC17A9 and LRRC37A2. In some embodiments, the two or more antigens are SLC17A9 and CD244. In some embodiments, the two or more antigens are SLC17A9 and EMB. In some embodiments, the two or more antigens are SLC17A9 and MLKL. In some embodiments, the two or more antigens are SLC17A9 and MYADM. In some embodiments, the two or more antigens are SLC17A9 and FLT3. In some embodiments, the two or more antigens are SLC17A9 and ADGRE2. In some embodiments, the two or more antigens are SLC17A9 and LILRA2. In some embodiments, the two or more antigens are SLC17A9 and LILRA1.

[0374] In some embodiments, the two or more antigens are CYBA and LRRC37A2. In some embodiments, the two or more antigens are CYBA and PRTN3. In some embodiments, the two or more antigens are CYBA and MLC1. In some embodiments, the two or more antigens are CYBA and LAT2. In some embodiments, the two or more antigens are CYBA and MS4A3. In some embodiments, the two or more antigens are CYBA and ICAM3. In some embodiments, the two or more antigens are CYBA and PIEZO1.

[0375] In some embodiments, the two or more antigens are CMTM7 and LRRC37A2. In some embodiments, the two or more antigens are CMTM7 and EMB. In some embodiments, the two or more antigens are CMTM7 and MLKL. In some embodiments, the two or more antigens are CMTM7 and MYADM. In some embodiments, the two or more antigens are CMTM7 and ADGRE2. In some embodiments, the two or more antigens are CMTM7 and LILRA2. In some embodiments, the two or more antigens are CMTM7 and LILRA1.

[0376] In some embodiments, the two or more antigens are LRRC37A2 and CD244. In some embodiments, the two or more antigens are LRRC37A2 and PRTN3. In some embodiments, the two or more antigens are LRRC37A2 and EMB. In some embodiments, the two or more antigens are LRRC37A2 and CARD9. In some embodiments, the two or more antigens are LRRC37A2 and MLC1. In some embodiments, the two or more antigens are LRRC37A2 and ITGB2. In some embodiments, the two or more antigens are LRRC37A2 and LAT2. In some embodiments, the two or more antigens are LRRC37A2 and SIGLEC5. ​​In some embodiments, the two or more antigens are LRRC37A2 and CD300LF. In some embodiments, the two or more antigens are LRRC37A2 and MLKL. In some embodiments, the two or more antigens are LRRC37A2 and INPP5D. In some embodiments, the two or more antigens are LRRC37A2 and MYADM. In some embodiments, the two or more antigens are LRRC37A2 and MS4A3. In some embodiments, the two or more antigens are LRRC37A2 and HCK. In some embodiments, the two or more antigens are LRRC37A2 and APBB1IP. In some embodiments, the two or more antigens are LRRC37A2 and ICAM3. In some embodiments, the two or more antigens are LRRC37A2 and CD33. In some embodiments, the two or more antigens are LRRC37A2 and PIEZO1. In some embodiments, the two or more antigens are LRRC37A2 and FLT3. In some embodiments, the two or more antigens are LRRC37A2 and ADGRE2. In some embodiments, the two or more antigens are LRRC37A2 and FMNL1. In some embodiments, the two or more antigens are LRRC37A2 and CLEC12A. In some embodiments, the two or more antigens are LRRC37A2 and LILRA2. In some embodiments, the two or more antigens are LRRC37A2 and LILRA1.In some embodiments, the two or more antigens are LRRC37A2 and CD93. In some embodiments, the two or more antigens are LRRC37A2 and C3AR1.

[0377] In some embodiments, the two or more antigens are CD244 and EMB. In some embodiments, the two or more antigens are CD244 and MLKL. In some embodiments, the two or more antigens are CD244 and ADGRE2.

[0378] In some embodiments, the two or more antigens are PRTN3 and SIGLEC5.

[0379] In some embodiments, the two or more antigens are EMB and MLKL. In some embodiments, the two or more antigens are EMB and MYADM. In some embodiments, the two or more antigens are EMB and FLT3. In some embodiments, the two or more antigens are EMB and ADGRE2. In some embodiments, the two or more antigens are EMB and LILRA2.

[0380] In some embodiments, the two or more antigens are CARD9 and MLC1. In some embodiments, the two or more antigens are CARD9 and LAT2. In some embodiments, the two or more antigens are CARD9 and SIGLEC5. ​​In some embodiments, the two or more antigens are CARD9 and CD300LF. In some embodiments, the two or more antigens are CARD9 and INPP5D. In some embodiments, the two or more antigens are CARD9 and MS4A3. In some embodiments, the two or more antigens are CARD9 and HCK. In some embodiments, the two or more antigens are CARD9 and ICAM3. In some embodiments, the two or more antigens are CARD9 and CD38. In some embodiments, the two or more antigens are CARD9 and CD33. In some embodiments, the two or more antigens are CARD9 and PIEZO1. In some embodiments, the two or more antigens are CARD9 and FMNL1. In some embodiments, the two or more antigens are CARD9 and CLEC12A. In some embodiments, the two or more antigens are CARD9 and CD93.

[0381] In some embodiments, the two or more antigens are MLC1 and SIGLEC5. ​​In some embodiments, the two or more antigens are MLC1 and MS4A3. In some embodiments, the two or more antigens are MLC1 and PIEZO1.

[0382] In some embodiments, the two or more antigens are ITGB2 and PIEZO1. In some embodiments, the two or more antigens are ITGB2 and MS4A3.

[0383] In some embodiments, the two or more antigens are LAT2 and MS4A3. In some embodiments, the two or more antigens are LAT2 and SIGLEC5. ​​In some embodiments, the two or more antigens are LAT2 and PIEZO1.

[0384] In some embodiments, the two or more antigens are CD300LF and PIEZO1. In some embodiments, the two or more antigens are CD300LF and MS4A3.

[0385] In some embodiments, the two or more antigens are MLKL and MYADM. In some embodiments, the two or more antigens are MLKL and FLT3. In some embodiments, the two or more antigens are MLKL and ADGRE2. In some embodiments, the two or more antigens are MLKL and LILRA2. In some embodiments, the two or more antigens are MLKL and LILRA1.

[0386] In some embodiments, the two or more antigens are INPP5D and PIEZO1. In some embodiments, the two or more antigens are INPP5D and MS4A3.

[0387] In some embodiments, the two or more antigens are MYADM and FLT3.

[0388] In some embodiments, the two or more antigens are MS4A3 and PIEZO1. In some embodiments, the two or more antigens are HCK and PIEZO1. In some embodiments, the two or more antigens are ICAM3 and PIEZO1. In some embodiments, the two or more antigens are CD38 and PIEZO1. In some embodiments, the two or more antigens are CD93 and CD38. In some embodiments, the two or more antigens are CD33 and PIEZO1.

[0389] In some embodiments, the two or more antigens are APBB1IP and C3AR1.

[0390] In some embodiments, the two or more antigens are FLT3 and ADGRE2. In some embodiments, the two or more antigens are ADGRE2 and LILRA2. In some embodiments, the two or more antigens are ADGRE2 and LILRA1.

[0391] In some embodiments, the two or more antigens are PIEZO1 and P2RY8.

[0392] In some embodiments, the two or more antigens are FLT3 and CD33. In some embodiments, the cells express two or more chimeric receptors of the present disclosure, where one chimeric receptor binds FLT3 and a second chimeric receptor binds CD33. In some embodiments, the chimeric receptor is a multispecific receptor comprising two or more antigen binding domains, such that one antigen binding domain binds FLT3 and a second antigen binding domain binds CD33. In some embodiments, the chimeric antigen receptor and / or antigen-binding domain that binds to FLT3 is selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 3 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 4 or a sequence at least 90% identical thereto; (c) a VH comprising the amino acid sequence of SEQ ID NO: 5 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 6 or a sequence at least 90% identical thereto; (d) a VH comprising the amino acid sequence of SEQ ID NO: 7 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 8 or a sequence at least 90% identical thereto; (e) a VH comprising the amino acid sequence of SEQ ID NO: 9 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 10 or a sequence at least 90% identical thereto; (f) a VH comprising the amino acid sequence of SEQ ID NO: 11 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 12 or a sequence at least 90% identical thereto; (g) a VH comprising the amino acid sequence of SEQ ID NO: 13 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 14 or a sequence at least 90% identical thereto; and (h) a VH comprising the amino acid sequence of SEQ ID NO: 15 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 16 or a sequence at least 90% identical thereto.In some embodiments, the chimeric antigen receptor and / or antigen binding domain that binds CD33 comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from (a) a VH comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 18, or a sequence at least 90% identical thereto, and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20, or a sequence at least 90% identical thereto.

[0393] In some embodiments, the two or more antigens are FLT3 and CLEC12A. In some embodiments, the cells express two or more chimeric receptors of the present disclosure, where one chimeric receptor binds FLT3 and a second chimeric receptor binds CLEC12A. In some embodiments, the chimeric receptor is a multispecific receptor comprising two or more antigen binding domains, such that one antigen binding domain binds FLT3 and a second antigen binding domain binds CLEC12A. In some embodiments, the chimeric receptor is a multispecific receptor comprising two or more antigen binding domains, such that one antigen binding domain binds FLT3 and a second antigen binding domain binds CD33.In some embodiments, the chimeric antigen receptor and / or antigen-binding domain that binds to FLT3 is selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 3 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 4 or a sequence at least 90% identical thereto; (c) a VH comprising the amino acid sequence of SEQ ID NO: 5 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 6 or a sequence at least 90% identical thereto; (d) a VH comprising the amino acid sequence of SEQ ID NO: 7 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 8 or a sequence at least 90% identical thereto; (e) a VH comprising the amino acid sequence of SEQ ID NO: 9 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 10 or a sequence at least 90% identical thereto; (f) a VH comprising the amino acid sequence of SEQ ID NO: 11 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 12 or a sequence at least 90% identical thereto; (g) a VH comprising the amino acid sequence of SEQ ID NO: 13 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 14 or a sequence at least 90% identical thereto; and (h) a VH comprising the amino acid sequence of SEQ ID NO: 15 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 16 or a sequence at least 90% identical thereto.In some embodiments, the chimeric antigen receptor and / or antigen binding domain that binds to CLEC12A comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from: (a) a VH comprising the amino acid sequence of SEQ ID NO: 21, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 22, or a sequence at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 23, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 24, or a sequence at least 90% identical thereto; and (c) a VH comprising the amino acid sequence of SEQ ID NO: 25, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 26, or a sequence at least 90% identical thereto.

[0394] In some embodiments, the two or more antigens are CLEC12A and CD33. In some embodiments, the cells express two or more chimeric receptors of the present disclosure, where one chimeric receptor binds CLEC12A and a second chimeric receptor binds CD33. In some embodiments, the chimeric receptor is a multispecific receptor comprising two or more antigen binding domains, such that one antigen binding domain binds CLEC12A and a second antigen binding domain binds CD33. In some embodiments, the chimeric antigen receptor and / or antigen binding domain that binds to CLEC12A comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from: (a) a VH comprising the amino acid sequence of SEQ ID NO: 21, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 22, or a sequence at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 23, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 24, or a sequence at least 90% identical thereto; and (c) a VH comprising the amino acid sequence of SEQ ID NO: 25, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 26, or a sequence at least 90% identical thereto. In some embodiments, the chimeric antigen receptor and / or antigen binding domain that binds CD33 comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from (a) a VH comprising the amino acid sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 18, or a sequence at least 90% identical thereto, and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19, or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20, or a sequence at least 90% identical thereto.

[0395] In some embodiments, the immunoresponsive cell comprises a bicistronic chimeric antigen receptor. In some embodiments, the bicistronic chimeric antigen receptor comprises a FLT3 CAR and a CD33 CAR. In some embodiments, the bicistronic chimeric antigen receptor comprises a FLT3 CAR and a CLEC12A CAR. In some embodiments, the bicistronic chimeric antigen receptor comprises a CLEC12A CAR and a CD33 CAR. In some embodiments, the bicistronic chimeric antigen receptor comprises any pair of antigens provided in Table 3.

[0396] Chimeric inhibitory receptors In some embodiments, a cell (e.g., an immunoresponsive cell) of the present disclosure comprises one or more chimeric inhibitory receptors of the present disclosure. In some embodiments, each of the one or more chimeric inhibitory receptors comprises an antigen-binding domain that binds to an antigen expressed on normal cells but not on tumor cells, such as AML cells. In some embodiments, the one or more chimeric inhibitory receptors bind to an antigen expressed on non-tumor cells derived from a tissue selected from brain, nervous tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

[0397] In some embodiments, a chimeric inhibitory receptor may be used with one or more chimeric receptors (e.g., chimeric TCRs or CARs) expressed on a cell (e.g., an immunoresponsive cell) of the present disclosure, e.g., as a non-logic gate to control, modulate, or otherwise inhibit one or more activities of the one or more chimeric receptors. In some embodiments, a chimeric receptor of the present disclosure may inhibit one or more activities of a cell (e.g., an immunoresponsive cell) of the present disclosure. In some embodiments, a chimeric inhibitory receptor is combined with one or more chimeric receptors of the present disclosure to combine OR logic gating with NOT logic gating and / or combine AND logic gating with NOT logic gating.

[0398] In some embodiments, the chimeric inhibitory receptor binds one or more antigens selected from EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR2, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, and TMEM200A.

[0399] In some embodiments, the chimeric receptor binds to the FLT3 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0400] In some embodiments, the chimeric receptor binds to the MS4A3 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0401] In some embodiments, the chimeric receptor binds to the CD33 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0402] In some embodiments, the chimeric receptor binds to the CLEC12A antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0403] In some embodiments, the chimeric receptor binds to the ADGRE2 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0404] In some embodiments, the chimeric receptor binds to the SLC22A16 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0405] In some embodiments, the chimeric receptor binds to a CD123 / IL3RA antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0406] In some embodiments, the chimeric receptor binds to the MLC1 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0407] In some embodiments, the chimeric receptor binds to an SPNS3 antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0408] In some embodiments, the chimeric receptor binds a GAPT antigen and the chimeric inhibitory receptor binds an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0409] In some embodiments, the chimeric receptor binds to the LAT2 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0410] In some embodiments, the chimeric receptor binds to a PIEZO1 antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0411] In some embodiments, the chimeric receptor binds to the CD38 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0412] In some embodiments, the chimeric receptor binds to an EMB antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0413] In some embodiments, the chimeric receptor binds to a CD131 / CSF2RB antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0414] In some embodiments, the chimeric receptor binds to the LILRA2 / CD85H antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0415] In some embodiments, the chimeric receptor binds to the SLC17A9 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0416] In some embodiments, the chimeric receptor binds to a MYADM antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0417] In some embodiments, the chimeric receptor binds to the CD300LF antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0418] In some embodiments, the chimeric receptor binds to the CD244 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0419] In some embodiments, the chimeric receptor binds to a CD93 antigen. In some embodiments, the chimeric receptor binds to a CD117 / CKIT antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0420] In some embodiments, the chimeric receptor binds to a CD117 / c-KIT antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0421] In some embodiments, the chimeric receptor binds to the CMTM7 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0422] In some embodiments, the chimeric receptor binds to a CYBA antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0423] In some embodiments, the chimeric receptor binds to an HCK antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0424] In some embodiments, the chimeric receptor binds to an ICAM3 antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0425] In some embodiments, the chimeric receptor binds to the LRRC37A3 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0426] In some embodiments, the chimeric receptor binds to an ITGAM antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0427] In some embodiments, the chimeric receptor binds to the ITGB2 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0428] In some embodiments, the chimeric receptor binds to the LILRA1 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0429] In some embodiments, the chimeric receptor binds to a PRTN3 antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0430] In some embodiments, the chimeric receptor binds to a CARD9 antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0431] In some embodiments, the chimeric receptor binds a SIGLEC5 antigen and the chimeric inhibitory receptor binds an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0432] In some embodiments, the chimeric receptor binds a SELL antigen and the chimeric inhibitory receptor binds an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0433] In some embodiments, the chimeric receptor binds to an MLKL antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0434] In some embodiments, the chimeric receptor binds to an INPP5D antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0435] In some embodiments, the chimeric receptor binds to an APBB1IP antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0436] In some embodiments, the chimeric receptor binds to the ITGA4 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0437] In some embodiments, the chimeric receptor binds to a C3AR1 antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0438] In some embodiments, the chimeric receptor binds to the ITGA5 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0439] In some embodiments, the chimeric receptor binds to the FMNL1 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0440] In some embodiments, the chimeric receptor binds to the VSTM1 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0441] In some embodiments, the chimeric receptor binds to a PRAM1 antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0442] In some embodiments, the chimeric receptor binds to an IL1RAP antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0443] In some embodiments, the chimeric receptor binds to a CCR1 / CD191 antigen and the chimeric inhibitory receptor binds to an EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0444] In some embodiments, the chimeric receptor binds to the LILRB2 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0445] In some embodiments, the chimeric receptor binds to the CD70 antigen and the chimeric inhibitory receptor binds to the EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen.

[0446] In some embodiments, the chimeric inhibitory receptor binds to the EMCN antigen and the chimeric receptor binds to the FLT3 antigen. In some embodiments, the chimeric inhibitory receptor binds to the EMCN antigen and the chimeric receptor binds to the MLC1 antigen.

[0447] In some embodiments, the chimeric inhibitory receptor binds to the JAM2 antigen and the chimeric receptor binds to the FLT3 antigen. In some embodiments, the chimeric inhibitory receptor binds to the JAM2 antigen and the chimeric receptor binds to the MLC1 antigen.

[0448] In some embodiments, the chimeric inhibitory receptor binds to the MS4A15 antigen and the chimeric receptor binds to the CLEC12A antigen.

[0449] In some embodiments, the chimeric inhibitory receptor binds to the SLC34A2 antigen and the chimeric receptor binds to the CLEC12A antigen.

[0450] In some embodiments, the chimeric inhibitory receptor binds to the C4BPA antigen and the chimeric receptor binds to the CD33 antigen.

[0451] In some embodiments, the chimeric inhibitory receptor binds to the TRPM1 antigen and the chimeric receptor binds to the CD33 antigen.

[0452] In some embodiments, the chimeric inhibitory receptor binds to the SCTR antigen and the chimeric receptor binds to the SLC22A16 antigen.

[0453] In some embodiments, the chimeric inhibitory receptor binds to the SLC2A2 antigen and the chimeric receptor binds to the IL1RAP antigen.

[0454] In some embodiments, the chimeric inhibitory receptor binds to the KCNQ2 antigen and the chimeric receptor binds to the PIEZO1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the KCNQ2 antigen and the chimeric receptor binds to the IL1RAP antigen.

[0455] In some embodiments, the chimeric inhibitory receptor binds to the PERP antigen and the chimeric receptor binds to the CD123 antigen. In some embodiments, the chimeric inhibitory receptor binds to the PERP antigen and the chimeric receptor binds to the IL3RA antigen.

[0456] In some embodiments, the chimeric inhibitory receptor binds to the WLS antigen. In some embodiments, the chimeric inhibitory receptor binds to the FFAR2 antigen. In some embodiments, the chimeric inhibitory receptor binds to the NCKAP1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the MPZL2 antigen. In some embodiments, the chimeric inhibitory receptor binds to the PLSCR4 antigen. In some embodiments, the chimeric inhibitory receptor binds to the TMEM47 antigen. In some embodiments, the chimeric inhibitory receptor binds to the ADGRL4 antigen. In some embodiments, the chimeric inhibitory receptor binds to the MET antigen. In some embodiments, the chimeric inhibitory receptor binds to the BACE2 antigen. In some embodiments, the chimeric inhibitory receptor binds to the ATP8B1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the LIFR antigen. In some embodiments, the chimeric inhibitory receptor binds to the ART4 antigen. In some embodiments, the chimeric inhibitory receptor binds to the CALCRL antigen. In some embodiments, the chimeric inhibitory receptor binds to the CNTNAP3 antigen. In some embodiments, the chimeric inhibitory receptor binds to the PCDH9 antigen. In some embodiments, the chimeric inhibitory receptor binds to the IL18R1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the SLC8A3 antigen. In some embodiments, the chimeric inhibitory receptor binds to the CDH26 antigen. In some embodiments, the chimeric inhibitory receptor binds to the TMEM163 antigen. In some embodiments, the chimeric inhibitory receptor binds to the ABCA13 antigen. In some embodiments, the chimeric inhibitory receptor binds to the CACHD1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the CYYR1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the ADGRG6 antigen. In some embodiments, the chimeric inhibitory receptor binds to the ATP9A antigen. In some embodiments, the chimeric inhibitory receptor binds to the CALN1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the CDCP1 antigen. In some embodiments, the chimeric inhibitory receptor binds to the IL12RB2 antigen.In some embodiments, the chimeric inhibitory receptor binds to the SLC16A14 antigen. In some embodiments, the chimeric inhibitory receptor binds to the TMEM136 antigen. In some embodiments, the chimeric inhibitory receptor binds to the TMEM200A antigen.

[0457] In some embodiments, the chimeric inhibitory receptor is selected from the group consisting of EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR2, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CA The chimeric receptor binds to the LN1, CDCP1, IL12RB2, SLC16A14, TMEM136, or TMEM200A antigen, and the chimeric receptor binds to the MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, or CD70 antigen.

[0458] In some embodiments, the chimeric inhibitory receptor binds to an EMCN antigen and the chimeric receptor binds to an MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, or CD70 antigen.

[0459] In some embodiments, the chimeric inhibitory receptor binds to the JAM2 antigen and the chimeric receptor binds to the MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, FLT3, CD33, CLEC12A, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, or CD70 antigen.

[0460] In some embodiments, the chimeric inhibitory receptor...

Claims

1. 1. An isolated immunoresponsive cell, comprising: (a) a first chimeric receptor comprising an extracellular antigen-binding domain that binds to a first antigen; (b) a second chimeric receptor comprising an extracellular antigen-binding domain that binds a second antigen; and Including, each antigen is selected from the group consisting of FLT3, CD33, CLEC12A, MS4A3, VSTM1, LAT2, MLC1, CD131, GAPT, PRAM1, SLC22A16, SLC17A9, SPNS3, ADGRE2, IL3RA, CD117, CD93, IL1RAP, CD244, CCR1, LILRB2, PIEZO1, CD38, EMB, MYADM, LILRA2, CD300LF, and CD70; the first antigen is different from the second antigen; The isolated immunoresponsive cell.

2. the first antigen is FLT3, and the extracellular antigen-binding domain of the first chimeric receptor is (a) a VH comprising the amino acid sequence of SEQ ID NO: 3 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 4 or a sequence at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 90% identical thereto; (c) a VH comprising the amino acid sequence of SEQ ID NO: 5 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 6 or a sequence at least 90% identical thereto; (d) a VH comprising the amino acid sequence of SEQ ID NO: 7 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 8 or a sequence at least 90% identical thereto; (e) a VH comprising the amino acid sequence of SEQ ID NO: 9 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 10 or a sequence at least 90% identical thereto; (f) a VH comprising the amino acid sequence of SEQ ID NO: 11 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 12 or a sequence at least 90% identical thereto; (g) a VH comprising the amino acid sequence of SEQ ID NO: 13 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 14 or a sequence at least 90% identical thereto; and (h) a VH comprising the amino acid sequence of SEQ ID NO: 15 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 16 or a sequence at least 90% identical thereto; 2. The isolated immunoresponsive cell of claim 1, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of:

3. i) the second antigen is CD33 and the extracellular antigen-binding domain of the second chimeric receptor is (a) a VH comprising the amino acid sequence of SEQ ID NO: 17 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 18 or a sequence at least 90% identical thereto; and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20 or a sequence at least 90% identical thereto; or comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: ii) the second antigen is CLEC12A; (a) a VH comprising the amino acid sequence of SEQ ID NO: 21 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 22 or a sequence at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 23 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 24 or a sequence at least 90% identical thereto; and (c) a VH comprising the amino acid sequence of SEQ ID NO: 25 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 26 or a sequence at least 90% identical thereto; a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: The isolated immunoresponsive cell of claim 2 .

4. the first antigen is CLEC12A, and the extracellular antigen-binding domain of the first chimeric receptor is (a) a VH comprising the amino acid sequence of SEQ ID NO: 21 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 22 or a sequence at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 23 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 24 or a sequence at least 90% identical thereto; and (c) a VH comprising the amino acid sequence of SEQ ID NO: 25 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 26 or a sequence at least 90% identical thereto; and a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: Optionally, the second antigen is CD33 and the extracellular antigen-binding domain of the second chimeric receptor is (a) a VH comprising the amino acid sequence of SEQ ID NO: 17 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 18 or a sequence at least 90% identical thereto; and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19 or a sequence at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20 or a sequence at least 90% identical thereto; a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: The isolated immunoresponsive cell of claim 1 .

5. i) binding of the first chimeric receptor to the first antigen can activate the immunoresponsive cell, and / or binding of the second chimeric receptor to the second antigen can stimulate the immunoresponsive cell, and / or ii) the immunoresponsive cells exhibit a greater degree of cytolytic activity against target cells that are positive for both the first antigen and the second antigen compared to cytolytic activity against target cells that are positive for only the first antigen or the second antigen; and / or iii) the first chimeric receptor binds the first antigen with a binding affinity that is lower than the binding affinity of the second chimeric receptor for the second antigen; and / or iv) the first chimeric receptor binds the first antigen with low avidity; The isolated immunoresponsive cell of any one of claims 1 to 4.

6. the first chimeric receptor is a first CAR; the second chimeric receptor is a second CAR; Each CAR is i) comprising a CD3 zeta chain intracellular signaling domain, and optionally each CAR further comprising one or more additional intracellular signaling domains selected from the group consisting of a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, and a 2B4 intracellular signaling domain; and / or ii) comprises a transmembrane domain, wherein the transmembrane domain is selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 zeta chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, and a BTLA transmembrane domain; and / or iii) comprising a spacer region between the antigen-binding domain and the transmembrane domain, wherein the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-64; The isolated immunoresponsive cell of any one of claims 1 to 5.

7. 7. The isolated immunoresponsive cell of any one of claims 1 to 6, wherein the cell further comprises an inhibitory chimeric receptor comprising an antigen binding domain, and optionally the inhibitory chimeric receptor inhibits one or more activities of the cell.

8. 8. The isolated immunoresponsive cell of claim 7, wherein the inhibitory chimeric receptor binds to an antigen expressed on a non-tumor cell, and optionally the antigen expressed on the non-tumor cell is derived from a tissue selected from the group consisting of brain, nervous tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, urinary bladder, male reproductive organs, female reproductive organs, adipose, soft tissue, and skin.

9. The inhibitory chimeric receptor is selected from the group consisting of EMCN, JAM2, MS4A15, C4BPA, TRPM1, SCTR, SLC2A2, KCNQ2, PERP, WLS, FFAR2, PTPRB, NCKAP1, MPZL2, PLSCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, and PCDH. 9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHD1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, and TMEM200A.

10. The isolated immunoresponsive cell of any one of claims 7 to 9, wherein the inhibitory chimeric receptor comprises an antigen-binding domain comprising a single-chain variable fragment (scFv), and the scFv is derived from an anti-EMCN antibody.

11. 11. The isolated immunoresponsive cell of any one of claims 7 to 10, wherein the antigen-binding domain of the first chimeric receptor, the antigen-binding domain of the second chimeric receptor, and / or the antigen-binding domain of the inhibitory chimeric receptor comprises one or more single-chain variable fragments (scFv), each of the one or more scFvs comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), optionally wherein the VH and VL are separated by a peptide linker, and optionally wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:

27.

12. 12. The isolated immunoresponsive cell of claim 11, wherein each of the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, where VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain.

13. 13. The isolated immunoresponsive cell of claim 11 or claim 12, wherein each of the one or more scFvs binds to a distinct epitope on the same antigen.

14. 14. The isolated immunoresponsive cell of any one of claims 11 to 13, wherein each of the one or more scFvs is separated by a peptide linker, and optionally the peptide linker comprises the amino acid sequence of GGGGSGGGGGSGGGGS (SEQ ID NO: 27) or EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 74).

15. the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell, a bone marrow cell, a macrophage, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, and an induced pluripotent stem cell (iPSC), and an iPSC-derived cell; Optionally, the immunoresponsive cells are allogeneic. The isolated immunoresponsive cell of any one of claims 1 to 14.

16. A pharmaceutical composition comprising an effective amount of the isolated immunoresponsive cell of any one of claims 1 to 15, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

17. 1. A method of providing anti-tumor immunity in a subject, comprising: Administering to a subject in need thereof a therapeutically effective amount of any of the isolated immunoresponsive cells of any one of claims 1 to 15 or the pharmaceutical composition of claim 16. The method.

18. 1. A method of treating or preventing bone marrow disorders in a subject, comprising: administering to the subject an effective amount of the isolated immunoresponsive cell of any one of claims 1 to 15 or the pharmaceutical composition of claim 16; Optionally, the bone marrow disorder is myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelomonocytic leukemia, acute myeloid leukemia (AML), acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, chronic myelogenous leukemia, and polycythemia vera. The method.

19. A kit for treating and / or preventing bone marrow disorders, comprising the isolated immunoresponsive cell of any one of claims 1 to 15 or the pharmaceutical composition of claim 16, Optionally, the kit further comprises written instructions for using the cells to treat and / or prevent a bone marrow disorder in a subject.

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