Chimeric Receptors and Methods of Use Thereof

Abstract

Provided herein are chimeric receptor acute myeloid leukemia antigen targets and methods of use thereof.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 841,128, filed Apr. 30, 2019; U.S. Provisional Application No. 62 / 854,151, filed May 29, 2019; and U.S. Provisional Application No. 62 / 893,106, filed Aug. 28, 2019, each of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing submitted via EFS - Web, which is hereby incorporated by reference in its entirety. The ASCII copy, created in XX month, 20XX, is named XXXXXUS_sequencelisting.txt and is X,XXX,XXX bytes in size.

Background Art

[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 remission 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 hematological malignancies such as acute myeloid leukemia (AML). Acute myeloid leukemia is the most common acute leukemia in adults. AML is a cancer of myeloid blood cells, characterized by the rapid proliferation of abnormal cells that accumulate in the bone marrow and blood, interfering with normal blood cells. Sometimes AML can spread to the brain, skin, or gums. Standard chemotherapy treatment for AML has not substantially changed over the past 40 years (Pulte et al., 2008), and the overall survival rate remains very low.

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

Prior Art Documents

Non - Patent Documents

[0005]

Non - Patent Document 1

Non - Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0006] Summary In one aspect, provided herein is an isolated immunoreactive 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 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 the first antigen is different from the second antigen, and the isolated immunoreactive cell is provided.

[0007] In some embodiments, the first antigen is FLT3, and the extracellular antigen-binding domain of the first 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: 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 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.

[0009] In some embodiments, the first antigen is CLEC12A, and the extracellular antigen-binding domain of the first 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: 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. Optionally, 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.

[0010] In some embodiments, binding of the first chimeric receptor to the first antigen can activate immune-responsive cells and / or binding of the second chimeric receptor to the second antigen can stimulate immune-responsive cells and / or the immune-responsive cells exhibit a higher 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 the first chimeric receptor binds to the first antigen with a binding affinity lower 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 low binding activity.

[0011] In some embodiments, the first chimeric receptor is a first CAR, the second chimeric receptor is a second CAR, each CAR comprises a CD3 zeta chain intracellular signaling domain, and optionally, each CAR further comprises one or more additional intracellular signaling domains, and the one or more additional intracellular signaling domains are selected from 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, a 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, 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, and the spacer region has an amino acid sequence selected from SEQ ID NOs: 55 - 64.

[0012] In some embodiments, 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.

[0013] In some embodiments, the inhibitory chimeric receptor binds to an antigen expressed on non-tumor cells, and optionally, the antigen expressed on non-tumor cells is derived from a tissue selected from the brain, nerve tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, digestive tract, kidney, bladder, male genitalia, female genitalia, fat, 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), and 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 comprises a heavy-chain variable domain (VH) and a light-chain variable domain (VL), optionally, VH and VL are separated by a peptide linker, and 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 a 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.

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

[0019] In some embodiments, each of the one or more scFvs is separated by a peptide linker, and optionally, the peptide linker comprises the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 27) or EAAAKEAAAKEAAAKEAAAK (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 as 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 antitumor immunity in a subject, the method comprising administering to a subject in need thereof any one of the isolated immunoresponsive cells described in any of the embodiments herein, or a pharmaceutical composition described in any of the embodiments herein, in a therapeutically effective amount.

[0023] In another aspect, provided herein is a method for treating or preventing bone marrow disorders in a subject, the method comprising administering to the subject an effective amount of the isolated immunoreactive cells 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 tumor, 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 bone marrow disorders, the kit comprising the isolated immunoreactive cells described in any of the embodiments herein, or a pharmaceutical composition described in any of the embodiments herein, optionally, wherein the kit further comprises written instructions for using the cells for treating and / or preventing bone marrow disorders 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, and the one or more intracellular signaling domains are selected from the group consisting of the intracellular signaling domain of the CD3 zeta chain, the intracellular signaling domain of CD97, the intracellular signaling domain of CD11a - CD18, the intracellular signaling domain of CD2, the intracellular signaling domain of ICOS, the intracellular signaling domain of CD27, the intracellular signaling domain of CD154, the intracellular signaling domain of CD8, the intracellular signaling domain of OX40, the intracellular signaling domain of 4 - 1BB, the intracellular signaling domain of CD28, the intracellular signaling domain of ZAP40, the intracellular signaling domain of CD30, the intracellular signaling domain of GITR, the intracellular signaling domain of HVEM, the intracellular signaling domain of DAP10, the intracellular signaling domain of DAP12, and the intracellular signaling domain of MyD88.

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

[0030] In some embodiments, the CAR comprises a spacer region between the antigen - binding domain and the transmembrane domain, and 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, 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 a chimeric receptor according to any one of the embodiments.

[0033] In some embodiments, the chimeric receptor is recombinantly expressed. In some embodiments, the chimeric receptor is expressed from a selected locus from a vector or the genome of a 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 myeloid 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 cell is autologous. In some embodiments, the cell is 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 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 the group consisting of the antigens listed in Table 1, or the first and second antigens are selected from the group consisting of the antigen pairs listed in Table 3, and the first antigen is different from the second, the isolated cell.

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

[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 the first antigen is different from the second, the isolated cell.

[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 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: 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 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is at least 90% identical thereto. In some embodiments that can be combined with any of the preceding embodiments, the second antigen is CD33.In some embodiments that can 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 can 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 can 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 can be combined with any of the preceding embodiments, the second antigen is CLECL12A. In some embodiments that can 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 can 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 that is 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 that is at least 90% identical thereto. In some embodiments that can 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 that is 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 that is at least 90% identical thereto. In some embodiments that can 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: 25 or a sequence that is 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 that is 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, 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: 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. In some embodiments, which can be combined with any of the preceding embodiments, the second antigen is CD33.In some embodiments that can 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 that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 18 or a sequence that is at least 90% identical thereto, and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20 or a sequence that is at least 90% identical thereto. In some embodiments that can 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 that is 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 that is at least 90% identical thereto. In some embodiments that can 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 that is 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 that is at least 90% identical thereto.

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

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

[0053] In some embodiments, the immunoresponsive cell exhibits a higher 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, the binding of the first chimeric receptor to the first antigen or the binding of the second chimeric receptor to the second antigen can activate immunoreactive cells.

[0055] In some embodiments, the first chimeric receptor binds to the first antigen with a 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 a low binding activity.

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

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

[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 includes a spacer region between the antigen-binding domain and the transmembrane domain, and 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 includes 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 includes a single-chain variable fragment (scFv). In some embodiments, the scFv includes a heavy-chain variable domain (VH) and a light-chain variable domain (VL). In some embodiments, VH and VL are separated by a peptide linker. In some embodiments, the peptide linker includes the amino acid sequence of SEQ ID NO: 27. In some embodiments, the scFv includes 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 a 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 a vector or the genome of a cell.

[0070] In some embodiments, the cell further includes an inhibitory chimeric receptor that includes 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 that is expressed on non-tumor cells.

[0074] In some embodiments, the inhibitory chimeric receptor binds to an antigen that is expressed on non-tumor cells derived from a tissue selected from the group consisting of brain, nervous tissue, endocrine, bone, bone marrow, immune system, muscle, lung, liver, gallbladder, pancreas, digestive tract, kidney, bladder, male genitalia, female genitalia, fat, 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, when the enzyme inhibitory domain is in proximity to an immunoreceptor, it inhibits the activation of 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, 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 myeloid 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 cell is autologous.

[0085] In some embodiments, the cell is allogeneic.

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

[0088] In some embodiments, one antigen-binding domain binds to MS4A3 and a 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 a 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 a 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 a 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 a 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 a 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 a 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 a 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 a 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 a 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 comprises (a) a VH comprising the amino acid sequence of SEQ ID NO: 1 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 2 or a sequence that is at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 3 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 4 or a sequence that is at least 90% identical thereto; (c) a VH comprising the amino acid sequence of SEQ ID NO: 5 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 6 or a sequence that is at least 90% identical thereto; (d) a VH comprising the amino acid sequence of SEQ ID NO: 7 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 8 or a sequence that is at least 90% identical thereto; (e) a VH comprising the amino acid sequence of SEQ ID NO: 9 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 10 or a sequence that is at least 90% identical thereto; (f) a VH comprising the amino acid sequence of SEQ ID NO: 11 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 12 or a sequence that is at least 90% identical thereto; (g) a VH comprising the amino acid sequence of SEQ ID NO: 13 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 14 or a sequence that is at least 90% identical thereto; and (h) a VH comprising the amino acid sequence of SEQ ID NO: 15 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 16 or a sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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: 9 or a sequence that is 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 that is 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: 11 or a sequence that is 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 that is 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: 13 or a sequence that is 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 that is 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: 15 or a sequence that is 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 that is at least 90% identical thereto. In some embodiments, which can be combined with any of the previous embodiments, the second antigen-binding domain binds to CD33.In some embodiments that can be combined with any of the previous embodiments, the second antigen-binding domain that binds to CD33 comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 17 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 18 or a sequence that is at least 90% identical thereto; and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20 or a sequence that is at least 90% identical thereto. In some embodiments that can be combined with any of the previous embodiments, the second antigen-binding domain that binds to CD33 comprises a variable heavy chain domain (VH) comprising the amino acid sequence of SEQ ID NO: 17 or a sequence that is at least 90% identical thereto, and a variable light chain domain (VL) comprising the amino acid sequence of SEQ ID NO: 18 or a sequence that is at least 90% identical thereto. In some embodiments that can be combined with any of the previous embodiments, the second antigen-binding domain that binds to CD33 comprises a variable heavy chain domain (VH) comprising the amino acid sequence of SEQ ID NO: 19 or a sequence that is at least 90% identical thereto, and a variable light chain domain (VL) comprising the amino acid sequence of SEQ ID NO: 20 or a sequence that is at least 90% identical thereto. In some embodiments that can be combined with any of the previous embodiments, the second antigen-binding domain binds to CLEC12A. In some embodiments that can be combined with any of the previous embodiments, the second antigen-binding domain that binds to CLEC12A comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 21 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 22 or a sequence that is at least 90% identical thereto; (b) a VH comprising the amino acid sequence of SEQ ID NO: 23 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 24 or a sequence that is at least 90% identical thereto; and (c) a VH comprising the amino acid sequence of SEQ ID NO: 25 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 26 or a sequence that is at least 90% identical thereto.In some embodiments that can be combined with any of the previous embodiments, the second 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 that is 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 that is at least 90% identical thereto. In some embodiments that can be combined with any of the previous embodiments, the second 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 that is 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 that is at least 90% identical thereto. In some embodiments that can be combined with any of the previous embodiments, the second antigen-binding domain that binds to CLEC12A comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 25 or a sequence that is 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 that is 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, the antigen-binding domain that binds to 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. In some embodiments, which can be combined with any of the preceding embodiments, the second antigen-binding domain binds to CD33.In some embodiments that can be combined with any of the preceding embodiments, the second antigen-binding domain that binds to CD33 comprises a variable heavy domain (VH) and a variable light domain (VL) selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 17 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 18 or a sequence that is at least 90% identical thereto; and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19 or a sequence that is at least 90% identical thereto, and a VL comprising the amino acid sequence of SEQ ID NO: 20 or a sequence that is at least 90% identical thereto. In some embodiments that can be combined with any of the preceding embodiments, the second antigen-binding domain that binds to CD33 comprises a variable heavy domain (VH) comprising the amino acid sequence of SEQ ID NO: 17 or a sequence that is at least 90% identical thereto, and a variable light domain (VL) comprising the amino acid sequence of SEQ ID NO: 18 or a sequence that is at least 90% identical thereto. In some embodiments that can be combined with any of the preceding embodiments, the second antigen-binding domain that binds to CD33 comprises a variable heavy domain (VH) comprising the amino acid sequence of SEQ ID NO: 19 or a sequence that is at least 90% identical thereto, and a variable light domain (VL) comprising the amino acid sequence of SEQ ID NO: 20 or a sequence that is 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, and the one or more intracellular signaling domains are selected from the group consisting of the intracellular signaling domain of the CD3 zeta chain, the intracellular signaling domain of CD97, the intracellular signaling domain of CD11a - CD18, the intracellular signaling domain of CD2, the intracellular signaling domain of ICOS, the intracellular signaling domain of CD27, the intracellular signaling domain of CD154, the intracellular signaling domain of CD8, the intracellular signaling domain of OX40, the intracellular signaling domain of 4 - 1BB, the intracellular signaling domain of CD28, the intracellular signaling domain of ZAP40, the intracellular signaling domain of CD30, the intracellular signaling domain of GITR, the intracellular signaling domain of HVEM, the intracellular signaling domain of DAP10, the intracellular signaling domain of DAP12, and the intracellular signaling domain of MyD88.

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

[0105] In some embodiments, the CAR comprises a spacer region between the antigen - binding domain and the transmembrane domain, and 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, 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 a 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 a chimeric receptor according to 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 to a different antigen.

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

[0112] In some embodiments, binding of either of the two antigens of the chimeric receptor can activate the immunoreactive cell.

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

[0114] In some embodiments, the binding of either of the two antigens of the chimeric receptor and the binding of the additional chimeric receptor to its cognate antigen are required to activate immune-responsive cells.

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

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

[0120] In some embodiments, the one or more intracellular signaling domains of the first CAR are different from the one or more intracellular signaling domains of the 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 the 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, a 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, and 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, and the spacer region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 55 - 64.

[0127] In some embodiments, the cell further comprises an inhibitory chimeric receptor comprising an antigen-binding domain.

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

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

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

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

[0132] In some embodiments, the inhibitory chimeric receptor binds to an antigen expressed on non-tumor cells derived from a tissue selected from the group consisting of brain, nerve tissue, endocrine, bone, bone marrow, immune system, muscle, lung, liver, gallbladder, pancreas, digestive tract, kidney, bladder, male genitalia, female genitalia, fat, 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, when the enzyme inhibitory domain is in proximity to the immune receptor, it inhibits the activation of the immune receptor.

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

[0137] In some embodiments, the enzymatic 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 domains of the additional chimeric receptor and / or inhibitory chimeric receptor include 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 includes a single-chain variable fragment (scFv). In some embodiments, the scFv includes a heavy-chain variable domain (VH) and a light-chain variable domain (VL). In some embodiments, VH and VL are separated by a peptide linker. In some embodiments, the peptide linker includes the amino acid sequence of SEQ ID NO: 27. In some embodiments, the scFv includes 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 a vector or the genome of the cell.

[0144] In some embodiments, the cell is selected from the group consisting of 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.

[0145] In some embodiments, the cell is autologous.

[0146] In some embodiments, the cell is 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 non-tumor cells.

[0160] In some embodiments, the antigen is expressed on non-tumor cells derived from a tissue selected from the group consisting of brain, nerve tissue, endocrine, bone, bone marrow, immune system, muscle, lung, liver, gallbladder, pancreas, digestive tract, kidney, bladder, male genitalia, female genitalia, fat, soft tissue, and skin.

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

[0162] In some embodiments, when the enzyme inhibitory domain is in proximity to the immune receptor, it inhibits activation of the immune receptor.

[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, 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 a chimeric inhibitory receptor according to 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 cell further comprises 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 to 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 to 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 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.

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

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

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

[0179] In some embodiments, the chimeric inhibitory receptor and / or the antigen-binding domain of the 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, 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 immunoreactive cell.

[0181] In some embodiments, binding of the chimeric inhibitory receptor to the first antigen can inhibit immunoreactive cells.

[0182] In some embodiments, binding of the chimeric receptor to the second antigen can activate immunoreactive cells.

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

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

[0185] In some embodiments, the chimeric receptor binds to the first antigen with a low binding activity.

[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 myeloid 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 cell is autologous.

[0190] In some embodiments, the cell is allogeneic.

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

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

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

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

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

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

[0197] In another aspect, provided herein is a genetically modified cell comprising the nucleic acid of the embodiment.

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

[0199] In another aspect, provided herein is a genetically modified cell comprising the nucleic acid of the embodiment.

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

[0201] In another aspect, provided herein is a method for 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 tumors in the subject.

[0205] In another aspect, provided herein is a method for treating or preventing myelodysplasia 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 myelodysplasia 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 myelodysplasia 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 isolated cell according to any one of the embodiments of the effective amount, 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 an isolated cell according to any one of the embodiments.

[0212] In some embodiments, the kit further comprises written instructions for using the cell for treating and / or preventing 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 for producing 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 for producing 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 of treating and / or preventing a myeloid disorder, 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 myeloid disorder is selected from the group consisting of myelodysplastic syndromes, myeloproliferative tumors, 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 myeloid disorder is acute myeloid leukemia (AML).

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

[0221] This patent or application documents include at least one drawing created in color. Copies of this patent or patent application publication, including color drawing(s), are provided by the Patent Office upon request and payment of the necessary fees.

[0222] These and other features, aspects, and advantages of the present disclosure will be better understood with reference to the following description and the accompanying drawings. [Invention 1001] An isolated immunoreactive 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 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 immunoreactive cell. [Invention 1002] 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 An isolated immunoreactive cell of the present invention 1001, 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 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, or 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, An isolated immunoreactive cell of the present invention 1002. [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 comprising 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 The isolated immunoreactive cell of the present invention 1001. [The present invention 1005] i) The binding of the first chimeric receptor to the first antigen can activate the immunoreactive cell and / or the binding of the second chimeric receptor to the second antigen can stimulate the immunoreactive cell, and / or ii) The immunoreactive cell exhibits a higher degree of cytolytic activity against target cells that are positive for both the first antigen and the second antigen as compared to the cytolytic activity against target cells that are positive only for the first antigen or the second antigen, and / or iii) The first chimeric receptor binds to the first antigen with a binding affinity lower than the binding affinity of the second chimeric receptor for the second antigen, and / or iv) The first chimeric receptor binds to the first antigen with a low binding activity, The isolated immunoreactive cell according to any one of the present inventions 1001 to 1004. [The present invention 1006] 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 comprises one or more additional intracellular signaling domains, said 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 GITR intracellular signaling domain, a 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) comprising a transmembrane domain, said transmembrane domain being 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 said antigen-binding domain and said transmembrane domain, said spacer region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 55 to 64 An isolated immunoreactive cell according to any one of aspects 1001 to 1005 of the present invention. [Aspect 1007 of the present invention] An isolated immunoreactive cell according to any one of aspects 1001 to 1006 of the present invention, wherein said cell further comprises an inhibitory chimeric receptor comprising an antigen-binding domain, and optionally, said inhibitory chimeric receptor inhibits one or more activities of said cell. [Aspect 1008 of the present invention] The inhibitory chimeric receptor binds to an antigen expressed on non-tumor cells, and optionally, the antigen expressed on the non-tumor cells is derived from a tissue selected from the group consisting of brain, nerve tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, digestive tract, kidney, bladder, male genitalia, female genitalia, fat, soft tissue, and skin. The isolated immunoreactive cell of the present invention 1007. [The present invention 1009] The inhibitory chimeric 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 isolated immunoreactive cell of the present invention 1007 or the present invention 1008. [The present invention 1010] The inhibitory chimeric receptor includes an antigen-binding domain containing a single-chain variable fragment (scFv), and the scFv is derived from an anti-EMCN antibody. The isolated immunoreactive cell of any one of the present inventions 1007 to 1009. [The present invention 1011] 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 includes one or more single-chain variable fragments (scFv). Each of the one or more scFvs includes 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 includes the amino acid sequence of SEQ ID NO: 27. The isolated immunoreactive cell of any one of the present inventions 1007 to 1010. [The present invention 1012] Each of the one or more scFvs includes a 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 isolated immunoreactive cell of the present invention 1011. [The present invention 1013] An isolated immunoreactive cell of the present invention 1011 or the present invention 1012, wherein each of the one or more scFvs binds to a distinct epitope on the same antigen. [The present invention 1014] An isolated immunoreactive cell according to any one of the present inventions 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 of GGGGSGGGGSGGGGS (SEQ ID NO: 27) or EAAAKEAAAKEAAAKEAAAK (SEQ ID NO: 74). [The present invention 1015] The cell is selected from the group consisting of 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 immunoreactive cell is allogeneic. An isolated immunoreactive cell according to any one of the present inventions 1001 to 1014. [The present invention 1016] A pharmaceutical composition comprising an effective amount of an isolated immunoreactive cell according to any one of the present inventions 1001 to 1015, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof. [The present invention 1017] A method for providing antitumor immunity in a subject, comprising the step of administering to a subject in need thereof a therapeutically effective amount of any one of the isolated immunoreactive cells according to any one of the present inventions 1001 to 1015 or the pharmaceutical composition of the present invention 1016. The method. [The present invention 1018] A method for treating or preventing bone marrow disorders in a subject, comprising the step of administering to the subject an effective amount of an isolated immunoreactive cell according to any one 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 myeloid leukemia, and polycythemia vera. The method. [The present invention 1019] A kit for treating and / or preventing bone marrow disorders, comprising an isolated immunoreactive cell according to 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 for treating and / or preventing bone marrow disorders in a subject.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0224] Detailed Description The practice of the present disclosure, unless otherwise indicated, employs conventional methods of molecular biology, chemistry, biochemistry, virology, and immunology within the scope of the relevant art. Such techniques are fully described in the literature. For example, see Hepatitis C Viruses: Genomes and Molecular Biology (S.L. Tan ed., Taylor & Francis, 2006), Fundamental Virology, 3 rd Edition, vols. I & II (B.N. Fields and D.M. Knipe, eds.), Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell eds., Blackwell Scientific Publications), A.L. Lehninger, 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] Definitions Unless otherwise defined, all terms, notations, and other scientific terms used in this specification are intended to have the meaning generally understood by those skilled in the art. In some cases, terms having a generally understood meaning are defined in this specification for clarity and / or immediate reference, and including such definitions in this specification should not necessarily be construed as representing a difference from what is generally understood in the art. The techniques and procedures described or referenced in this specification are generally well-known and are commonly used using conventional methodologies by those skilled in the art, 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. Procedures involving the use of commercially available kits and reagents are generally carried out in accordance with the protocols and conditions defined by the manufacturer, unless otherwise stated.

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

[0227] As used in this specification, the term "including" also specifically includes embodiments "consisting of" and "consisting essentially of" the recited elements, unless otherwise indicated.

[0228] The term "about" indicates the value shown and the range above and below that value, and encompasses it. In certain embodiments, the term "about" indicates ±10%, ±5%, or ±1% of the specified value. In certain specific embodiments, where applicable, the term "about" indicates ± one standard deviation of the specified value(s).

[0229] As used herein, the term "activating an immune-responsive cell" refers to inducing signal transduction or a change in protein expression in a cell that results in the initiation of an immune response. For example, when CD3 chains cluster in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs), a signal transduction cascade is generated. In certain embodiments, when an endogenous TCR or exogenous CAR binds to an antigen, formation of an immunological synapse occurs that includes 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 signal transduction molecules enables the ITAM motifs contained within the CD3 chains to be phosphorylated. This phosphorylation then initiates the T cell activation pathway and ultimately activates transcription factors such as NF-κB and AP-1. These transcription factors increase IL-2 production for proliferation, induce expression of major regulatory factor T cell proteins, and induce overall gene expression in T cells to initiate a T cell-mediated immune response.

[0230] As used herein, the term "stimulating an immune-responsive cell" refers to a signal that results in a robust and sustained immune response. In various embodiments, this is mediated simultaneously, following activation of immune cells (e.g., T cells), or via receptors including but not limited to CD28, CD137 (4-1BB), OX40, CD40, and ICOS. Without being bound by particular theory, receiving multiple stimulatory signals is important for initiating a robust and long-term T cell-mediated immune response. Without receiving these stimulatory signals, T cells are rapidly inhibited and become unresponsive to antigen. The effects of these co-stimulatory signals are diverse and are only partially understood, but they generally increase gene expression to generate long-lived, proliferative, and anti-apoptotic T cells that respond robustly to antigen 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 includes a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, the domains within the CAR polypeptide construct are within the same polypeptide chain, for example, comprising a chimeric fusion protein. In some embodiments, the domains within the CAR polypeptide construct are not adjacent to each other and are, for example, within different polypeptide chains as provided by the RCAR described herein. In one aspect, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain includes a primary signaling domain (e.g., the primary signaling domain of CD3-zeta). In one aspect, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In one aspect, the co-stimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, ICOS, and / or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain that includes 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 that includes a functional signaling domain derived from a co-stimulatory molecule 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 that includes two functional signaling domains derived from one or more co-stimulatory molecules 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 co-stimulatory 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, and 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 the functional portion of a protein that acts by transmitting information intracellularly through a defined signaling pathway to regulate cellular activity by generating a second messenger or by functioning as an effector in response to such a messenger. In some embodiments, the signaling domain of the chimeric receptor of the present disclosure is derived from a stimulatory or co-stimulatory molecule described herein, or is a synthetic or engineered signaling domain.

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

[0234] As used herein, the term "antibody fragment" refers to at least one portion of a full-length antibody or a recombinant variant thereof, and refers to an antigen-binding domain such as the antigen-determining variable region of a full-length antibody that is sufficient to confer recognition and specific binding of the antibody fragment to a target such as an antigen. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments, scFv antibody fragments, linear antibodies, single-domain antibodies such as sdAb (either VL or VH), camelid VHH domains, as well as two Fab fragments linked by disulfide bridges in the hinge region, and multispecific antibodies formed from antibody fragments such as bivalent fragments that include isolated CDRs or other epitope-binding fragments of an antibody, but are not limited thereto. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetra-bodies, v-NAR, 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. Patent 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 that includes at least one antibody fragment that includes the variable region of a light chain and at least one antibody fragment that includes the variable region of a heavy chain, wherein the light chain and heavy chain variable regions are linked via a short flexible polypeptide linker and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the full-length antibody from which it is derived. As used herein, unless specified otherwise, an scFv can have the VL and VH variable regions in either order with respect to the N-terminus and C-terminus of the polypeptide, and the scFv can include VL-linker-VH or can include VH-linker-VL.

[0236] As used herein, the term "complementary determining region" or "CDR" refers to the amino acid sequences within the antibody variable regions that confer antigen specificity and binding affinity. For example, generally, each heavy chain variable region has three CDRs (e.g., HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). The exact 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 combinations 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 VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In the combined Kabat and Chothia numbering scheme, in some embodiments, the CDR corresponds to the amino acid residues that are Kabat CDR, Chothia CDR, or a portion of both of them.For example, in some embodiments, the CDR corresponds to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of VH, such as mammalian VH, such as human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of VL, such as mammalian VL, such as human VL.

[0237] The portion of the chimeric receptor of the present disclosure that includes an antibody or an antibody fragment thereof may exist in various forms in which the antigen-binding domain is expressed as part of an adjacent polypeptide chain including, for example, an scFv antibody fragment, a linear antibody, a single-domain antibody such as sdAb (either VL or VH), a camelid VHH domain, a humanized antibody, a bispecific antibody, an antibody complex (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 aspect, the antigen-binding domain of the chimeric receptor of the present disclosure includes an antibody fragment. In a further aspect, the chimeric receptor includes an antibody fragment that includes an scFv.

[0238] As used herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains that exist in an antibody molecule in its native conformation and that typically 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 that exist in an antibody molecule in its native conformation. 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 an antibody expressed by a bacteriophage or yeast expression system. This term should also be construed to mean an antibody produced by the synthesis of a DNA molecule encoding the antibody, where the DNA molecule expresses the antibody protein, or an amino acid sequence that identifies the antibody, and the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence techniques that are 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 be accompanied by either antibody production, or activation of cells having specific immunological capabilities, or both. One of ordinary skill in the art will understand that virtually any macromolecule, including all proteins or peptides, 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 manifest itself by various means, including, but not limited to, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in lifespan, a decrease in tumor cell proliferation, a decrease in tumor cell survival rate, or an improvement in various physiological symptoms associated with the cancerous condition. The "anti-tumor effect" can also manifest itself by the ability of the peptides, polynucleotides, cells, and antibodies of the present disclosure to prevent the occurrence of tumors in the first place.

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

[0244] As used herein, the term "allogeneic" refers to any substance derived from a different animal of the same species as the individual into which the substance 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 substances derived from individuals of the same species can be sufficiently different to interact antigenically with each other.

[0245] As used herein, the term "affinity" refers to a measure of binding strength. Without being bound by theory, affinity depends on the proximity of stereochemical fit between the antibody binding site and the epitope, the size of the contact area between them, and the distribution of charged and hydrophobic groups. Affinity also includes the term "binding activity," which refers to the strength of antigen-antibody binding after the formation of a reversible complex. Methods for calculating the affinity of an antibody for an antigen are known in the art and include 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 signal transduction or changes in protein expression in cells such as activated immune response cells that result in a decrease in the immune response. Non-limiting examples of polypeptides known to suppress or reduce the immune response 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 can be present in the tumor microenvironment and can inhibit the immune response against tumor cells. In various embodiments, inhibiting, blocking, or antagonizing the interaction of immunosuppressive polypeptides and / or their ligands can enhance the immune response of immune-responsive cells.

[0247] As used herein, the term "enzymatic inhibitory domain" refers to a protein domain that inhibits an intracellular signaling cascade, such as a native T cell activation cascade. In some embodiments, the enzymatic 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 enzymatic 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, for example, 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, a portion of the enzyme comprises an enzyme domain(s), an enzyme fragment(s), or a variant(s) thereof. In some embodiments, a 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 signal transduction or a change in protein expression in cells, such as activated immunoreactive cells, that results in an increase in the immune response. Immunostimulatory activity may include pro-inflammatory activity. Non-limiting examples of polypeptides known to stimulate or increase the immune response 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 may be present in the tumor microenvironment and may activate the immune response against tumor cells. In various embodiments, promoting, stimulating, or a ligand stimulating a receptor of a pro-inflammatory polypeptide and / or its ligand may enhance the immune response of immunoreactive cells.

[0249] The 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% identical or the same as the endogenous nucleic acid sequence, but typically exhibit substantial identity. Nucleic acids having "substantial identity" or "substantial homology" to an endogenous sequence can typically hybridize to at least one strand of a double-stranded nucleic acid molecule. As used herein, "hybridization" refers to base pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., the genes 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. Low stringency hybridization can be achieved in the absence of an organic solvent, such as formamide, while 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, concentration of a detergent, such as sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA, are well known to those of skill 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 with 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 sequences are 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 nucleic acid level to the sequences used for comparison. Sequence identity is typically measured using sequence analysis software (e.g., the 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 a degree of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine. In an exemplary method of measuring the degree of identity, the BLAST program, which shows sequences with probability scores of e-3 to e-100 for closely related sequences, may be used.

[0251] As used herein, the term "encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA that functions as a template for the synthesis of other polymers and macromolecules in a biological process having a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, or the biological property derived therefrom. Thus, a gene, cDNA, or RNA encodes a protein when transcription and translation of the rRNA corresponding to that gene produces the protein in a cell or other biological system. Both a nucleotide sequence that is identical to the mRNA sequence, the coding strand that is typically provided in a sequence listing, and the non-coding strand that is used as a template for transcription of a gene or cDNA can be said to encode the protein or other product of that gene or cDNA. Unless otherwise indicated, the term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may include introns to the extent that the nucleotide sequence encoding the protein may include introns (s) in some versions.

[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, enabling cell-cell 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 target disease or disorder, e.g., a myeloid disorder.

[0254] As used herein, the term "immunoresponsive cell" refers to a cell that functions as an immune response (e.g., an immune effector response) or a precursor, or a 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 myeloid-derived phagocytes.

[0255] As used herein, the term "immune effector response" or "immune effector function" refers to a function or response of an immunoresponsive cell, for example, that enhances or promotes an immune attack on 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 of a target cell or the inhibition of its growth or proliferation. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0256] As used herein, the term "flexible polypeptide linker" or "linker" refers to a peptide linker composed of amino acids such as glycine and / or serine residues that are 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 and has the amino acid sequence (Gly-Gly-Gly-Ser) n wherein n is a positive integer greater than 1 (Array 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 comprises (Gly4Ser)4 (Array No. 225) or (Gly4Ser)3 (Array No. 226) but is not limited thereto. In other embodiments, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (Array No. 229) . Also within the scope of the present disclosure are, for example, the linkers described in WO2012 / 138475.

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

[0258] As used herein, the terms "treat", "treatment", and "treating" refer to a reduction or alleviation in the progression, severity and / or duration of a proliferative disorder, or alleviation of one or more symptoms (preferably, one or more distinguishable symptoms) of a proliferative disorder resulting from administration of one or more therapies (e.g., one or more therapeutic agents such as the CARs 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 may not necessarily be distinguishable by the patient. In other embodiments, the terms "treat", "treatment", and "treating" refer to inhibition of the progression of a proliferative disorder, either physically, e.g., by stabilization of distinguishable symptoms, physiologically, e.g., by stabilization of physical parameters, or both. In some embodiments, the terms "treat", "treatment", and "treating" refer to a decrease or stabilization in tumor size or the number of cancerous cells.

[0259] As used herein, the term "subject" is intended to include a living organism (e.g., a mammal, a human) in which an immune response can be induced.

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

[0261] Other interpretation rules The ranges recited in this specification are to be understood to include all subranges of values within 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, references to compounds having one or more stereocenters are intended to include each stereoisomer and all combinations of their stereoisomers.

[0263] Bone marrow antigen Certain aspects of the present disclosure relate to chimeric receptors and cells such as immunoreactive cells that have been genetically modified to express one or more of such chimeric receptors that bind to an antigen of interest, and methods of treating and / or preventing myeloid malignancies such as AML and other conditions where 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 bone marrow cells useful for the treatment of bone marrow malignancies, and immune-responsive cells genetically modified to express such chimeric receptors. Bone marrow malignancies are clonal diseases caused by dysfunction of hematopoietic stem cells or progenitor cells and result from genetic and epigenetic modifications that interfere with major processes such as cell proliferation and differentiation. Bone marrow malignancies can be chronic or acute. Chronic diseases include myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDS), 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 infections, and an increased tendency to bruise or bleed. Most cases of AML are de novo, although some cases can be secondary to chronic diseases. There are eight different subtypes of AML based on the cell type from which the leukemia originates and the maturity of the cells. AML subtypes include myeloblastic undifferentiated (M0), myeloblastic minimally differentiated (M1), myeloblastic fully differentiated (M2), promyelocytic (M3), myelomonocytic (M4), monocytic (M5), erythroleukemia (M6), and megakaryocytic (M7).

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

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

[0268] (Table 1) AML Antigens TIFF0007711945000001.tif229163TIFF0007711945000002.tif241163TIFF0007711945000003.tif246163TIFF0007711945000004.tif235163TIFF0007711945000005.tif164163

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

[0270] Chimeric receptor Certain aspects of the present disclosure relate to chimeric receptors that bind to an antigen 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 the antibody heavy or light chains. The CDRs are 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, the 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) TIFF0007711945000006.tif193163TIFF0007711945000007.tif180163

[0273] (Table B) TIFF0007711945000008.tif234163TIFF0007711945000009.tif246163TIFF0007711945000010.tif217163TIFF0007711945000011.tif248163TIFF0007711945000012.tif213163

[0274] (Table A2) TIFF0007711945000013.tif208170

[0275] Certain aspects of the present disclosure relate to chimeric receptors (e.g., CARs or chimeric TCRs) that include 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is at least 90% identical thereto.

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

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

[0279] In some embodiments, antibodies and derivatives thereof that can 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 phage display libraries), and functional binding fragments of antibodies. For example, antibody fragments or portions thereof that can bind to a myeloid (e.g., AML) antigen include, but are not limited to, Fv, Fab, Fab’, and F(ab’)2 fragments. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, 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 cleavage forms using an antibody gene in which one or more stop codons have been introduced upstream of the natural termination site. For example, a chimeric gene encoding the F(ab’)2 heavy chain portion can be designed to include a DNA sequence encoding the CH domain and hinge region of the heavy chain.

[0280] Methods of raising antibodies that target specific antigens are generally known in the art. Synthetic and engineered antibodies are described, for example, in US4816567, EP0125023Bl, US4816397, EP0120694Bl, WO86 / 01533, EP0194276Bl, US5225539, EP0239400Bl, EP0451216Bl, EP0519596Al, and US4946778.

[0281] In some embodiments, commercially available antibodies can be used to bind to a myeloid (e.g., AML) antigen. The CDRs of commercially available antibodies are readily accessible to 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 antibody 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 antibody 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 antibody described in U.S. Patent Publication No. 2017 / 0037149, published on 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 on June 28, 2018. The antigen-binding domain can be a scFv comprising a variable light chain domain (VL) and a variable heavy chain domain (VH). In some embodiments, the chimeric receptor can have a multispecific antigen-binding domain. For example, the chimeric receptor can be specific for FLT ’ 3 and one or more additional antigens, such as 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 rituximab described in U.S. Patent Publication No. 2018 / 0002397, published on 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 can be a scFv comprising a variable light chain domain (VL) and a variable heavy chain domain (VH). In some embodiments, the chimeric receptor can have a multispecific antigen-binding domain. For example, the chimeric receptor can 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 antibody described in U.S. Patent No. 7,741,443. The antigen-binding domain can be a single-chain variable fragment (scFv) that includes a light-chain variable domain (VL) and a heavy-chain variable domain (VH). In some embodiments, the chimeric receptor can have a multispecific antigen-binding domain. For example, the chimeric receptor can 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 that include two variable chains expressed as part of a complex with invariant CD3 chain molecules. TCRs are found on the surface of T cells and play a role in recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, the TCRs of the present disclosure include an alpha chain encoded by TRA and a beta chain encoded by TRB. In certain embodiments, the TCR includes 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 of the variable regions 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. When the TCR complex associates with its antigen and MHC (peptide / MHC), the T cell expressing the TCR complex is activated.

[0288] In some embodiments, the TCRs of the present disclosure are recombinant TCRs. In certain embodiments, the TCR is a TCR of non-natural origin. In certain embodiments, the TCR differs from a TCR of natural origin by at least one amino acid residue. In some embodiments, the TCR differs from a TCR of natural origin by at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least twenty, at least twenty-five, at least thirty, at least forty, at least fifty, at least sixty, at least seventy, at least eighty, at least ninety, at least one hundred, or more amino acid residues. In certain embodiments, the TCR is modified from a TCR of natural origin by at least one amino acid residue. In some embodiments, the TCR is modified from a TCR of natural origin by at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least twenty, at least twenty-five, at least thirty, at least forty, at least fifty, at least sixty, at least seventy, at least eighty, at least ninety, at least one hundred, or more amino acid residues.

[0289] Chimeric TCR In some embodiments, the TCRs of the present disclosure can include one or more antigen-binding domains that can be transplanted into one or more constant domains of a TCR chain, such as the TCR alpha chain or the TCR beta chain, to create a chimeric TCR that specifically binds to a target antigen (e.g., an AML antigen) of the present disclosure. Without wishing to be bound by theory, it is believed that the chimeric TCR can transmit signals through the TCR complex upon antigen binding. For example, an antibody or antibody fragment (e.g., scFv) can be transplanted into at least a portion of a constant domain of a TCR chain, such as the TCR alpha chain and / or the TCR beta chain, e.g., an extracellular constant domain, a transmembrane domain, and a cytoplasmic domain. As another example, the CDRs of an antibody or antibody fragment can be transplanted into the TCR alpha chain and / or beta chain to create a chimeric TCR that specifically binds to an antigen (e.g., an AML antigen) of the present disclosure. Such chimeric TCRs can be made by methods known in the art (see, e.g., 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, a CAR is an engineered receptor that confers or imparts the desired specificity to immune effector cells. In certain embodiments, a CAR can be used to confer the specificity of an antibody to an immune-responsive cell such as a T cell. 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 the immune-responsive cell. In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce stimulation of the immune-responsive cell. In some embodiments, activation of the immune-responsive cell results in killing of the target cell. In some embodiments, activation of the immune-responsive cell results in cytokine or chemokine expression and / or secretion by the immune-responsive cell. In some embodiments, stimulation of the immune-responsive cell results in cytokine or chemokine expression and / or secretion by the immune-responsive cell. In some embodiments, stimulation of the immune-responsive cell induces differentiation of the immune-responsive cell. In some embodiments, stimulation of the immune-responsive cell induces proliferation of the immune-responsive cell.

[0293] The CARs of the present disclosure can be first, second, or third generation CARs. A "first generation" CAR generally comprises 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 a major transducer of signals from the endogenous TCR. A "first generation" CAR provides novel antigen recognition that is independent of HLA-mediated antigen presentation and CD4 via the CD3ζ chain signaling domain within a single fusion molecule + and CD8 +It can cause the activation of both 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 antitumor activity of immune-responsive 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 the CARs of the present disclosure binds to one or more antigens expressed on myeloid cells such as AML cells with a dissociation constant (Kd) of about 2×10 -7 M or less, about 1×10 -7 M or less, about 9×10 -8 M or less, about 1×10 -8 M or less, about 9×10 -9 M or less, about 5×10 -9 M or less, about 4×10 -9 M or less, about 3×10 -9 M or less, about 2×10 -9 M or less, or about 1×10 -9 M or less. In some embodiments, the Kd ranges from about 2×10 -7 M to about 1×10 -9 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 by using a specific labeling reagent (e.g., an antibody or scFv) for the complex of interest. For example, the scFv can be radiolabeled and used in an RIA assay. The radioisotope 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 proteins (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent proteins (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent proteins (e.g., YFP, Citrine, Venus, and YPet).

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

[0298] The 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, as well as single-domain antibodies (sdAbs), such as heavy-chain variable domains (VH), light-chain variable domains (VL), and variable domains of camelid-derived nanobodies (VHH), but are not limited thereto, and recombinant fibronectin domains, T-cell receptors (TCRs), affinity-improved recombinant TCRs, or fragments thereof, such as single-chain TCRs, and any domain that functions as an antigen-binding domain and binds to alternative scaffolds known in the art. 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 use in humans, the antigen-binding domain of the CAR may beneficially 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 includes 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 includes an antigen-binding fragment of an antibody.

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

[0301] In some embodiments, the extracellular antigen-binding domain comprises a 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 distinct epitopes 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, 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 comprises the sequence of SEQ ID NO: 54, 220, 221, or 223 is encoded by a nucleic acid comprising the sequence. 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 one or more scFvs 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. When two or more scFvs are linked together, each scFv can be linked to the next scFv to which a peptide is linked. In some embodiments, each of one or more scFvs is separated by a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 27) or EAAAKEAAAKEAAAKEAAAK (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 suitable antigen-binding domains 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 (sdAb). 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 scFv 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, the CARs of the present disclosure can 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 logical gating, such as OR logical gating, to the CAR.

[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 independently be selected from an antibody, an antigen-binding fragment of an antibody, scFv, sdAb, a recombinant fibronectin domain, a T cell receptor (TCR), an affinity-improved 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, the bispecific CAR or tanCAR comprises an antigen-binding domain comprising a bispecific antibody or an antibody fragment (e.g., scFv). In some embodiments, within each antibody or antibody fragment (e.g., scFv) of the bispecific antibody molecule, the VH can be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH1) upstream of its VL (VL1), and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL2) upstream of its VH (VH2), such that the overall bispecific antibody molecule has the arrangement 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), and the overall bispecific antibody molecule has the arrangement VL1-VH1-VH2-VL2. In some embodiments, the linker is arranged between the two antibodies or antibody fragments (e.g., scFv), 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, for example, a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6 (Array No. 227). Generally, the linker between two scFvs must be long enough to avoid mispairing between the domains of the two scFvs. In some embodiments, the linker is disposed between the VL and VH of the first scFv. In some embodiments, the linker is disposed between the VL and VH of the second scFv. In constructs having multiple linkers, any two or more linkers may be the same or different. Thus, in some embodiments, the bispecific CAR or tanCAR may include VL, VH, and further include one or more linkers in the arrangements described herein.

[0308] In some embodiments, the chimeric receptor includes a bivalent CAR. In some embodiments, the bivalent CAR is a FLT3 bivalent CAR. In some embodiments, the bivalent FLT3 CAR includes the NC7 scFv and the 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 includes a bivalent chimeric antigen receptor. In some embodiments, the bivalent chimeric receptor includes a FLT3 CAR and a CD33 CAR. In some embodiments, the bivalent chimeric receptor includes a FLT3 CAR and a CLEC12A CAR. In some embodiments, the bivalent chimeric receptor includes a CLEC12A CAR and a CD33 CAR. In some embodiments, the bivalent chimeric receptor includes an EMCN CAR. In some embodiments, the bivalent chimeric receptor includes a CAR having an antigen-binding domain that targets any of the antigens provided in Table 1. In some embodiments, the bivalent chimeric receptor includes a CAR having an antigen-binding domain that targets any of the antigens provided in Table 2. In some embodiments, the bivalent chimeric receptor includes 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 includes a CAR having any combination of two or more of the 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 an FLT3 CAR and a CD33 CAR. In some embodiments, the bicistronic chimeric antigen receptor comprises an 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 of the antigen-binding domains described herein.

[0311] Transmembrane domain In some embodiments, the transmembrane domain of the CARs 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 signals are transmitted to the cell. In some embodiments, the transmembrane domain of the CARs 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 can 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 can 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 can 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 can 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 can 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 can 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 can 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 can 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 can 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 can 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 can 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 can 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 comprises a polypeptide or a fragment thereof having 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% identical to the sequence of NCBI reference number 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. In some embodiments, the homology can be determined using standard software such as BLAST or FASTA. In some embodiments, the polypeptide can comprise 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 continuous portion of NCBI reference number 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, and 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.

[0314] Further examples of suitable polypeptides from which the transmembrane domain may be derived include the transmembrane domain(s) of the alpha, beta, or zeta chain of the 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, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), 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, but are not limited thereto.

[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 CARs of the present disclosure can also include a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region can be flexible enough to allow the antigen-binding domain to be oriented 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 localized between the antigen-binding domain and the transmembrane domain. In some embodiments, the spacer region can include 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, the nucleic acid encoding any of the spacer regions of the present disclosure can include 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) TIFF0007711945000014.tif123163

[0318] (Table D) TIFF0007711945000015.tif190161

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

[0320] In some embodiments, the CAR of the present disclosure is 2 to 10 amino acid residues in length and may further comprise a short oligopeptide or polypeptide linker capable of forming 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 double strand. In some embodiments, the linker comprises the amino sequence of GGCKJSGGCKJS (Array No. 228) and contains.

[0321] Intracellular signaling domain In some embodiments, the CARs of the present disclosure include one or more cytoplasmic domains or regions. The cytoplasmic domain or region of the CAR may include an intracellular signaling domain. The intracellular signaling domain typically participates in the activation of one or more effector functions of immune cells (e.g., T cells or NK cells) engineered to express the CARs of the present disclosure. For example, the effector functions of T cells can be cytolytic activity or helper activity such as cytokine secretion. Thus, in some embodiments, the term “intracellular signaling domain” refers to a part of a protein that transmits an effector function signal and instructs the cell to perform a specific function. Usually, the entire intracellular signaling domain can be used, but often it is not necessary to use the entire chain. In embodiments where a truncated portion of the intracellular signaling domain is used, such truncated portion can be used in place of the corresponding intact chain as long as the truncated portion transmits the effector function signal.

[0322] Examples of suitable intracellular signaling domains that can 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 signaling after antigen receptor engagement, as well as any derivatives or variants of these sequences, and any recombinant sequences having the same functional capabilities.

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

[0324] In some embodiments, the primary signaling domain controls the primary activation of the TCR complex either in a stimulatory or inhibitory manner. A primary intracellular signaling domain that acts in a stimulatory manner may include a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). 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, the CARs of the present disclosure include an intracellular signaling domain, for example, the primary signaling domain of a CD3-zeta polypeptide. The CD3-zeta polypeptides of the present disclosure can 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% identical to the sequence of NCBI reference number NP_932170 or NP_001106864.2. In some embodiments, the CD3-zeta polypeptide can include 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 continuous portion of NCBI reference number 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 includes a modified ITAM domain (e.g., increased or decreased in activity) compared to the native ITAM domain, such as a mutant ITAM domain. In one embodiment, the primary signaling domain includes a modified ITAM-containing primary intracellular signaling domain, such as an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In one embodiment, the primary signaling domain includes one, two, three, four, or more ITAM motifs.

[0327] In some embodiments, the intracellular signaling domain of the CAR of the present disclosure may itself include the CD3-zeta signaling domain, or it may be combined with any other desired intracellular signaling domain(s) useful in the context of the CAR of the present disclosure. For example, the intracellular signaling domain of the CAR can include a portion of the CD3 zeta chain and a co-stimulatory signaling domain. The co-stimulatory signaling domain may refer to a portion of the CAR that includes the intracellular domain of a co-stimulatory molecule. The co-stimulatory molecules of the present disclosure are cell surface molecules other than antigen receptors or their ligands 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, ligands that specifically bind to CD83, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated 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, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), 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., but are not limited thereto.

[0328] In some embodiments, the intracellular signaling sequences within a portion of the cytoplasm of the CARs of the present disclosure can be linked to each other in a random or specific order. In some embodiments, for example, short oligopeptides or polypeptide linkers having a length of 2 to 10 amino acids (e.g., 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids) can form a bond with the intracellular signaling sequence. In one embodiment, a glycine-serine double strand can be used as a suitable linker. In one embodiment, a single amino acid, such as alanine or glycine, can be used as a suitable linker.

[0329] In some embodiments, the intracellular signaling domain comprises two or more co-stimulatory signaling domains, such as 2 co-stimulatory signaling domains, 3 co-stimulatory signaling domains, 4 co-stimulatory signaling domains, 5 co-stimulatory signaling domains, 6 co-stimulatory signaling domains, 7 co-stimulatory signaling domains, 8 co-stimulatory signaling domains, 9 co-stimulatory signaling domains, 10 co-stimulatory signaling domains, or more co-stimulatory signaling domains. In one embodiment, the intracellular signaling domain comprises 2 co-stimulatory signaling domains. In some embodiments, two or more co-stimulatory 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, the cells of the present disclosure express a CAR comprising an antigen-binding domain that binds to the target antigen of the present disclosure, a transmembrane domain, a primary signaling domain, and one or more co-stimulatory signaling domains.

[0331] In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 153 or a sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 19 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 195 or a sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 156 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 158 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 160 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 162 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 164 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 166 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 168 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 170 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 172 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 174 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 176 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 178 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 180 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 182 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 184 or a sequence that is at least 90% identical thereto.In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 186 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 188 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 190 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 192 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 194 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 196 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 198 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 200 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 202 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 204 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 213 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 215 or a sequence that is at least 90% identical thereto. In some embodiments, the CAR comprises the nucleotide sequence set forth in SEQ ID NO: 217 or a sequence that is at least 90% identical thereto.

[0333] Natural killer cell receptor (NKR) CAR In some embodiments, the CARs of the present disclosure include one or more components of natural killer cell receptors (NKRs), thereby forming NKR-CARs. The NKR components can be transmembrane domains, hinge domains, or cytoplasmic domains from any suitable natural killer cell receptor, including but not limited to killer cell immunoglobulin-like receptors (KIRs) such as KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIRS DPI; natural cytotoxicity receptors (NCRs) such as NKp30, NKp44, and NKp46; signaling lymphocyte activation molecule (SLAM) family of immune cell receptors such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10; Fc receptors (FcRs) such as CD16 and CD64; and Ly49 receptors such as Ly49A and Ly49C. In some embodiments, the NKR-CAR can interact with an intracellular signaling domain such as an adaptor molecule or DAP12. Exemplary constructs and sequences of CARs containing NKR components are described in International Patent Publication WO2014 / 145252, published September 18, 2014.

[0334] Chimeric inhibitory receptor Certain aspects of the present disclosure relate to chimeric inhibitory receptors. Chimeric inhibitory receptors are not useful, for example, as logic gates for controlling cellular activities 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, the chimeric inhibitory receptor includes an antigen-binding domain, a transmembrane domain of the present disclosure (e.g., any suitable transmembrane domain used in conjunction with the chimeric receptors of the present disclosure), and an intracellular domain. In some embodiments, the chimeric inhibitory receptor can inhibit one or more activities of cells such as immune-responsive cells.

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

[0337] In some embodiments, the enzyme inhibitory domain of the chimeric inhibitory receptor of the present disclosure includes at least a portion of an extracellular domain, a transmembrane domain, and / or an intracellular domain. In some embodiments, the enzyme inhibitory domain includes 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, for example, 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, a portion of the enzyme includes an enzyme domain(s), an enzyme fragment(s), or a variant(s) thereof. In some embodiments, a 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 regulate basal inhibition. Examples of modifications include, but are not limited to, cleavage mutations, amino acid substitutions, introduction of positions for post-translational modifications (these examples are known to those skilled 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, one or more modifications reduce basal inhibition. In other embodiments, one or more modifications increase basal inhibition.

[0339] In some embodiments, the enzyme inhibitory domain inhibits immune receptor activation, for example, upon mobilization of the chimeric inhibitory receptor of the present disclosure that is proximal to the immune receptor. In some embodiments, the immune receptor is a naturally occurring immune receptor. In some embodiments, the immune receptor is a naturally occurring antigen receptor. In some embodiments, the immune receptor is selected from a T cell receptor, a pattern recognition receptor (PRR), a NOD-like receptor (NLR), a Toll-like receptor (TLR), a killer activation 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 immune receptor is a T cell receptor. In some embodiments, the immune receptor is a chimeric immune receptor. In some embodiments, the chimeric immune receptor is a chimeric TCR or CAR.

[0340] In some embodiments, the chimeric inhibitory receptor of the present disclosure may also include one or more intracellular inhibitory co-signaling domains. In some embodiments, the intracellular inhibitory co-signaling domain includes an inhibitory domain. In some embodiments, the one or more intracellular inhibitory co-signaling domains include one or more ITIM-containing proteins, or fragments thereof. ITIM is a conserved amino acid sequence found in the cytoplasmic tails of many inhibitory immune receptors. 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 include one or more non-ITIM scaffold proteins, or fragments thereof. In some embodiments, the one or more non-ITIM scaffold 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, ΤIΜ3, 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 inhibitory chimeric receptor binds to an antigen expressed on non-tumor cells. Exemplary antigens for use with chimeric inhibitory receptors are listed in Table 2.

[0342] (Table 2) TIFF0007711945000016.tif239161TIFF0007711945000017.tif228161TIFF0007711945000018.tif240161TIFF0007711945000019.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 comprising two or more antigen-binding domains, such that the chimeric inhibitory receptor can bind to two or more antigens. Alternatively, the cell can be engineered to express two or more chimeric inhibitory receptors that bind to 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] Immune-responsive cells Certain aspects of the disclosure relate to cells genetically engineered to contain one or more chimeric receptors of the disclosure or one or more nucleic acids encoding such chimeric receptors, e.g., immune-responsive cells, and methods of using such cells to treat myeloid malignancies (e.g., AML).

[0347] In some embodiments, the cells are mammalian cells. In some embodiments, the mammalian cells are primary cells. In some embodiments, the mammalian cells are cell lines. In some embodiments, the mammalian cells are bone marrow cells, blood cells, skin cells, bone cells, muscle cells, nerve cells, fat cells, hepatocytes, or heart cells. In some embodiments, the cells are stem cells. 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 cells are cells derived from or differentiated from the stem cells of the present disclosure. In some embodiments, the cells are immune cells. 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 cells are nerve cells. The nerve 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 neuron 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 cells are immune-responsive cells. The immune-responsive 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-responsive cells of the present disclosure include, but are not limited to, cells of the lymphocyte lineage. Lymphoid lineages, including B cells, T cells, and natural killer (NK) cells, provide antibody production, regulation of the cellular immune system, detection of foreign substances in the blood, detection of cells foreign to the host, and the like. Examples of immune-responsive 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 that can be derived from or differentiated into lymphocytes). T cells can be lymphocytes that mature in the thymus and are mainly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. In some embodiments, the T cells of the present disclosure are 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 cells, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosa-associated invariant T cells, and any type of T cell including, but not limited to, 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 cells or tumor cells. A 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 can be lymphocytes that are part of the 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 immune-responsive cells of the present disclosure are T cells. The T cells of the present disclosure can be autologous, allogeneic, or induced in vitro from engineered precursors or stem cells.

[0351] In some embodiments, the immune-responsive cells of the present disclosure are universal T cells having a 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 immune-responsive cells of the present disclosure are isolated immune-responsive cells comprising one or more chimeric receptors of the present disclosure. In some embodiments, the immune-responsive 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 immune-responsive cells are T cells. In some embodiments, the immune-responsive cells are natural killer (NK) cells.

[0354] Cells expressing multiple chimeric receptors In some embodiments, the cells of the present disclosure (e.g., immune-responsive cells) comprise two or more chimeric receptors of the present disclosure. In some embodiments, the cells comprise two or more chimeric receptors, and one of the two or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cells comprise 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 cells comprise 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 cells comprise 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 an antigen-binding domain that binds to a different antigen, e.g., an antigen-binding domain that binds to the same antigen or different antigens. In some embodiments, each antigen bound by two or more chimeric receptors is expressed in the same myeloid cell type (e.g., the same AML cell type). In one embodiment, the cell comprises a first chimeric receptor that targets a first antigen and comprises an intracellular signaling domain that has a co-stimulatory signaling domain but is not a primary signaling domain, and a second chimeric receptor that targets a second different antigen and comprises an intracellular signaling domain that has a primary signaling domain but is not a co-stimulatory signaling domain. Without wishing to be bound by theory, placing a co-stimulatory signaling domain (e.g., 4-1BB, CD28, or OX-40) on the first chimeric receptor and a primary signaling domain (e.g., the CD3-zeta chain) on the second chimeric receptor may limit chimeric receptor activity against cells in which both targets are expressed. Thus, in some embodiments, the cells of the present disclosure (e.g., immunoreactive cells) comprise: (a) a first chimeric receptor comprising an antigen-binding domain that binds to a first antigen, a transmembrane domain, and a co-stimulatory signaling domain; and (b) a second chimeric receptor comprising an antigen-binding domain that binds to a second antigen, a transmembrane domain, and a primary signaling domain. In some embodiments, the cells of the present disclosure (e.g., immunoreactive cells) comprise: (a) a first chimeric receptor comprising an antigen-binding domain that binds to a first antigen, a transmembrane domain, and a primary signaling domain; and (b) a second chimeric receptor comprising an antigen-binding domain that binds to a second antigen, a transmembrane domain, and a co-stimulatory signaling domain. In some embodiments, the cells of the present disclosure (e.g., immunoreactive cells) comprise: (a) a first chimeric receptor comprising an antigen-binding domain that binds to a first antigen, a transmembrane domain, a primary signaling domain, and a co-stimulatory domain; and (b) a second chimeric receptor comprising an antigen-binding domain that binds to a second antigen, a transmembrane domain, a primary signaling domain, and a co-stimulatory domain.In embodiments where both the first chimeric receptor and the second chimeric receptor each include a co-stimulatory signaling domain, the co-stimulatory signaling domain of the first chimeric receptor and the co-stimulatory signaling domain of the second chimeric receptor can be derived from the same protein, such as 4-1BB, CD28, or OX40. Alternatively, the co-stimulatory signaling domain of the first chimeric receptor can be derived from a protein different from the co-stimulatory signaling domain of the second chimeric receptor.

[0356] In embodiments where the cells of the present disclosure (e.g., immunoreactive cells) express two or more distinct chimeric receptors, the respective antigen-binding domains of the different chimeric receptors can be designed such that the antigen-binding domains do not interact with each other. For example, the cells of the present disclosure (e.g., immunoreactive cells) that express a first chimeric receptor and a second chimeric receptor can include a first chimeric receptor that includes 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 include an antibody fragment such as an scFv, while the antigen-binding domain of the second chimeric receptor can include a VHH.

[0357] Without wishing to be bound by theory, in cells having multiple chimeric transmembrane receptors each including an antigen-binding domain, an interaction between the antigen-binding domains of each receptor may undesirably have the potential to inhibit the ability of one or more of those antigen-binding domains to bind their cognate antigen. Thus, in embodiments where the cells of the present disclosure (e.g., immunoreactive cells) express two or more chimeric receptors, the chimeric receptors include antigen-binding domains that minimize such inhibitory interactions. In one embodiment, the antigen-binding domain of one chimeric receptor includes an scFv and the antigen-binding domain of the second chimeric receptor includes a single VH domain, such as 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, the binding of the antigen-binding domain of the first chimeric receptor to its cognate antigen is not substantially reduced by the presence of the second chimeric receptor. In some embodiments, the binding of the antigen-binding domain of the 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 where the cells of the present disclosure (e.g., immunoreactive cells) comprise two or more distinct chimeric receptors of the present disclosure that bind to different antigens, the two or more chimeric receptors provide logical gating to the cells, such as OR logical gating, AND logical gating, NOT logical gating, or any combination of such logical gating. Thus, in certain embodiments, the cells of the present disclosure (e.g., immunoreactive cells) comprise two or more chimeric receptors, and binding of the first chimeric receptor to a first antigen can activate the cell. In some embodiments, the cells of the present disclosure (e.g., immunoreactive cells) comprise two or more chimeric receptors, and binding of the second chimeric receptor to a second antigen can stimulate the cell. In some embodiments, the cells of the present disclosure (e.g., immunoreactive cells) comprise two or more chimeric receptors, and binding of the first chimeric receptor to a first antigen and binding of the second chimeric receptor to a second antigen are required to activate the cell. In some embodiments, the cells of the present disclosure (e.g., immunoreactive cells) comprise two or more chimeric receptors, and binding of the first chimeric receptor to a first antigen and binding of the second chimeric receptor to a second antigen are required to stimulate the cell. In some embodiments, the cells of the present disclosure (e.g., immunoreactive cells) comprise two or more chimeric receptors, and the cells exhibit a higher degree of cytolytic activity against cells that are positive for both the first antigen and the second antigen compared to cytolytic activity against cells that are positive for only the first antigen or only the second antigen. In some embodiments, the cells of the present disclosure (e.g., immunoreactive cells) comprise two or more chimeric receptors, and binding of the first chimeric receptor to a first antigen or binding of the second chimeric receptor to a second antigen can activate the immunoreactive cell.

[0360] In some embodiments, the cells of the present disclosure (e.g., immunoreactive cells) include 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 includes a first chimeric receptor having a first antigen-binding domain and a co-stimulatory 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), and cells that express the same. In such embodiments, when the cell encounters a first antigen, the co-stimulatory domain is activated and the cell proliferates. In addition, when the cell encounters a second antigen, the intracellular signaling domain is activated and cell killing activity is induced. Thus, in some embodiments, the cells of the present disclosure (e.g., immunoreactive cells) are fully activated only in the presence of both antigens.

[0361] In certain embodiments, the cells of the present disclosure (e.g., immunoreactive cells) exhibit a higher degree of cytolytic activity against cells that are positive for both a first antigen and a second antigen compared to cells that are positive for the first antigen alone. In certain embodiments, the first chimeric receptor binds to the first antigen with a low binding affinity or a low binding activity. In certain embodiments, the first chimeric receptor binds to the first antigen with a low accessibility 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 compared to the binding affinity with which the second chimeric receptor binds to the second antigen.

[0362] In some embodiments, the paired selection should favor the redundant expression of two target antigens in the tumor in order to minimize the risk of antigen avoidance. Thus, in some embodiments, the cells of the present disclosure (e.g., immunoreactive cells) 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 antigen 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 / IL3RA 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 / IL3RA 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, and as a result, the chimeric receptor can bind to two or more antigens.

[0365] In some embodiments, the immune-responsive cell can comprise one or more tumor-targeting chimeric receptors and one or more inhibitory chimeric receptors that target an antigen not expressed on the tumor. The combination of the tumor-targeting chimeric receptor and the inhibitory chimeric receptor in the same immune-responsive cell can be used to reduce off-target toxicity. For example, if healthy cells express both an antigen recognized by a tumor-targeting chimeric receptor and an antigen recognized by an inhibitory chimeric receptor, an immune-responsive cell expressing a tumor antigen can bind to the healthy cells. In such a case, the inhibitory chimeric antigen can also bind to its cognate ligand on the healthy cells, and the inhibitory function of the inhibitory chimeric receptor reduces, diminishes, prevents, or inhibits the activation of the immune-responsive cell 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 cell expresses two or more chimeric receptors that bind to different antigens. Exemplary pairs of antigens are shown in Table 3.

[0368] (Table 3) TIFF0007711945000020.tif144128TIFF0007711945000021.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 cell expresses two or more chimeric receptors of the present disclosure, one chimeric receptor binds to FLT3, and the second chimeric receptor binds to 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 to FLT3 and the second antigen-binding domain binds to CD33. In some embodiments, the chimeric antigen receptor and / or 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; (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 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 cell expresses two or more chimeric receptors of the present disclosure, wherein one chimeric receptor binds to FLT3 and the second chimeric receptor binds to 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 to FLT3 and the second antigen-binding domain binds to 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 to FLT3 and the second antigen-binding domain binds to CD33.In some embodiments, the chimeric antigen receptor and / or antigen-binding domain that binds to FLT3 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: 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 cell expresses two or more chimeric receptors of the present disclosure, one chimeric receptor binds to CLEC12A, and the second chimeric receptor binds to 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 to CLEC12A and the second antigen-binding domain binds to 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 to 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 immune-responsive cells comprise a bicistronic chimeric antigen receptor. In some embodiments, the bicistronic chimeric antigen receptor comprises an FLT3 CAR and a CD33 CAR. In some embodiments, the bicistronic chimeric antigen receptor comprises an 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 receptor In some embodiments, the cells of the disclosure (e.g., immune-responsive cells) comprise one or more chimeric inhibitory receptors of the disclosure. In some embodiments, each of the one or more chimeric inhibitory receptors comprises an antigen-binding domain that binds to an antigen that is 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 that is expressed on non-tumor cells 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, bladder, male genitalia, female genitalia, fat, soft tissue, and skin.

[0397] In some embodiments, the chimeric inhibitory receptor can be used with one or more chimeric receptors (e.g., chimeric TCR or CAR) expressed on the cells of the disclosure (e.g., immune-responsive cells), such as not being a logic gate for controlling, regulating, or otherwise inhibiting the activity of one or more of the one or more chimeric receptors. In some embodiments, the chimeric receptors of the disclosure can inhibit the activity of one or more of the cells of the disclosure (e.g., immune-responsive cells). In some embodiments, the chimeric inhibitory receptor is combined with one or more of the chimeric receptors of the disclosure, combining OR logic gating with NOT logic gating, and / or combining AND logic gating with NOT logic gating.

[0398] In some embodiments, the chimeric inhibitory receptor binds to 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 the CD123 / IL3RA 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.

[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 the SPNS3 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.

[0408] In some embodiments, the chimeric receptor binds to the GAPT 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.

[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 the PIEZO1 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.

[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 the EMB 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.

[0413] In some embodiments, the chimeric receptor binds to the CD131 / CSF2RB 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.

[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 the MYADM 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.

[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 the CD93 antigen. In some embodiments, the chimeric receptor binds to the CD117 / CKIT 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.

[0420] In some embodiments, the chimeric receptor binds to the CD117 / c-KIT 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.

[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 the CYBA 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.

[0423] In some embodiments, the chimeric receptor binds to the HCK 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.

[0424] In some embodiments, the chimeric receptor binds to the ICAM3 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.

[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 the ITGAM 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.

[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 the PRTN3 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.

[0430] In some embodiments, the chimeric receptor binds to the CARD9 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.

[0431] In some embodiments, the chimeric receptor binds to the SIGLEC5 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.

[0432] In some embodiments, the chimeric receptor binds to the SELL 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.

[0433] In some embodiments, the chimeric receptor binds to the MLKL 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.

[0434] In some embodiments, the chimeric receptor binds to the INPP5D 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.

[0435] In some embodiments, the chimeric receptor binds to the APBB1IP 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.

[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 the C3AR1 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.

[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 the PRAM1 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.

[0442] In some embodiments, the chimeric receptor binds to the IL1RAP 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.

[0443] In some embodiments, the chimeric receptor binds to the CCR1 / CD191 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.

[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 chime...

Claims

**Claim 1** An isolated immunoreactive cell, wherein the isolated immunoreactive cell comprises (a) a first chimeric receptor comprising an extracellular antigen-binding domain that binds to a first antigen and a second chimeric receptor comprising an extracellular antigen-binding domain that binds to a second antigen; or (b) a chimeric receptor comprising two or more antigen-binding domains, wherein each of the two or more antigen-binding domains binds to an antigen, and each antigen-binding domain binds to a distinct antigen ; the first antigen is FLT3, and the second antigen is CD33, and the isolated immunoreactive cell further comprises an inhibitory chimeric receptor comprising an antigen-binding domain, the inhibitory chimeric 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, PLCR4, TMEM47, ADGRL4, MET, BACE2, ATP8B1, LIFR, ART4, CALCRL, CNTNAP3, PCDH9, IL18R1, SLC8A3, CDH26, TMEM163, ABCA13, CACHED1, CYYR1, ABCB1, ADGRG6, ATP9A, CALN1, CDCP1, IL12RB2, SLC16A14, TMEM136, and TMEM200A, the isolated immunoreactive cell. **Claim 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 VH comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3 and comprising the three amino acid sequences of SEQ ID NOs: 81, 82, and 83, and a VL comprising the amino acid sequence of SEQ ID NO: 4, or a VL comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4 and comprising the three amino acid sequences of SEQ ID NOs: 84, 85, and 86; (b) a VH comprising the amino acid sequence of SEQ ID NO: 1, or a VH comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 and comprising the three amino acid sequences of SEQ ID NOs: 75, 76, and 77, and a VL comprising the amino acid sequence of SEQ ID NO: 2, or a VL comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 and comprising the three amino acid sequences of SEQ ID NOs: 78, 79, and 80; (c) VH comprising the amino acid sequence of SEQ ID NO: 5, or VH comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5 and comprising the three amino acid sequences of SEQ ID NOs: 87, 88, and 89, and VL comprising the amino acid sequence of SEQ ID NO: 6, or VL comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 and comprising the three amino acid sequences of SEQ ID NOs: 90, 91, and 92; (d) VH comprising the amino acid sequence of SEQ ID NO: 7, or VH comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 and comprising the three amino acid sequences of SEQ ID NOs: 93, 94, and 95, and VL comprising the amino acid sequence of SEQ ID NO: 8, or VL comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and comprising the three amino acid sequences of SEQ ID NOs: 96, 97, and 98; (e) VH comprising the amino acid sequence of SEQ ID NO: 9, or VH comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9 and comprising the three amino acid sequences of SEQ ID NOs: 99, 100, and 101, and VL comprising the amino acid sequence of SEQ ID NO: 10, or VL comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10 and comprising the three amino acid sequences of SEQ ID NOs: 102, 103, and 104; (f) VH comprising the amino acid sequence of SEQ ID NO: 11, or VH comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11 and comprising the three amino acid sequences of SEQ ID NOs: 105, 106, and 107, and VL comprising the amino acid sequence of SEQ ID NO: 12, or VL comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12 and comprising the three amino acid sequences of SEQ ID NOs: 108, 109, and 110; (g) VH comprising the amino acid sequence of SEQ ID NO: 13, or VH comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13 and comprising the three amino acid sequences of SEQ ID NOs: 111, 112, and 113, and VL comprising the amino acid sequence of SEQ ID NO: 14, or VL comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14 and comprising the three amino acid sequences of SEQ ID NOs: 114, 115, and 116; and (h) VH comprising the amino acid sequence of SEQ ID NO: 15, or VH comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15 and comprising the three amino acid sequences of SEQ ID NOs: 117, 118, and 119, and A VL comprising the amino acid sequence of SEQ ID NO: 16, or a VL comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16 and comprising the three amino acid sequences of SEQ ID NOs: 120, 121, and 122 The isolated immunoreactive cell according to claim 1, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of **Claim 3** wherein 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 VH comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17 and comprising the three amino acid sequences of SEQ ID NOs: 123, 124, and 125, and a VL comprising the amino acid sequence of SEQ ID NO: 18, or a VL comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 18 and comprising the three amino acid sequences of SEQ ID NOs: 126, 127, and 128; and (b) a VH comprising the amino acid sequence of SEQ ID NO: 19, or a VH comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19 and comprising the three amino acid sequences of SEQ ID NOs: 129, 130, and 131, and a VL comprising the amino acid sequence of SEQ ID NO: 20, or a VL comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20 and comprising the three amino acid sequences of SEQ ID NOs: 132, 133, and 134 The isolated immunoreactive cell according to claim 2, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of **Claim 4** where each chimeric receptor is a CAR, and each CAR is i) comprising a CD3 zeta chain intracellular signaling domain, and / or ii) comprising a transmembrane domain, the transmembrane domain being selected from the group consisting of the CD8 transmembrane domain, the CD28 transmembrane domain, the CD3 zeta chain transmembrane domain, the CD4 transmembrane domain, the 4-1BB transmembrane domain, the OX40 transmembrane domain, the ICOS transmembrane domain, the CTLA-4 transmembrane domain, the PD-1 transmembrane domain, the LAG-3 transmembrane domain, the 2B4 transmembrane domain, and the BTLA transmembrane domain, and / or iii) comprising a spacer region between the antigen-binding domain and the transmembrane domain, the spacer region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 55-64, The isolated immunoreactive cell according to any one of claims 1 to 3. **Claim 5** ​ In (i), each CAR further comprises one or more additional intracellular signaling domains, and the one or more additional intracellular signaling domains are 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. The isolated immunoreactive cell according to claim 4.

6. The isolated immunoreactive cell according to any one of claims 1 to 5, wherein the inhibitory chimeric receptor inhibits one or more activities of the cell.

7. The isolated immunoreactive cell according to claim 6, wherein the inhibitory chimeric receptor binds to an antigen expressed on non-tumor cells.

8. The isolated immunoreactive cell according to claim 7, wherein the antigen expressed on the non-tumor cells is derived from a tissue selected from the group consisting of brain, nerve tissue, endocrine, bone, bone marrow, immune system, endothelial tissue, muscle, lung, liver, gallbladder, pancreas, digestive tract, kidney, bladder, male genitalia, female genitalia, fat, soft tissue, and skin.

9. The isolated immunoreactive cell according to any one of claims 6 to 8, 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.

10. The isolated immunoreactive cell according to any one of claims 6 to 9, 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), and each of the one or more scFv comprises a heavy-chain variable domain (VH) and a light-chain variable domain (VL).

11. The isolated immunoreactive cell according to claim 10, wherein the VH and the VL are separated by a peptide linker.

12. The isolated immunoreactive cell according to claim 11, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:

27.

13. The isolated immunoreactive cell according to any one of claims 10 to 12, wherein each of the one or more scFvs is separated by a peptide linker.

14. The isolated immunoreactive cell according to claim 13, wherein the peptide linker comprises the amino acid sequence of GGGGSGGGGGSGGGGGS (SEQ ID NO: 27) or EAAAK EAAAK EAAAK EAAAK (SEQ ID NO: 74).

15. The cell is selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, natural killer T (NKT) cells, myeloid cells, macrophages, human embryonic stem cells (ESC), ESC-derived cells, pluripotent stem cells, and induced pluripotent stem cells (iPSC), and iPSC-derived cells. The isolated immunoreactive cell according to any one of claims 1 to 14.

16. The isolated immunoreactive cell according to claim 15, wherein the immunoreactive cell is allogeneic.

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

18. A medicament for providing anti-tumor immunity in a subject, comprising a therapeutically effective amount of the isolated immunoreactive cell according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 17.

19. A medicament for treating or preventing myelodysplasia in a subject, comprising an effective amount of the isolated immunoreactive cell according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 17.

20. The medicament according to claim 19, wherein the myelodysplasia is myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelomonocytic leukemia, acute myeloid leukemia (AML), acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, chronic myeloid leukemia, and polycythemia vera.

21. The medicament according to claim 19 or 20, wherein the myelodysplasia is AML.

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