Chimeric receptors targeting ADGRE2 and / or CLEC12A and uses thereof

Abstract

The subject matter of the present disclosure provides a chimeric receptor that targets ADGRE2 and a chimeric receptor that targets CLEC12A. The subject matter of the present disclosure also provides a cell comprising an ADGRE2-targeted chimeric receptor, a cell comprising a CLEC12A-targeted chimeric receptor, and a cell comprising an ADGRE2-targeted chimeric receptor and a CLEC12A-targeted chimeric receptor. The subject matter of the present disclosure further provides a use of such a cell for treating a tumor, e.g., AML.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 179,799, filed April 26, 2021, and U.S. Provisional Patent Application No. 63 / 287,655, filed December 9, 2021, the entire contents of each of which are incorporated by reference and claim priority to each of which.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy above was created on April 21, 2022, is named 087108_0110_SL.txt, and is 150,762 bytes in size.

[0003] 1. Introduction The subject matter of the present disclosure provides methods and compositions for immunotherapy.The subject matter of the present disclosure relates to chimeric receptors that target ADGRE2 and chimeric receptors that target CLEC12A, cells containing such chimeric receptors, and methods of using such cells in therapy, for example, in the treatment of acute myeloid leukemia (AML). [Background technology]

[0004] 2. Background technology Cell-based immunotherapy is a potentially curative therapy for cancer treatment. Immune cells, such as T cells, can be modified to target tumor antigens by the introduction of genetic material encoding artificial or synthetic antigen receptors, called chimeric antigen receptors (CARs), specific for selected antigens. Targeted T cell therapy using CARs has recently achieved clinical success in the treatment of hematological malignancies.

[0005] The prognosis of relapsed and refractory acute myeloid leukemia (R / R AML) is very poor. The only treatment option is allogeneic hematopoietic stem cell transplantation, which is often associated with treatment failure and significant treatment-related toxicity and mortality. Thus, new therapeutic approaches are desperately needed for R / R AML. Over the past few years, autologous T cells genetically modified to express chimeric antigen receptors (CARs) targeting CD19 have revolutionized the treatment of patients with R / RB cell hematological malignancies, improving outcomes and leading to the FDA approval of three CD19 CARs (tisagenlecleucel, axicabtagene ciloleucel, and brexcabtagene autoleucel) for R / R acute lymphoblastic leukemia and / or certain B cell non-Hodgkin lymphomas. In the case of AML, clinical studies of CAR T cell therapy are still in their early stages, and clinical results using mainly CD33 and CD123 CAR T cells suggest challenges in terms of both safety and efficacy, which are due to the abundant expression of CD33 and CD123 in normal hematopoiesis and the phenotypic heterogeneity in AML tumor cells. Therefore, there is a need for novel combination CAR formats for R / R AML that may offer improved safety and efficacy compared to alternative CAR therapies currently under clinical investigation. Summary of the Invention

[0006] 3. Overview of the Invention The subject matter of the present disclosure provides chimeric receptors that target ADGRE2, and chimeric receptors that target CLEC12A, cells containing such chimeric receptors, and methods of using such cells in therapy, e.g., for the treatment of acute myeloid leukemia (AML).

[0007] In one aspect, the subject matter of the present disclosure provides a chimeric receptor that targets ADGRE2. In an embodiment, the ADGRE2-targeted chimeric receptor comprises an extracellular antigen-binding domain that binds to ADGRE2, a transmembrane domain, and an intracellular domain. In an embodiment, the extracellular antigen-binding domain comprises a) a heavy chain variable region comprising a CDR1 comprising an amino acid sequence represented by SEQ ID NO: 33 or a conservative modification thereof, a CDR2 comprising an amino acid sequence represented by SEQ ID NO: 34 or a conservative modification thereof, and a CDR3 comprising an amino acid sequence represented by SEQ ID NO: 35 or a conservative modification thereof, and / or b) a light chain variable region comprising a CDR1 comprising an amino acid sequence represented by SEQ ID NO: 36 or a conservative modification thereof, a CDR2 comprising an amino acid sequence represented by SEQ ID NO: 37 or a conservative modification thereof, and a CDR3 comprising an amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof.

[0008] In certain embodiments, the extracellular antigen-binding domain is a single chain variable fragment (scFv), Fab, or F(ab) 2 In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the scFv is a humanized scFv.

[0009] In one embodiment, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35. In one embodiment, the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38. In one embodiment, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38.

[0010] In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical or homologous to an amino acid sequence represented by SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, or SEQ ID NO: 146. In certain embodiments, the heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:46, or SEQ ID NO:49. In certain embodiments, the heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO:39.

[0011] In certain embodiments, the light chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical or homologous to an amino acid sequence represented by SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, or SEQ ID NO: 147. In certain embodiments, the light chain variable region comprises an amino acid sequence represented by SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:47, or SEQ ID NO:50. In certain embodiments, the light chain variable region comprises an amino acid sequence represented by SEQ ID NO:40.

[0012] In one embodiment, the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical or homologous to an amino acid sequence set forth in SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, or SEQ ID NO:55. In one embodiment, the heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:46, or SEQ ID NO:49, and the light chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to an amino acid sequence represented by SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, or SEQ ID NO:56. In one embodiment, the heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:46, or SEQ ID NO:49, and the light chain variable region comprises an amino acid sequence represented by SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:47, or SEQ ID NO:50.

[0013] In one embodiment, a) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 39, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 40; b) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 43 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 44; c) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 46, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 47; d) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 49 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 50; e) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 52 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 53; f) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO:55 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:56; or g) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO:146, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:147.

[0014] In one embodiment, a) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 39, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 40; b) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 43 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 44; c) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 46 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 47; or d) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO:49, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:50.

[0015] In one embodiment, the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO:39, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:40.

[0016] In some embodiments, the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region, hi some embodiments, the linker consists of the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:149.

[0017] In one embodiment, the heavy chain variable region and the light chain variable region are, from N-terminus to C-terminus, V H -V LIn one embodiment, the extracellular antigen binding domain comprises or is an scFv, which comprises or consists of the amino acid sequence represented by SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:148. In one embodiment, the extracellular antigen binding domain comprises or is an scFv, which comprises or consists of the amino acid sequence represented by SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:48, or SEQ ID NO:51. In one embodiment, the extracellular antigen binding domain comprises or is an scFv, which comprises or consists of the amino acid sequence represented by SEQ ID NO:41.

[0018] In one embodiment, the extracellular antigen binding domain comprises about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 Dissociation constant (K D ) to ADGRE2. In some embodiments, the extracellular antigen-binding domain binds to ADGRE2 with an EC50 of between about 1 and about 100 nM. In some embodiments, the EC50 is about 10 to about 95 nM. In some embodiments, the EC50 is about 25 to about 75 nM.

[0019] In some embodiments, the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ 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, or a BTLA polypeptide. In some embodiments, the transmembrane domain comprises a CD28 polypeptide.

[0020] In one embodiment, the intracellular domain comprises a CD3ζ polypeptide. In one embodiment, the CD3ζ polypeptide is a modified CD3ζ polypeptide. In one embodiment, the modified CD3ζ polypeptide comprises a native ITAM1, an ITAM2 mutant comprising two loss-of-function mutations, and an ITAM3 mutant comprising two loss-of-function mutations. In one embodiment, the native ITAM1 comprises the amino acid sequence represented by SEQ ID NO: 15. In one embodiment, the ITAM2 mutant comprises the amino acid sequence represented by SEQ ID NO: 21. In one embodiment, the ITAM3 mutant comprises the amino acid sequence represented by SEQ ID NO: 25. In one embodiment, the modified CD3ζ polypeptide comprises or consists of the amino acid sequence represented by SEQ ID NO: 27.

[0021] In some embodiments, the intracellular domain further comprises at least one costimulatory signaling region.In some embodiments, at least one costimulatory signaling region comprises CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, DAP-10 polypeptide, or combinations thereof.In some embodiments, at least one costimulatory signaling region comprises CD28 polypeptide.

[0022] In some embodiments, the ADGRE2-targeted chimeric receptor is a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), or a TCR-like fusion molecule. In some embodiments, the ADGRE2-targeted chimeric receptor is a CAR.

[0023] In one aspect, the presently disclosed subject matter provides a chimeric receptor that targets CLEC12A. In one embodiment, the CLEC12A-targeted chimeric receptor comprises an extracellular antigen-binding domain that binds to CLEC12A, a transmembrane domain, and an intracellular domain. In one embodiment, the extracellular antigen-binding domain is i) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 69 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 70 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 71 or a conservative modification thereof; ii) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83 or a conservative modification thereof; iii) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 89 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 90 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 91 or a conservative modification thereof; iv) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 89 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 90 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 98 or a conservative modification thereof; v) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 103 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83 or a conservative modification thereof; vi) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 109 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 103 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83 or a conservative modification thereof; vii) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 89 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 90 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 114 or a conservative modification thereof; viii) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 120 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 121 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 122 or a conservative modification thereof; or ix) a heavy chain variable region comprising CDR1 having the amino acid sequence represented by SEQ ID NO: 129 or a conservative modification thereof, CDR2 having the amino acid sequence represented by SEQ ID NO: 130 or a conservative modification thereof, and CDR3 having the amino acid sequence represented by SEQ ID NO: 131 or a conservative modification thereof, and / or i) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 72 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 74 or a conservative modification thereof; ii) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 85 or a conservative modification thereof; iii) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 92 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 93 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 94 or a conservative modification thereof; iv) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 99 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 93 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 151 or a conservative modification thereof; v) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 104 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 105 or a conservative modification thereof; vi) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 110 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 85 or a conservative modification thereof; vii) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 115 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 93 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 116 or a conservative modification thereof; viii) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 123 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 124 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 125 or a conservative modification thereof; or ix) a light chain variable region comprising: CDR1 having the amino acid sequence represented by SEQ ID NO: 132 or a conservative modification thereof; CDR2 having the amino acid sequence represented by SEQ ID NO: 133 or a conservative modification thereof; and CDR3 having the amino acid sequence represented by SEQ ID NO: 134 or a conservative modification thereof.

[0024] In certain embodiments, the extracellular antigen-binding domain is a single chain variable fragment (scFv), Fab, or F(ab) 2 In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the scFv is a human scFv.

[0025] In one embodiment, the heavy chain variable region comprises: a) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 69, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 70, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 71; b) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83; c) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 89, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 90, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 91; d) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 89, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 90, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 98; e) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 103, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83; f) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 109, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 103, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83; g) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 89, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 90, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 114; h) a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 120, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 121, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 122; or i) comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 129, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 130, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 131.

[0026] In one embodiment, the heavy chain variable region comprises: a) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 69, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 70, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 71; b) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83; c) a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 89, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 91; or d) comprising a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 89, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 98.

[0027] In one embodiment, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO:69, a CDR2 comprising the amino acid sequence represented by SEQ ID NO:70, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO:71.

[0028] In one embodiment, the light chain variable region comprises: a) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 72, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 74; b) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 85; c) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 92, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 93, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 94; d) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 99, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 93, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 151; e) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 104, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 105; f) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 110, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 85; g) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 115, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 93, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 116; h) a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 123, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 124, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 125; or i) comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 132, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 133, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 134.

[0029] In one embodiment, the light chain variable region comprises: a) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 72, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 74; b) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 85; c) a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 92, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 93, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 94; or d) comprising a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 99, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 93, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 151.

[0030] In one embodiment, the light chain variable region comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO:72, a CDR2 comprising the amino acid sequence represented by SEQ ID NO:73, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO:74.

[0031] In one embodiment, the chimeric receptor comprises: a) the heavy chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 69, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 70, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 71, and the light chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 72, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 74; b) the heavy chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83, and the light chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 85; c) the heavy chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 89, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 90, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 91, and the light chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 92, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 93, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 94; d) the heavy chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 89, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 90, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 98, and the light chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 99, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 93, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 151; e) the heavy chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 103, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83; and the light chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 104, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 105; f) the heavy chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 109, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 103, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83, and the light chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 110, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 85; g) the heavy chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 89, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 90, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 114, and the light chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 115, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 93, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 116; h) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 120, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 121, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 122, and the light chain variable region comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 123, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 124, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 125, or i) the heavy chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 129, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 130, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 131, and the light chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 132, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 133, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 134.

[0032] In one embodiment, a) the heavy chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 69, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 70, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 71, and the light chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 72, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 74; b) the heavy chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83, and the light chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 85; c) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 89, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 91, and the light chain variable region comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 92, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 93, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 94, or d) The heavy chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 89, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 90, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 98, and the light chain variable region comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 99, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 93, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 151.

[0033] In one embodiment, the heavy chain variable region comprises a CDR1 having an amino acid sequence represented by SEQ ID NO:69, a CDR2 having an amino acid sequence represented by SEQ ID NO:70, and a CDR3 having an amino acid sequence represented by SEQ ID NO:71, and the light chain variable region comprises a CDR1 having an amino acid sequence represented by SEQ ID NO:72, a CDR2 having an amino acid sequence represented by SEQ ID NO:73, and a CDR3 having an amino acid sequence represented by SEQ ID NO:74.

[0034] In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical or homologous to an amino acid sequence represented by SEQ ID NO:75, SEQ ID NO:86, SEQ ID NO:95, SEQ ID NO:100, SEQ ID NO:106, SEQ ID NO:111, SEQ ID NO:117, SEQ ID NO:126, or SEQ ID NO:135. In certain embodiments, the heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO:75, SEQ ID NO:86, SEQ ID NO:95, SEQ ID NO:100, SEQ ID NO:106, SEQ ID NO:111, SEQ ID NO:117, SEQ ID NO:126, or SEQ ID NO:135. In certain embodiments, the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 75, SEQ ID NO: 86, SEQ ID NO: 95, or SEQ ID NO: 100. In certain embodiments, the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 75.

[0035] In certain embodiments, the light chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical or homologous to an amino acid sequence represented by SEQ ID NO:76, SEQ ID NO:87, SEQ ID NO:96, SEQ ID NO:101, SEQ ID NO:107, SEQ ID NO:112, SEQ ID NO:118, SEQ ID NO:127, or SEQ ID NO:136. In certain embodiments, the light chain variable region comprises an amino acid sequence represented by SEQ ID NO:76, SEQ ID NO:87, SEQ ID NO:96, SEQ ID NO:101, SEQ ID NO:107, SEQ ID NO:112, SEQ ID NO:118, SEQ ID NO:127, or SEQ ID NO:136. In certain embodiments, the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:76, SEQ ID NO:87, SEQ ID NO:96, or SEQ ID NO: 101. In certain embodiments, the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:76.

[0036] In one embodiment, a) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 75 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 76; b) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 86 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 87; c) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 95 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 96; d) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 100 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 101; e) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 106 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 107; f) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 111 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 112; g) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 117 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 118; h) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 126 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 127; or i) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 135, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 136.

[0037] In one embodiment, the chimeric receptor comprises: a) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 75 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 76; b) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 86 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 87; c) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 95 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 96; or d) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO:100, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:101.

[0038] In one embodiment, the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO:75, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:76.

[0039] In some embodiments, the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region, hi some embodiments, the linker consists of the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:149.

[0040] In one embodiment, the heavy chain variable region and the light chain variable region are, from N-terminus to C-terminus, V H -V L In one embodiment, the extracellular antigen binding domain comprises or is an scFv, which comprises or consists of the amino acid sequence represented by SEQ ID NO:79, SEQ ID NO:88, SEQ ID NO:97, SEQ ID NO:102, SEQ ID NO:108, SEQ ID NO:113, SEQ ID NO:119, SEQ ID NO:128, or SEQ ID NO:137. In one embodiment, the extracellular antigen binding domain comprises or is an scFv, which comprises or consists of the amino acid sequence represented by SEQ ID NO:79, SEQ ID NO:88, SEQ ID NO:97, or SEQ ID NO:102. In one embodiment, the extracellular antigen binding domain comprises or is an scFv, which comprises or consists of the amino acid sequence represented by SEQ ID NO:79.

[0041] In one embodiment, the extracellular antigen binding domain comprises about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 Dissociation constant (K DIn one embodiment, the binding to CLEC12A at K D In one embodiment, K D In one embodiment, K is between about 0.05 pM and about 0.5 pM. D In one embodiment, K is between about 0.1 nM and about 5.0 nM. D is between about 0.3 nM and about 3.5 nM. In one embodiment, the extracellular antigen-binding domain binds to CLEC12A with an EC50 of between about 1 nM and about 100 nM.

[0042] In some embodiments, the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ 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, or a BTLA polypeptide. In some embodiments, the transmembrane domain comprises a CD8 polypeptide.

[0043] In some embodiments, the CLEC12A-targeting chimeric receptor is a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), or a TCR-like fusion molecule. In some embodiments, the CLEC12A-targeting chimeric receptor is a chimeric costimulatory receptor (CCR).

[0044] In some embodiments, the intracellular domain does not include a CD3ζ polypeptide. In some embodiments, the intracellular domain includes at least one costimulatory signaling region. In some embodiments, the at least one costimulatory signaling region includes a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof. In some embodiments, the at least one costimulatory signaling region includes a 4-1BB polypeptide.

[0045] In some embodiments, the chimeric receptor is expressed from a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector.

[0046] The subject matter of the present disclosure further provides a cell comprising the chimeric receptor disclosed herein. In an embodiment, the cell comprises the ADGRE2-targeted chimeric receptor disclosed herein. In an embodiment, the cell comprises the CLEC12A-targeted chimeric receptor disclosed herein. In an embodiment, the cell comprises a) the ADGRE2-targeted chimeric receptor disclosed herein, and b) the CLEC12A-targeted chimeric receptor disclosed herein. In an embodiment, the ADGRE2-targeted chimeric receptor is a chimeric antigen receptor (CAR) and the CLEC12A-targeted chimeric receptor is a chimeric costimulatory receptor (CCR).

[0047] In one embodiment, the CAR binds to ADGRE2, and a) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 33 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 34 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 35 or a conservative modification thereof, and / or b) an extracellular antigen-binding domain comprising a light chain variable region comprising: CDR1 comprising the amino acid sequence represented by SEQ ID NO: 36 or a conservative modification thereof; CDR2 comprising the amino acid sequence represented by SEQ ID NO: 37 or a conservative modification thereof; and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof.

[0048] In one embodiment, the heavy chain variable region comprises a CDR1 having an amino acid sequence represented by SEQ ID NO: 33, a CDR2 having an amino acid sequence represented by SEQ ID NO: 34, and a CDR3 having an amino acid sequence represented by SEQ ID NO: 35, and the light chain variable region comprises a CDR1 having an amino acid sequence represented by SEQ ID NO: 36, a CDR2 having an amino acid sequence represented by SEQ ID NO: 37, and a CDR3 having an amino acid sequence represented by SEQ ID NO: 38.

[0049] In one embodiment, a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO:39; and / or b) the light chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO:40.

[0050] In one embodiment, the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO:39, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:40.

[0051] In an embodiment, the CCR comprises an extracellular antigen-binding domain, which binds to CLEC12A; a) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 69 or a conservative modification thereof, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 70 or a conservative modification thereof, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 71 or a conservative modification thereof, and / or b) a light chain variable region comprising: CDR1 having the amino acid sequence represented by SEQ ID NO: 72 or a conservative modification thereof; CDR2 having the amino acid sequence represented by SEQ ID NO: 73 or a conservative modification thereof; and CDR3 having the amino acid sequence represented by SEQ ID NO: 74 or a conservative modification thereof.

[0052] In one embodiment, the heavy chain variable region comprises a CDR1 having an amino acid sequence represented by SEQ ID NO:69, a CDR2 having an amino acid sequence represented by SEQ ID NO:70, and a CDR3 having an amino acid sequence represented by SEQ ID NO:71, and the light chain variable region comprises a CDR1 having an amino acid sequence represented by SEQ ID NO:72, a CDR2 having an amino acid sequence represented by SEQ ID NO:73, and a CDR3 having an amino acid sequence represented by SEQ ID NO:74.

[0053] In one embodiment, a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO:75; and / or b) the light chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical or homologous to the amino acid sequence set forth in SEQ ID NO:76.

[0054] In one embodiment, the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO:75, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:76.

[0055] In some embodiments, the cell is transduced with an ADGRE2-targeted chimeric receptor and / or a CLEC12A-targeted chimeric receptor. In some embodiments, the ADGRE2-targeted chimeric receptor and / or the CLEC12A-targeted chimeric receptor is constitutively expressed on the surface of the cell. In some embodiments, the cell is an immunoresponsive cell. In some embodiments, the cell is a lymphoid cell or a myeloid cell. In some embodiments, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a stem cell from which a lymphoid cell can differentiate, and a stem cell from which a myeloid cell can differentiate. In some embodiments, the cell is a T cell. In some embodiments, the T cell is selected from the group consisting of a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a tumor-infiltrating lymphocyte (TIL), a natural killer T cell, a mucosal-associated invariant T cell, and a gamma delta T cell. In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the NK cell is derived from a stem cell. In some embodiments, the stem cell is a pluripotent stem cell. In certain embodiments, the pluripotent stem cells are embryonic-like stem cells or induced pluripotent stem cells.

[0056] Furthermore, the subject matter of the present disclosure provides a nucleic acid molecule that encodes the chimeric receptor disclosed herein.In an embodiment, the nucleic acid molecule encodes the ADGRE2-targeted chimeric receptor disclosed herein.In an embodiment, the nucleic acid molecule encodes the CLEC12A-targeted chimeric receptor disclosed herein.

[0057] In some embodiments, the nucleic acid molecule further comprises a promoter operably linked to the chimeric receptor. In some embodiments, the promoter is endogenous or exogenous. In some embodiments, the exogenous promoter is selected from the group consisting of elongation factor (EF)-1 promoter, cytomegalovirus immediate early promoter (CMV) promoter, simian virus 40 early promoter (SV40) promoter, phosphoglycerate kinase (PGK) promoter, metallothionein promoter, and ubiquitin C promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is selected from the group consisting of NFAT transcription response element (TRE) promoter, CD69 promoter, CD25 promoter, IL-2 promoter, 4-1BB promoter, PD1 promoter, and LAG3 promoter. In some embodiments, the promoter is an endogenous promoter. In some embodiments, the endogenous promoter is selected from TCR alpha promoter, TCR beta promoter, and beta2-microglobulin promoter.

[0058] The presently disclosed subject matter also provides a nucleic acid composition comprising a first nucleic acid molecule encoding an ADGRE2-targeted chimeric receptor disclosed herein, and a second nucleic acid molecule encoding a CLEC12A-targeted chimeric receptor disclosed herein.

[0059] The subject matter of the present disclosure also provides a vector comprising the nucleic acid molecule disclosed herein or the nucleic acid composition disclosed herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector.

[0060] The subject matter of the present disclosure further provides a cell expressing the nucleic acid molecule disclosed herein or the nucleic acid composition disclosed herein. In some embodiments, the cell is a T cell.

[0061] The subject matter of the present disclosure provides a composition comprising the cells disclosed herein. In some embodiments, the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier. In some embodiments, the composition comprises about 25×10 6 ~Approx. 150×10 6 In one embodiment, the composition comprises about 25×10 6 ~Approx. 50×10 6 In one embodiment, the composition comprises about 2.5×10 cells. 6 Contains cells.

[0062] The subject matter of the present disclosure further provides various methods of using the cells of the present disclosure. The subject matter of the present disclosure provides a method of reducing tumor burden in a subject. In some embodiments, the method comprises administering to a subject a cell or composition disclosed herein. In some embodiments, the method reduces the number of tumor cells, reduces tumor size, and / or eradicates tumors in the subject.

[0063] The presently disclosed subject matter provides a method for increasing or prolonging the survival of a subject bearing a tumor, in some embodiments, the method comprises administering to the subject a cell or composition disclosed herein.

[0064] The presently disclosed subject matter provides a method of treating and / or preventing a tumor in a subject, hi some embodiments, the method comprises administering to the subject a cell or composition disclosed herein.

[0065] In some embodiments, the tumor expresses ADGRE2 and / or CLEC12A. In some embodiments, the tumor is cancer. In some embodiments, the tumor is a hematological cancer. In some embodiments, the tumor is selected from the group consisting of multiple myeloma, leukemia, lymphoma, and myeloid malignancies. In some embodiments, the leukemia is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia. In some embodiments, the leukemia is acute myeloid leukemia (AML). In some embodiments, the AML is relapsed / refractory acute myeloid leukemia (R / R AML). In certain embodiments, the myeloid malignancy is selected from the group consisting of myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), myeloid / lymphoid neoplasms (e.g., myeloid / lymphoid neoplasms with eosinophilia and platelet-derived growth factor receptor alpha (PDGFRA), platelet-derived growth factor receptor beta (PDGFRB), or fibroblast growth factor receptor 1 (FGFR1) rearrangements, or PCM1-JAK2), acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm, B lymphoblastic leukemia / lymphoma, and T lymphoblastic leukemia / lymphoma. In certain embodiments, the myeloid malignancy comprises myelodysplastic syndromes (MDS). In certain embodiments, the subject is a human subject.

[0066] Additionally, the presently disclosed subject matter provides a method of making a cell comprising a chimeric receptor disclosed herein, hi some embodiments, the method comprises introducing into a cell a nucleic acid molecule encoding a chimeric receptor disclosed herein.

[0067] The presently disclosed subject matter further provides a method of making a cell comprising an ADGRE2-targeted chimeric receptor disclosed herein, and a CLEC12A-targeted chimeric receptor disclosed herein, in one embodiment, the method comprises introducing into a cell a nucleic acid molecule encoding an ADGRE2-targeted chimeric receptor and a nucleic acid molecule encoding a CLEC12A-targeted chimeric receptor disclosed herein.

[0068] 4. Brief drawing description The following detailed description is given by way of example, but is not intended to limit the invention to the specific embodiments described, and can be understood in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0069] [Figure 1A] A and B show target antigen expression profiles of ADGRE2, CD33, and CD123 assessed by flow cytometry in fresh AML patient samples (peripheral blood or bone marrow). n=16 r / r adult AML patients with morphological disease. Target expression was compared between bulk AML cells (CD45dim / SSClow gate) and leukemic stem cells [LSC; Lin-CD34+CD38-(CD45RA+) in bulk AML cell gate). Target positivity gates were set based on negative control populations (T cells or B cells) of the same samples. A shows the percentage of target positive AML cells within the bulk or LSC populations of individual patient samples represented by each point. In the majority of AML patients evaluated, >90% of AML cells were positive for ADGRE2 in both bulk and LSC. B shows the percentage of evaluated AML patient populations with >70% target positive AML cells. All AML patients evaluated had >70% ADGRE2 positive AML cells in both bulk and LSC. [Figure 1B]A and B show target antigen expression profiles of ADGRE2, CD33, and CD123 assessed by flow cytometry in fresh AML patient samples (peripheral blood or bone marrow). n=16 r / r adult AML patients with morphological disease. Target expression was compared between bulk AML cells (CD45dim / SSClow gate) and leukemic stem cells [LSC; Lin-CD34+CD38-(CD45RA+) in bulk AML cell gate). Target positivity gates were set based on negative control populations (T cells or B cells) of the same samples. A shows the percentage of target positive AML cells within the bulk or LSC populations of individual patient samples represented by each point. In the majority of AML patients evaluated, >90% of AML cells were positive for ADGRE2 in both bulk and LSC. B shows the percentage of evaluated AML patient populations with >70% target positive AML cells. All AML patients evaluated had >70% ADGRE2 positive AML cells in both bulk and LSC. [Diagram 2] Schematic diagrams of CAR, CCR, and CAR+CCR are shown. [Diagram 3] We present the rationale for the ADCLEC.syn1 approach, which provides improved anti-leukemic efficacy compared to CD33 and CD123-CARs. ADGRE2-CAR alone provides some anti-leukemic activity, but may be limited by ADGRE2-low escape mechanisms. CLEC12A-CCR alone does not mediate cell lysis. By combining the low-affinity ADGRE2-CAR with the high-affinity CLEC12A-CCR, ADCLEC.syn1 enhances AML-directed avidity and reduces the risk of ADGRE2-low AML escape. Furthermore, CLEC12A-CCR-dependent trans-costimulation via 4-1BB further enhances T cell function. In comparison, single-targeted CD33-CAR or CD123-CAR approaches may be limited in efficacy by low antigen escape and phenotypic heterogeneity (see target profile in Figure 1). [Figure 4A]A-C show protein expression profiles of ADGRE2, CLEC12A (CD371), CD33, and CD123 in normal hematopoietic and non-hematopoietic tissues, showing the largely non-overlapping expression profiles of ADGRE2 and CLEC12A. A and B show flow cytometry co-expression profiles of ADGRE2 / CLEC12A versus CD33 / CD123 in representative adult normal donor bone marrow cell populations (gating: monocytes-CD45hi / SSCmed / CD14+, granulocytes-CD45dim / SSChi, HSC-CD45dim / SSClow / CD34+ / CD38- / CD45RA- / CD90+, T cells-CD45hi / SSClow / CD3+, B cells-CD45hi / SSClow / CD19+). C shows a heatmap outlining immunohistochemical co-staining of ADGRE2 and CLEC12A in formalin-fixed paraffin-embedded normal human tissues. Staining intensity refers to cellular positivity, excluding fluid, where nonspecific staining is likely. ADGRE2 / CLEC12A was expected to be co-expressed in myeloid cells of immune-related tissues (mainly monocytic lineage), but showed no or limited overlapping expression in other tissues evaluated. [Figure 4B]A-C show protein expression profiles of ADGRE2, CLEC12A (CD371), CD33, and CD123 in normal hematopoietic and non-hematopoietic tissues, showing the largely non-overlapping expression profiles of ADGRE2 and CLEC12A. A and B show flow cytometry co-expression profiles of ADGRE2 / CLEC12A versus CD33 / CD123 in representative adult normal donor bone marrow cell populations (gating: monocytes-CD45hi / SSCmed / CD14+, granulocytes-CD45dim / SSChi, HSC-CD45dim / SSClow / CD34+ / CD38- / CD45RA- / CD90+, T cells-CD45hi / SSClow / CD3+, B cells-CD45hi / SSClow / CD19+). C shows a heatmap outlining immunohistochemical co-staining of ADGRE2 and CLEC12A in formalin-fixed paraffin-embedded normal human tissues. Staining intensity refers to cellular positivity, excluding fluid, where nonspecific staining is likely. ADGRE2 / CLEC12A was expected to be co-expressed in myeloid cells of immune-related tissues (mainly monocytic lineage), but showed no or limited overlapping expression in other tissues evaluated. [Figure 4C]A-C show protein expression profiles of ADGRE2, CLEC12A (CD371), CD33, and CD123 in normal hematopoietic and non-hematopoietic tissues, showing the largely non-overlapping expression profiles of ADGRE2 and CLEC12A. A and B show flow cytometry co-expression profiles of ADGRE2 / CLEC12A versus CD33 / CD123 in representative adult normal donor bone marrow cell populations (gating: monocytes-CD45hi / SSCmed / CD14+, granulocytes-CD45dim / SSChi, HSC-CD45dim / SSClow / CD34+ / CD38- / CD45RA- / CD90+, T cells-CD45hi / SSClow / CD3+, B cells-CD45hi / SSClow / CD19+). C shows a heatmap outlining immunohistochemical co-staining of ADGRE2 and CLEC12A in formalin-fixed paraffin-embedded normal human tissues. Staining intensity refers to cellular positivity, excluding fluid, where nonspecific staining is likely. ADGRE2 / CLEC12A was expected to be co-expressed in myeloid cells of immune-related tissues (mainly monocytic lineage), but showed no or limited overlapping expression in other tissues evaluated. [Diagram 5] We present the rationale for the ADCLEC.syn1 approach to improve safety profile compared to CD33-CAR and CD123-CAR. The ADGRE2-CAR alone, with optimized affinity and fine-tuned CD3ζ signaling strength, spares cells with low or very low levels of ADGRE2 (HSCs or granulocytes, respectively). The high-affinity CLEC12A-CCR alone does not mediate cell lysis. ADCLEC.syn1 combines the low-affinity ADGRE2-CAR with the high-affinity CLEC12A-CCR. Normal hematopoietic cells have little overlapping expression profiles between ADGRE2 and CLEC12A, and CLEC12A is negative for HSCs. Thus, the addition of the CLEC12A-CCR does not increase the risk of HSC toxicity. [Figure 6] 1 shows the ADGRE2 scFv binder selection scheme. [Figure 7A]A-C show 18 h in vitro CAR cytotoxicity assays associated with different ADGRE2 target expression levels. A favorable profile of the leading humanized ADGRE2 scFv was observed, showing maximum cytotoxicity at high ADGRE2 levels and minimum cytotoxicity at very low ADGRE2 levels. The different humanized ADGRE2 scFv candidates and the original 2A1 scFv were tested in the SFG retroviral 28z1XX CAR vector backbone. For each ADGRE2 scFv, two signal peptides were studied; one containing the established CD8α signal peptide and the other containing an alternative IgHV1-4 signal peptide. Shown with colored lines are the six scFvs with cytotoxic characteristics. The remaining scFv candidates (not included in the legend) are shown in grey. CAR T cells were co-cultured for 18 hours with MOLM13 AML cell lines expressing various levels of ADGRE2: high (WT, A), low (clone 1E8, B), and very low (clone 9D6, C). Cytotoxicity was measured based on the luciferase signal released from MOLM13 cells. [Figure 7B]A-C show 18 h in vitro CAR cytotoxicity assays associated with different ADGRE2 target expression levels. A favorable profile of the leading humanized ADGRE2 scFv was observed, showing maximum cytotoxicity at high ADGRE2 levels and minimum cytotoxicity at very low ADGRE2 levels. The different humanized ADGRE2 scFv candidates and the original 2A1 scFv were tested in the SFG retroviral 28z1XX CAR vector backbone. For each ADGRE2 scFv, two signal peptides were studied; one containing the established CD8α signal peptide and the other containing an alternative IgHV1-4 signal peptide. Shown with colored lines are the six scFvs with cytotoxic characteristics. The remaining scFv candidates (not included in the legend) are shown in grey. CAR T cells were co-cultured for 18 hours with MOLM13 AML cell lines expressing various levels of ADGRE2: high (WT, A), low (clone 1E8, B), and very low (clone 9D6, C). Cytotoxicity was measured based on the luciferase signal released from MOLM13 cells. [Figure 7C]A-C show 18 h in vitro CAR cytotoxicity assays associated with different ADGRE2 target expression levels. A favorable profile of the leading humanized ADGRE2 scFv was observed, showing maximum cytotoxicity at high ADGRE2 levels and minimum cytotoxicity at very low ADGRE2 levels. The different humanized ADGRE2 scFv candidates and the original 2A1scFv were tested in the SFG retroviral 28z1XX CAR vector backbone. For each ADGRE2 scFv, two signal peptides were studied; one containing the established CD8α signal peptide and the other containing an alternative IgHV1-4 signal peptide. Shown with colored lines are the six scFvs with cytotoxic characteristics. The remaining scFv candidates (not included in the legend) are shown in grey. CAR T cells were co-cultured for 18 hours with MOLM13 AML cell lines expressing various levels of ADGRE2: high (WT, A), low (clone 1E8, B), and very low (clone 9D6, C). Cytotoxicity was measured based on the luciferase signal released from MOLM13 cells. [Figure 8-1]We show the in vivo antitumor CAR efficacy of six humanized ADGRE2 scFvs at different ADGRE2 target expression levels. The in vivo antitumor CAR efficacy model showed a good profile for the lead humanized ADGRE2 scFv, with potent efficacy at high ADGRE2 levels, reduced cytotoxicity at low ADGRE2 levels, and no cytotoxicity at very low ADGRE2 levels. Six different humanized ADGRE2 scFv candidates and the original 2A1scFv were tested in the SFG-based retroviral 28z1XX CAR backbone. In vivo MOLM13 AML cell line xenograft experiments with 6-8 week old NSG mice. On day -5, mice were injected via tail vein with the indicated MOLM13 cell line clones (dose: 1E6 cells per mouse). On day -1, AML engraftment was confirmed by ffLuc-based in vivo bioluminescence imaging. On day 0, mice were injected via tail vein with CAR T cells (dose: 3E5 CAR-positive cells per mouse). AML burden was subsequently quantified by bioluminescence imaging and expressed as total flux (p / s). [Figure 8-2]We show the in vivo antitumor CAR efficacy of six humanized ADGRE2 scFvs at different ADGRE2 target expression levels. The in vivo antitumor CAR efficacy model showed a good profile for the lead humanized ADGRE2 scFv, with potent efficacy at high ADGRE2 levels, reduced cytotoxicity at low ADGRE2 levels, and no cytotoxicity at very low ADGRE2 levels. Six different humanized ADGRE2 scFv candidates and the original 2A1scFv were tested in the SFG-based retroviral 28z1XX CAR backbone. In vivo MOLM13 AML cell line xenograft experiments with 6-8 week old NSG mice. On day -5, mice were injected via tail vein with the indicated MOLM13 cell line clones (dose: 1E6 cells per mouse). On day -1, AML engraftment was confirmed by ffLuc-based in vivo bioluminescence imaging. On day 0, mice were injected via tail vein with CAR T cells (dose: 3E5 CAR-positive cells per mouse). AML burden was subsequently quantified by bioluminescence imaging and expressed as total flux (p / s). [Figure 8-3]We show the in vivo antitumor CAR efficacy of six humanized ADGRE2 scFvs at different ADGRE2 target expression levels. The in vivo antitumor CAR efficacy model showed a good profile for the lead humanized ADGRE2 scFv, with potent efficacy at high ADGRE2 levels, reduced cytotoxicity at low ADGRE2 levels, and no cytotoxicity at very low ADGRE2 levels. Six different humanized ADGRE2 scFv candidates and the original 2A1scFv were tested in the SFG-based retroviral 28z1XX CAR backbone. In vivo MOLM13 AML cell line xenograft experiments with 6-8 week old NSG mice. On day -5, mice were injected via tail vein with the indicated MOLM13 cell line clones (dose: 1E6 cells per mouse). On day -1, AML engraftment was confirmed by ffLuc-based in vivo bioluminescence imaging. On day 0, mice were injected via tail vein with CAR T cells (dose: 3E5 CAR-positive cells per mouse). AML burden was subsequently quantified by bioluminescence imaging and expressed as total flux (p / s). [Figure 9] CLEC12A scFv binder selection scheme. [Figure 10] A and B show in vitro CAR cytotoxicity of CLEC12A scFv in TRAC CAR-28z1XX format in the context of AML cell lines U937 and MOLM13. The in vitro 18-hour CAR cytotoxicity assay shows a good profile of the leading CLEC12A scFv with strong efficacy at high and low CLEC12A levels. Different CLEC12A scFv candidates were tested on the TRAC-AAV 28z1XX CAR backbone. Shown in colored lines are the nine scFvs with the highest efficacy in the context of various CLEC12A levels. A shows cytotoxicity with the CLEC12A-high cell line U937. B shows cytotoxicity with the CLEC12A-low cell line MOLM13. The remaining scFv candidates are shown in grey. T cells were co-cultured with AML cell lines for 18 hours. Cytotoxicity was measured based on the luciferase signal from the AML cell lines. [Figure 11] We present an in vivo antitumor CAR efficacy model showing high efficacy for leading CLEC12A scFvs. Nine different CLEC12A scFv candidates were tested in the TRAC-AAV 28z1XX CAR backbone. In vivo U937 AML cell line xenograft experiment with 6-8 week old NSG mice. On day -4, mice were injected via tail vein with the indicated U937 cell line clones (dose: 1E6 cells per mouse). On day -1, AML engraftment was confirmed by ffLuc-based in vivo bioluminescence imaging. On day 0, mice were injected via tail vein with CAR T cells (dose: 4E5 CAR positive cells per mouse). Subsequently, AML burden was quantified by bioluminescence imaging and expressed by total flux (p / s). [Figure 12A] A-C show in vitro and in vivo CAR efficacy assays validating the ADCLEC.syn1 concept (shown as a schematic in Figure 3). A shows an in vitro 18-h CAR cytotoxicity assay. ADCLEC.syn1-transduced T cells were co-cultured with EL4 mouse lymphoma cell lines expressing no target (ADGRE2- / CLEC12A-), or overexpressing CAR target alone (ADGRE2+ / CLEC12A-), CCR target alone (ADGRE2- / CLEC12A+), or both CAR and CCR targets (ADGRE2+ / CLEC12A+). A CD19-targeting 1928z1XX CAR construct was used as a negative control. % cytotoxicity indicates target-specific killing efficacy at a particular effector:target (E:T) ratio. B and C show in vivo MOLM13 AML cell line xenograft experiments using 6-8 week old NSG mice. On day -5, mice were injected with the indicated MOLM13 cell line clones via the tail vein (dose: 1x106 cells per mouse). On day -1, AML engraftment was confirmed by ffLuc-based in vivo bioluminescence imaging. On day 0, mice were injected with CAR T cells via the tail vein (dose: 5x105 CAR-positive cells per mouse). AML burden was subsequently quantified by bioluminescence imaging and expressed by total flux (p / s). [Figure 12B] A-C show in vitro and in vivo CAR efficacy assays validating the ADCLEC.syn1 concept (shown as a schematic in Figure 3). A shows an in vitro 18-h CAR cytotoxicity assay. ADCLEC.syn1-transduced T cells were co-cultured with EL4 mouse lymphoma cell lines expressing no target (ADGRE2- / CLEC12A-), or overexpressing CAR target alone (ADGRE2+ / CLEC12A-), CCR target alone (ADGRE2- / CLEC12A+), or both CAR and CCR targets (ADGRE2+ / CLEC12A+). A CD19-targeting 1928z1XX CAR construct was used as a negative control. % cytotoxicity indicates target-specific killing efficacy at a particular effector:target (E:T) ratio. B and C show in vivo MOLM13 AML cell line xenograft experiments using 6-8 week old NSG mice. On day -5, mice were injected with the indicated MOLM13 cell line clones via the tail vein (dose: 1x106 cells per mouse). On day -1, AML engraftment was confirmed by ffLuc-based in vivo bioluminescence imaging. On day 0, mice were injected with CAR T cells via the tail vein (dose: 5x105 CAR-positive cells per mouse). AML burden was subsequently quantified by bioluminescence imaging and expressed by total flux (p / s). [Figure 12C]A-C show in vitro and in vivo CAR efficacy assays validating the ADCLEC.syn1 concept (shown as a schematic in Figure 3). A shows an in vitro 18-h CAR cytotoxicity assay. ADCLEC.syn1-transduced T cells were co-cultured with EL4 mouse lymphoma cell lines expressing no target (ADGRE2- / CLEC12A-), or overexpressing CAR target alone (ADGRE2+ / CLEC12A-), CCR target alone (ADGRE2- / CLEC12A+), or both CAR and CCR targets (ADGRE2+ / CLEC12A+). A CD19-targeting 1928z1XX CAR construct was used as a negative control. % cytotoxicity indicates target-specific killing efficacy at a particular effector:target (E:T) ratio. B and C show in vivo MOLM13 AML cell line xenograft experiments using 6-8 week old NSG mice. On day -5, mice were injected with the indicated MOLM13 cell line clones via the tail vein (dose: 1x106 cells per mouse). On day -1, AML engraftment was confirmed by ffLuc-based in vivo bioluminescence imaging. On day 0, mice were injected with CAR T cells via the tail vein (dose: 5x105 CAR-positive cells per mouse). AML burden was subsequently quantified by bioluminescence imaging and expressed by total flux (p / s). [Figure 13] The pSFG-ADCLEC.syn1 restriction map (8940 bp) is shown. [Figure 14] 1 shows the gamma retroviral vector design for the ADCLEC.syn1 bicistronic construct containing ADGRE2ADGRE2-A-CAR and CLEC12ACLEC12A-A-CCR. Abbreviations: LTR=long terminal repeat, SD=splice donor site, SA=splice acceptor site, SP=signal peptide, scFv=single chain variable fragment, H=hinge, TM=transmembrane, C=co-stimulatory domain, S=stimulatory domain. [Figure 15-1] A representative ADCLEC.syn1CART cell production scheme is shown. [Figure 15-2] A representative ADCLEC.syn1CART cell production scheme is shown. [Figure 15-3] A representative ADCLEC.syn1CART cell production scheme is shown. [Figure 16] An algorithm for guiding the course of treatment following D30 disease assessment is presented. [Figure 17] A and B show soluble membrane protein (SMP) assays. [Figure 18A-1] A and B show the combinatorial gating strategy. A shows the combinatorial CAR gating strategy previously described. [Figure 18A-2] A and B show the combinatorial gating strategy. A shows the combinatorial CAR gating strategy previously described. [Figure 18B] A and B show the combinatorial gating strategy. B shows the "IF-BETTER" gating strategy. [Figure 19A] A and B show an in vitro CAR efficacy assay validating the "IF-BETTER" gating strategy in the context of ADCLEC.syn1-mediated ADGRE2 / CLEC12A co-targeting. A shows cytotoxicity induced by ADCLEC.syn1-transformed T cells co-cultured with EL4 mouse lymphoma cell lines overexpressing either no target (ADGRE2- / CLEC12A-), CAR target alone (ADGRE2+ / CLEC12A-), CCR target alone (ADGRE2- / CLEC12A+), or both CAR and CCR targets (ADGRE2+ / CLEC12A+). 1928z1XX CAR was used as a negative control. [Figure 19B] A and B show in vitro CAR efficacy assays validating the "IF-BETTER" gating strategy in the context of ADCLEC.syn1-mediated ADGRE2 / CLEC12A co-targeting. B shows cytotoxicity induced by untransduced, ADGRE2-CAR-, CLEC12A-CCR-, or ADCLEC.syn1-transduced T cells co-cultured with MOLM13 target cells engineered to express different ADGRE2 levels (i.e., ADGRE2-high (WT), ADGRE2-low, and ADGRE2-very-low). [Figure 20-1] In vivo validation of the "IF-BETTER" gating strategy using ADCLEC.syn1 T cells. Tumor growth and survival were measured in NSG AML xenograft models infused with non-transduced, ADGRE2-CAR-, ADGRE2-CAR+CLEC12A-CAR-, CLEC12A-CCR-, or ADCLEC.syn1-transduced T cells. [Figure 20-2] In vivo validation of the "IF-BETTER" gating strategy using ADCLEC.syn1 T cells. Tumor growth and survival were measured in NSG AML xenograft models infused with non-transduced, ADGRE2-CAR-, ADGRE2-CAR+CLEC12A-CAR-, CLEC12A-CCR-, or ADCLEC.syn1-transduced T cells. [Figure 20-3] In vivo validation of the "IF-BETTER" gating strategy using ADCLEC.syn1 T cells. Tumor growth and survival were measured in NSG AML xenograft models infused with non-transduced, ADGRE2-CAR-, ADGRE2-CAR+CLEC12A-CAR-, CLEC12A-CCR-, or ADCLEC.syn1-transduced T cells. [Figure 21]

[0023] Figure 1 shows in vivo stress testing of ADCLEC.syn1 with T cell dose titration and AML re-challenge. Tumor growth and survival were measured in an NSG AML xenograft model injected with different doses of ADCLEC.syn1 transduced T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] 5. MODE FOR CARRYING OUT THEINVENTION The subject matter disclosed herein provides a chimeric receptor targeting ADGRE2 and a chimeric receptor targeting CLEC12A. The subject matter disclosed herein further provides a cell comprising the chimeric receptor targeting ADGRE2 of the present disclosure, a cell comprising the chimeric receptor targeting CLEC12A of the present disclosure, and a cell comprising the chimeric receptor targeting ADGRE2 of the present disclosure and the chimeric receptor targeting CLEC12A of the present disclosure. The cell can be an immunoresponsive cell, for example, a genetically modified immunoresponsive cell (e.g., a T cell or a NK cell). The subject matter disclosed herein also provides a method of using such cells for treatment, for example, treatment and / or prevention of tumors associated with ADGRE2 and / or CLEC12A (e.g., AML).

[0071] Non-limiting embodiments of the presently disclosed subject matter are illustrated in the specification and by way of examples.

[0072] For purposes of clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections.

[0073] 5.1. Definition 5.2. Chimeric receptors 5.3. Extracellular Antigen-Binding Domains of ADGRE2-Targeted Chimeric Receptors 5.4. Examples of ADGRE2-targeted chimeric receptors 5.5. Extracellular Antigen-Binding Domain of CLEC12A-Targeted Chimeric Receptors 5.6. Examples of CLEC12A-targeted chimeric receptors 5.7. Cells 5.8. Nucleic Acid Molecules, Vectors and Genetic Modifications 5.9. Formulation and Administration 5.10. Treatment method

[0074] 5.1. Definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the subject matter of this disclosure belongs. The following references, Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991), provide those of ordinary skill in the art with general definitions of many of the terms used in the subject matter of this disclosure. As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified.

[0075] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by a person skilled in the art, which depends in part on the method of measuring or determining the value, i.e., the limit of the measurement system.For example, "about" can mean within 3 standard deviations or more than 3 standard deviations, per one practice in the art.Alternatively, "about" can mean within a range of up to 20%, preferably within a range of up to 10%, more preferably within a range of up to 5%, and even more preferably within a range of up to 1% of a given value.Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 times, more preferably within 2 times, of a value.

[0076] By "immunoresponsive cell" is meant a cell that functions in an immune response, or a precursor or progeny thereof. In some embodiments, the immunoresponsive cell is a lymphoid cell. Non-limiting examples of lymphoid cells include T cells, natural killer (NK) cells, B cells, and stem cells that can differentiate into lymphoid cells. In some embodiments, the immunoresponsive cell is a myeloid cell.

[0077] "Activating an immunoresponsive cell" refers to the induction of signal transduction or change in protein expression in a cell that results in the initiation of an immune response. For example, clustering of CD3 chains in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs) generates a signal transduction cascade. In one embodiment, binding of an endogenous TCR or an exogenous CAR to an antigen leads to the formation of an immunological synapse that includes the clustering of many molecules near the bound receptor (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.). This clustering of membrane-bound signal transduction molecules allows the ITAM motifs contained within the CD3 chains to be phosphorylated. This phosphorylation initiates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce global gene expression in T cells to increase IL-2 production for proliferation and expression of key regulator T cell proteins to initiate a T cell-mediated immune response.

[0078] "Stimulating immunoresponsive cells" refers to signals that result in a strong and sustained immune response. In various embodiments, the signals occur after activation of immune cells (e.g., T cells) or are mediated simultaneously through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, and ICOS. Receiving multiple stimulatory signals is important to develop a strong and long-lasting T cell-mediated immune response. T cells are quickly suppressed and become unresponsive to antigens. The effects of these costimulatory signals can vary, but generally result in increased gene expression to generate long-lived, proliferative, anti-apoptotic T cells that respond strongly to antigens for complete and sustained eradication.

[0079] The term "antibody" as used herein refers not only to intact antibody molecules, but also to fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are routinely used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules, but also to the well-known active fragments F(ab') 2 and Fab. F(ab') 2 , and Fab fragments, which lack the Fe fragment of an intact antibody, are cleared more rapidly from the circulation and may have less nonspecific tissue binding than intact antibodies (Wahl et al., Nucl Med (1983); 24:316-325). As used herein, the term includes intact native antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and non-conventional antibodies. In certain embodiments, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (herein referred to as V H ) and heavy chain constant (C H The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (herein referred to as V L and light chain constant CL The light chain constant region consists of one domain, C L It consists of: V H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0080] As used herein, "CDR" is defined as the complementarity determining region amino acid sequences of an antibody that are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th US Department of Health and Human Services, National Institutes of Health (1987), or the IMGT numbering system (Lefranc, The Immunologist (1999);7:132-136; Lefranc et al., Dev.Comp.Immunol. (2003);27:55-77). Generally, an antibody contains three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for the antibody to bind to an antigen or epitope. In certain embodiments, the CDR regions are distinguished using the IMGT numbering system. In certain embodiments, the CDR regions are distinguished using the IMGT numbering system available at http: / / www.imgt.org / IMGT_vquest / input.

[0081] As used herein, the term "single chain variable fragment" or "scFv" refers to a V H Heavy chains (VL) of immunoglobulins (e.g., mouse or human) covalently linked to form a VL heterodimer. H ) and light chain (V L ) fusion protein of the variable region of the heavy chain (V H ) and light chain (V L ) is directly bonded or V H N-terminus of V L or at the C-terminus of V H The C-terminus of V L The linker is linked to the N-terminus of the heavy chain variable region of the extracellular antigen-binding domain by a peptide-encoded linker (e.g., 10, 15, 20, 25 amino acids). The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO2014 / 087010, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the linker is a G4S linker.

[0082] In one embodiment, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:1, provided below. GGGGSGGGGSGGGSGGGGS [SEQ ID NO: 1]

[0083] In one embodiment, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:2, provided below. GGGGSGGGGSGGGGS [SEQ ID NO: 2]

[0084] In one embodiment, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:3, provided below. GGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 3]

[0085] In one embodiment, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:4, provided below. GGGGSGGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 4]

[0086] In one embodiment, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:5, provided below. GGGGS [SEQ ID NO:5]

[0087] In one embodiment, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:6, provided below. GGGGSGGGGS [SEQ ID NO: 6]

[0088] In one embodiment, the linker comprises the first three amino acids of the heavy chain constant region, hi one embodiment, the linker comprises or consists of the amino acid sequence represented by SEQ ID NO: 149, provided below. ASTGGGGSGGGGSGGGGS [SEQ ID NO: 149]

[0089] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be synthesized using the VFv polypeptide as described by Huston, et al. Proc. Nat. Acad. Sci. USA, (1988); 85:5879-5883; U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. H and V LIt can be expressed from a nucleic acid containing the coding sequence. Antagonistic scFvs with inhibitory activity have been described (see, e.g., Zhao et al., Hybridoma (Larchmt) (2008);27(6):445-51; Peter et al., J Cachexia Sarcopenia Muscle (2012);August 12; Shieh et al., J Imunol (2009);183(4):2277-85; Giomarelli et al., Thromb Haemost (2007);97(6):955-63; Fife et al., J Clin Invst (2006);116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40)). Agonistic scFvs with stimulatory activity have been described (Peter et al., J Biol Chem (2003);25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol (1997);17(5-6):427-55; Ho et al., BioChim Biophys Acta (2003);1638(3):257-66).

[0090] The term "chimeric antigen receptor" or "CAR" as used herein refers to a molecule that comprises an extracellular antigen-binding domain fused to an intracellular signaling domain that can activate or stimulate an immunoresponsive cell. In an embodiment, the CAR also comprises a transmembrane domain. In an embodiment, the extracellular antigen-binding domain of the CAR comprises an scFv. The scFv can be obtained from the fusion of the variable heavy and variable light regions of an antibody. Alternatively, or additionally, the scFv can be derived from a Fab (e.g., not from an antibody obtained from a Fab library). In an embodiment, the scFv is fused to a transmembrane domain, which is then fused to an intracellular signaling domain.

[0091] The term "chimeric costimulatory receptor" or "CCR" refers to a chimeric receptor that binds to an antigen to provide a costimulatory signal, but does not provide an activation signal by itself. CCRs are described in Krause, et al., J. Exp.Med.(1998);188(4):619-626 and US20020018783, the entire contents of which are incorporated by reference. CCRs mimic costimulatory signals, but unlike CARs, do not provide an activation signal by themselves. For example, CCRs lack the CD3ζ polypeptide.

[0092] By "substantially identical" or "substantially homologous" is meant a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or a reference nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). In certain embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous or identical to the amino acid or nucleic acid sequence used for comparison.

[0093] Sequence identity may be measured using sequence analysis software (e.g., Sequence Analysis Software Package, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine. An exemplary method for measuring the degree of identity may use the BLAST program, in which a probability score of e-3 to e-100 indicates closely related sequences.

[0094] As used herein, the percentage of homology between two amino acid sequences corresponds to the percentage of identity between the two sequences. The percentage of identity between two sequences is a function of the number of gaps that need to be introduced for optimal alignment of the two sequences and the number of identical positions shared by the sequences, taking into account the length of each gap (i.e., % homology = number of identical positions / total number of positions x 100). The comparison of sequences and the determination of the percentage of identity between two sequences can be accomplished using a mathematical algorithm.

[0095] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as implemented in the ALIGN program (version 2.0) using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm as implemented in the GAP program of the GCG software package (available at www.gcg.com) using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0096] Additionally or alternatively, the amino acid sequences of the presently disclosed subject matter can further be used as "query sequences" to perform searches against public databases, for example to identify related sequences. Such searches may be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to specific sequences disclosed herein (e.g., heavy and light chain variable region sequences of scFv m903, m904, m905, m906, and m900). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) may be used. An "effective amount" is an amount sufficient to affect a beneficial or desired clinical outcome upon treatment. An effective amount may be administered to a subject in one or more doses. In some embodiments, an effective amount may be an amount sufficient to palliate, ameliorate, stabilize, reverse or delay the progression of a disease, or reduce the pathological consequences of a disease. An effective amount can be determined by a physician on a case-by-case basis and is within the knowledge of one of ordinary skill in the art. In determining the appropriate dosage to achieve an effective amount, several factors are usually taken into consideration. These factors include the age, sex and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the cells to be administered.

[0097] As used herein, the term "endogenous" refers to a nucleic acid molecule or polypeptide that is normally expressed within a cell or tissue.

[0098] As used herein, the term "exogenous" refers to a nucleic acid molecule or polypeptide that is not endogenously present in a cell. Thus, the term "exogenous" will encompass any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and overexpressed nucleic acid molecules and polypeptides. By "exogenous" nucleic acid is meant a nucleic acid that is not present in a natural wild-type cell. For example, an exogenous nucleic acid may differ from its endogenous counterpart by sequence, position / location, or both. For clarity, an exogenous nucleic acid may have the same sequence or a different sequence compared to its natural endogenous counterpart. It can be introduced into the cell itself or its precursors by genetic engineering, and in some cases can be linked to alternative control sequences, such as non-native promoters or secretion sequences.

[0099] By "heterologous nucleic acid molecule or polypeptide" is meant a nucleic acid molecule (e.g., a cDNA, DNA or RNA molecule) or polypeptide that is not normally present in a cell or a sample obtained from a cell. The nucleic acid may be from another organism or may be, for example, an mRNA molecule that is not normally expressed in the cell or sample.

[0100] By "modulate" is meant to change, either positively or negatively. Exemplary modulations include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0101] By "increase" is meant a positive change of at least about 5%. The change can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

[0102] By "reduce" is meant to alter negatively by at least about 5%. The alteration can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0103] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components that normally accompany it in its natural state. "Isolation" indicates a degree of separation from the original source or surroundings. "Purification" indicates a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other substances so that impurities do not significantly affect the biological properties of the protein or cause other adverse effects. That is, a nucleic acid or peptide is purified when it is substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, e.g., polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may mean that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In the case of proteins that may undergo modifications, e.g., phosphorylation or glycosylation, different modifications may produce different isolated proteins, which can be purified separately.

[0104] By "isolated cell" is meant a cell that has been separated from molecules and / or cellular components that naturally accompany the cell.

[0105] As used herein, the term "antigen-binding domain" refers to a domain capable of specifically binding to a particular antigenic determinant or set of antigenic determinants present on a cell.

[0106] By "receptor" is meant a polypeptide or portion thereof present on a cell membrane that selectively binds to one or more ligands.

[0107] By "signal sequence" or "leader sequence" is meant a peptide sequence (eg, 5, 10, 15, 20, 25 or 30 amino acids) present at the N-terminus of a newly synthesized protein that directs its entry into the secretory pathway.

[0108] The terms "comprises" and "comprising" are intended to have the broad meaning ascribed to such terms under U.S. patent law and may mean "includes," "including," etc.

[0109] As used herein, "treatment" refers to a clinical intervention that attempts to change the disease course of the individual or cell being treated, and can be performed for prophylaxis or during the clinical pathology course. The therapeutic effect of treatment includes, but is not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or initially alleviating the disease state, and improving remission or prognosis. Preventing the progression of a disease or disorder can prevent the deterioration of the disorder in subjects who are affected or diagnosed or suspected of the disorder, while treatment can prevent the onset or symptoms of the disorder in subjects at risk of or suspected of the disorder.

[0110] An "individual" or "subject" herein is a vertebrate, such as a human or non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, hamsters, and guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, and non-human primates, such as apes and monkeys.

[0111] As used herein, the term "conservative sequence modifications" refers to amino acid modifications that do not substantially affect or alter the binding properties of the chimeric receptors of the present disclosure that contain the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the extracellular antigen-binding domain of the chimeric receptors of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties, such as their charge and polarity. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine, negatively charged amino acids include aspartic acid and glutamic acid, and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Furthermore, amino acids can be classified according to polarity. Polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine, and non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues in the CDR region can be replaced with other amino acid residues from the same group, and the modified antibody can be tested for retained functionality (i.e., the functions described in (c) to (l) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues in a particular sequence or CDR region are modified.

[0112] Other aspects of the presently disclosed subject matter are described in the disclosure that follows and are within the scope of the presently disclosed subject matter.

[0113] 5.2. Chimeric receptors In one embodiment, the chimeric receptor of the present disclosure comprises an extracellular antigen-binding domain that binds to ADGRE2 or CLEC12A. The extracellular antigen-binding domain can be an antigen-binding fragment of an antibody, a heavy chain variable region (V H ) antigen-binding fragment of an antibody light chain variable region (V L ), single chain variable fragment (scFv), Fab, or F(ab) 2 In some embodiments, the extracellular antigen-binding fragment is a single chain variable fragment (scFv). In some embodiments, the scFv is a human scFv. In some embodiments, the scFv is a humanized scFv. In some embodiments, the scFv is a mouse scFv. In some embodiments, the Fabs are cross-linked.

[0114] In some embodiments, the chimeric receptor of the present disclosure is a chimeric antigen receptor (CAR). In some embodiments, the chimeric receptor of the present disclosure is a chimeric costimulatory receptor (CCR). In some embodiments, the chimeric receptor is a TCR-like fusion molecule.

[0115] Chimeric Antigen Receptors (CARs) In one embodiment, the chimeric receptor is a CAR. A CAR is an engineered receptor that transfers or confers a specificity of interest to immune effector cells. CAR can be used to transfer the specificity of monoclonal antibodies to T cells, and the transfer of its coding sequence is facilitated by retroviral vectors.

[0116] There are three generations of CARs. "First generation" CARs are typically composed of an extracellular antigen binding domain (e.g., scFv), which is fused to a transmembrane domain, which is fused to a cytoplasmic / intracellular signaling domain. "First generation" CARs provide de novo antigen recognition, independent of HLA-mediated antigen presentation, and are directed to CD4 ζ chain signaling domains via the CD3ζ chain signaling domain in a single fusion molecule. + and CD8 +The chimeric receptor can induce both activation of T cells and activation of T cells. "Second generation" CARs add intracellular signaling domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide additional signals to T cells. "Second generation" CARs include CARs that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3zeta). "Third generation" CARs include CARs that provide multiple costimulations (e.g., CD28 and 4-1BB) and activation (CD3zeta). In some embodiments, the chimeric receptor is a second generation CAR. In some embodiments, the chimeric receptor is a CAR that includes the intracellular domain of a costimulatory molecule or a fragment thereof.

[0117] 5.2.1.1. Extracellular antigen-binding domain of the CAR In some embodiments, the extracellular antigen-binding domain of the CAR is a single chain variable fragment (scFv). In some embodiments, the scFv is a human scFv. In some embodiments, the scFv is a humanized scFv. In some embodiments, the scFv is a mouse scFv. In some embodiments, the scFv is identified by screening an scFv phage library with an antigen-Fc fusion protein.

[0118] In some embodiments, the extracellular antigen-binding domain is a Fab. In some embodiments, the Fab is cross-linked. In some embodiments, the extracellular antigen-binding domain is a F(ab) 2 Any of the above molecules can be included in a fusion protein with a heterologous sequence to form an extracellular antigen-binding domain.

[0119] The binding of the extracellular antigen-binding domain of the chimeric receptor, e.g., CAR, can be confirmed, 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 protein-antibody complex of particular interest by using a labeling reagent (e.g., antibody, or scFv) specific to the complex of interest. For example, scFv can be radioactively labeled and used in radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassay, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated herein by reference). Radioisotopes can be detected, for example, by means of a gamma counter or scintillation counter, or by autoradiography. In an embodiment, the extracellular antigen-binding domain of CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet).

[0120] 5.2.1.2. CAR transmembrane domain In one embodiment, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. After antigen recognition, the receptors assemble and transmit a signal to the cell. In accordance with the subject matter of the present disclosure, the transmembrane domain of the CAR may comprise a native or modified transmembrane domain of CD8 or a fragment thereof, a native or modified transmembrane domain of CD28 or a fragment thereof, a native or modified transmembrane domain of CD3ζ or a fragment thereof, a native or modified transmembrane domain of CD4 or a fragment thereof, a native or modified transmembrane domain of 4-1BB or a fragment thereof, a native or modified transmembrane domain of OX40 or a fragment thereof, a native or modified transmembrane domain of ICOS or a fragment thereof, a native or modified transmembrane domain of CD84 or a fragment thereof, a native or modified transmembrane domain of CD166 or a fragment thereof, a native or modified transmembrane domain of CD8a or a fragment thereof, a native or modified transmembrane domain of CD8b or a fragment thereof, a native or modified transmembrane domain of ICAM-1 or a fragment thereof, a native or modified transmembrane domain of CTLA-4 or a fragment thereof, a native or modified transmembrane domain of CD27 or a fragment thereof, a native or modified transmembrane domain of CD40 or a fragment thereof, NKGD2 or a fragment thereof, or a combination thereof.

[0121] In one embodiment, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., the transmembrane domain of CD8 or a fragment thereof).

[0122] In an embodiment, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., the transmembrane domain of CD8 or a fragment thereof). In an embodiment, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., the transmembrane domain of human CD8 or a fragment thereof). In an embodiment, the CD8 polypeptide comprises or consists of an amino acid sequence or a fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence having NCBI reference number NP_001139345.1 (SEQ ID NO: 7), and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In an embodiment, the CD8 polypeptide comprises or consists of an amino acid sequence that is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, and up to about 235 amino acids in length. In certain embodiments, the CD8 polypeptide comprises or consists of amino acids 1-235, 1-50, 50-100, 100-150, 150-200, 137-207, or 200-235 of SEQ ID NO: 7. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 137-207 of SEQ ID NO: 7. SEQ ID NO: 7 is provided below.

[0123] MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV [SEQ ID NO: 7]

[0124] An exemplary nucleotide sequence encoding amino acids 137-207 of SEQ ID NO:7 is represented as SEQ ID NO:8 and is provided below. Cccaccacgacgccagcgccgcgaccaccaaccccggcgcccacgatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgca cacgaggggctggacttcgcctgtgatatctacatctgggcgcccctggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaac [SEQ ID NO: 8]

[0125] In an embodiment, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., the transmembrane domain of mouse CD8 or a fragment thereof). In an embodiment, the CD8 polypeptide comprises or consists of an amino acid sequence or a fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence having NCBI reference number AAA92533.1 (SEQ ID NO: 9), and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In an embodiment, the CD8 polypeptide comprises or consists of an amino acid sequence that is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, and up to about 247 amino acids in length. In certain embodiments, the CD8 polypeptide comprises or consists of amino acids 1-247, 1-50, 50-100, 100-150, 150-200, 151-219, or 200-247 of SEQ ID NO: 9. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 151-219 of SEQ ID NO: 9. SEQ ID NO: 9 is provided below. 1 MASPLTRFLS LNLLLMGESI ILGSGEAKPQ APELRIFPKK MDAELGQKVD LVCEVLGSVS 61 QGCSWLFQNS SSKLPQPTFV VYMASSHNKI TWDEKLNSSK LFSAVRDTNN KYVLTLNKFS 121 KENEGYYFCS VISNSVMYFS SVVPVLQKVN STTTKPVLRT PSPVHPTGTS QPQRPEDCRP 181 RGSVKGTGLD FACDIYIWAP LAGICVAPLL SLIITLICYH RSRKRVCKCP RPLVRQEGKP 241 RPSEKIV [SEQ ID NO: 9]

[0126] In an embodiment, the transmembrane domain of a CAR of the present disclosure comprises a CD28 polypeptide (e.g., the transmembrane domain of CD28 or a fragment thereof).

[0127] In an embodiment, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a fragment thereof). In an embodiment, the CD28 polypeptide comprises or consists of an amino acid sequence or a fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the amino acid sequence having NCBI reference number NP_006130 (SEQ ID NO: 10), and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In an embodiment, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 10 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 220 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of amino acids 1-220, 1-50, 50-100, 100-150, 150-200, 153-179, or 200-220 of SEQ ID NO: 9. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 10. SEQ ID NO: 10 is provided below. 1 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD 61 SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP 121 PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR 181 SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS [SEQ ID NO: 10]

[0128] An exemplary nucleotide sequence encoding amino acids 153-179 of SEQ ID NO:10 is represented as SEQ ID NO:11 and is provided below. ttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg [SEQ ID NO: 11]

[0129] In an embodiment, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., the transmembrane domain of mouse CD28 or a fragment thereof). In an embodiment, the CD28 polypeptide comprises or consists of an amino acid sequence or a fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous or identical to the amino acid sequence having NCBI reference number NP_031668.3 (SEQ ID NO: 12), and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In an embodiment, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 12 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 218 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of amino acids 1-220, 1-50, 50-100, 100-150, 150-200, 151-177, or 200-218 of SEQ ID NO: 12. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 151-177 of SEQ ID NO: 12. SEQ ID NO: 12 is provided below. 1 MTLRLLFLAL NFFSVQVTEN KILVKQSPLL VVDSNEVSLS CRYSYNLLAK EFRASLYKGV 61 NSDVEVCVGN GNFTYQPQFR SNAEFNCDGD FDNETVTFRL WNLHVNHTDI YFCKIEFMYP 121 PPYLDNERSN GTIIHIKEKH LCHTQSSPKL FWALVVVAGV LFCYGLLVTV ALCVIWTNSR 181 RNRLLQSDYM NMTPRRPGLT RKPYQPYAPA RDFAAYRP [SEQ ID NO: 12]

[0130] In some embodiments, the CAR further comprises a spacer region linking 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 while maintaining the activation capacity of the CAR.

[0131] In an embodiment, the hinge / spacer region of the CAR is selected from the group consisting of a native or modified hinge region of CD8 or a fragment thereof, a native or modified hinge region of CD28 or a fragment thereof, a native or modified hinge region of CD3ζ or a fragment thereof, a native or modified hinge region of CD40 or a fragment thereof, a native or modified hinge region of 4-1BB or a fragment thereof, a native or modified hinge region of OX40 or a fragment thereof, a native or modified hinge region of CD84 or a fragment thereof, a native or modified hinge region of CD166 or a fragment thereof, a native or modified hinge region of CD8a ...8a or a fragment thereof, a native or modified hinge region of CD8a or a fragment thereof, a native or modified hinge region of CD28 or The hinge / spacer region may be a hinge region derived from an IgG1 or a CH of an immunoglobulin, a natural or modified hinge region or fragment thereof, a natural or modified hinge region of CD8b or a fragment thereof, a natural or modified hinge region of ICOS or a fragment thereof, a natural or modified hinge region of ICAM-1 or a fragment thereof, a natural or modified hinge region of CTLA-4 or a fragment thereof, a natural or modified hinge region of CD27 or a fragment thereof, a natural or modified hinge region of CD40 or a fragment thereof, a natural or modified hinge region of NKGD2 or a fragment thereof, a synthetic polypeptide (not based on a protein associated with an immune response), or a combination thereof. 2 CH 3The region may be a portion of CD3, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO: 10 or 12), a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO: 7 or 9), a variant thereof that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% homologous or identical to any of the foregoing, or a synthetic spacer sequence.

[0132] In one embodiment, the hinge / spacer region of the CAR comprises a CD28 polypeptide. In one embodiment, the hinge / spacer region of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 114 to 152 of SEQ ID NO:10.

[0133] An exemplary nucleotide sequence encoding amino acids 114-152 of SEQ ID NO:10 is represented as SEQ ID NO:13 and is provided below. attgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccc [SEQ ID NO: 13]

[0134] 5.2.1.3. Intracellular signaling domain of CAR In some embodiments, the CAR comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide. CD3ζ can activate or stimulate cells (e.g., lymphoid cells, e.g., T cells). Wild-type ("native") CD3ζ comprises three functional immunoreceptor tyrosine-based activation motifs (ITAMs), three functional basic-rich stretch (BRS) regions (BRS1, BRS2, ​​and BRS3). CD3ζ transmits activation signals to cells (e.g., lymphoid cells, e.g., T cells) after antigen binding. The intracellular signaling domain of the CD3ζ chain is the main transmitter of signals from endogenous TCR.

[0135] In some embodiments, the intracellular signaling domain of the CAR comprises native CD3ζ. In some embodiments, the native CD3ζ polypeptide comprises or consists of an amino acid sequence or a fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence having NCBI reference number NP_932170 (SEQ ID NO: 14) and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In some embodiments, the native CD3ζ polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 14 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, at least about 100, at least about 110, and up to about 164 amino acids in length. In certain embodiments, a native CD3ζ polypeptide comprises or consists of amino acids 1-164, 1-50, 50-100, 52-164, 100-150, or 150-164 of SEQ ID NO: 14. In certain embodiments, the intracellular signaling domain of a CAR comprises a CD3ζ polypeptide comprising or consisting of amino acids 52-164 of SEQ ID NO: 14. SEQ ID NO: 14 is provided below. 1 MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD 61 APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA 121 EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPR [SEQ ID NO: 14]

[0136] In some embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide. In some embodiments, the modified CD3ζ polypeptide comprises one, two or three ITAMs. In some embodiments, the modified CD3ζ polypeptide comprises a native ITAM1. In some embodiments, the native ITAM1 comprises or consists of the amino acid sequence represented by SEQ ID NO: 15. QNQLYNELNLGRREEYDVLDKR [SEQ ID NO: 15]

[0137] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:15 is represented as SEQ ID NO:16 and is provided below. Cagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagaga [SEQ ID NO: 16]

[0138] In some embodiments, the modified CD3ζ polypeptide comprises an ITAM1 mutant comprising one or more loss-of-function mutations. In some embodiments, the ITAM1 mutant comprises or consists of two loss-of-function mutations. In some embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM1. In some embodiments, the ITAM1 mutant consists of two loss-of-function mutations. In some embodiments, the ITAM1 mutant comprises or consists of the amino acid sequence represented by SEQ ID NO: 17, as provided below. QNQLFNELNLGRREEFDVLDKR [SEQ ID NO: 17]

[0139] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:17 is represented as SEQ ID NO:18 and is provided below. CAGAACCAGCTCTTTAACGAGCTCAATCTAGGACGAAGAGAGGAGTTCGATGTTTTGGACAAGAGA [SEQ ID NO: 18]

[0140] In one embodiment, the modified CD3ζ polypeptide comprises a native ITAM2. In one embodiment, the native ITAM2 comprises or consists of the amino acid sequence represented by SEQ ID NO: 19, as provided below. QEGLYNELQKDKMAEAYSEIGMK [SEQ ID NO: 19]

[0141] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:19 is represented as SEQ ID NO:20 and is provided below. CAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAA [SEQ ID NO: 20]

[0142] In some embodiments, the modified CD3ζ polypeptide comprises an ITAM2 mutant. In some embodiments, the ITAM2 mutant comprises or consists of one or more loss-of-function mutations. In some embodiments, the ITAM2 mutant comprises or consists of two loss-of-function mutations. In some embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM2. In some embodiments, the ITAM2 mutant consists of two loss-of-function mutations. In some embodiments, the ITAM2 mutant comprises or consists of the amino acid sequence represented by SEQ ID NO:21, as provided below. QEGLFNELQKDKMAEAFSEIGMK [SEQ ID NO: 21]

[0143] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:21 is represented as SEQ ID NO:22 and is provided below. Caggaaggcctgttcaatgaactgcagaaagataagatggcggaggccttcagtgagattgggatgaaa [SEQ ID NO: 22]

[0144] In one embodiment, the modified CD3ζ polypeptide comprises a native ITAM3. In one embodiment, the native ITAM3 comprises or consists of the amino acid sequence represented by SEQ ID NO:23, as provided below. HDGLYQGLSTATKDTYDALHMQ [SEQ ID NO: 23]

[0145] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:23 is represented as SEQ ID NO:24 and is provided below. CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG [SEQ ID NO: 24]

[0146] In some embodiments, the modified CD3ζ polypeptide comprises an ITAM3 mutant. In some embodiments, the ITAM3 mutant comprises or consists of two loss-of-function mutations. In some embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM3. In some embodiments, the ITAM3 mutant comprises or consists of two loss-of-function mutations. In some embodiments, the ITAM3 mutant comprises or consists of the amino acid sequence represented by SEQ ID NO: 25, as provided below. HDGLFQGLSTATKDTFDALHMQ [Sequence number 25]

[0147] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:25 is represented as SEQ ID NO:26 and is provided below. Cacgatggccttttccagggtctcagtacagccaccaaggacaccttcgacgcccttcacatgcag [SEQ ID NO: 26]

[0148] Various modified CD3ζ polypeptides and CARs comprising modified CD3ζ polypeptides are disclosed in International Patent Application Publication No. WO2019 / 133969, the entire contents of which are incorporated by reference herein.

[0149] In one embodiment, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1, an ITAM2 mutant comprising or consisting of one or more (e.g., two) loss-of-function mutations, and an ITAM3 mutant comprising or consisting of one or more (e.g., two) loss-of-function mutations. In one embodiment, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1, an ITAM2 mutant consisting of two loss-of-function mutations, and an ITAM3 mutant consisting of two loss-of-function mutations. In one embodiment, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1 consisting of the amino acid sequence represented by SEQ ID NO: 15, an ITAM2 mutant consisting of the amino acid sequence represented by SEQ ID NO: 21, and an ITAM3 mutant consisting of the amino acid sequence represented by SEQ ID NO: 25. In one embodiment, the modified CD3ζ polypeptide is represented as "1XX". In one embodiment, the modified CD3ζ polypeptide comprises or consists of the amino acid sequence represented by SEQ ID NO: 27. SEQ ID NO: 27 is provided below. RVKFSRSADA PAYQQGQNQL YNELNLGRRE EYDVLDKRRG RDPEMGGKPR RKNPQEGLFN ELQKDKMAEA FSEIGMKGER RRGKGHDGLF QGLSTATKDT FDALHMQALP PR [SEQ ID NO: 27]

[0150] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide that comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical to SEQ ID NO:27, or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions.

[0151] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:27 is represented as SEQ ID NO:28 and is provided below. agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcc tgttcaatgaactgcagaaagataagatggcggaggccttcagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggccttttccagggtctcagtacagccaccaaggacaccttcgacgcccttcacatgcaggccctgccccctcgc [SEQ ID NO: 28]

[0152] In some embodiments, the CAR is a second generation CAR. In some embodiments, the intracellular signaling domain of the CAR further comprises at least a costimulatory signaling domain. In some embodiments, the costimulatory signaling region comprises the intracellular domain of at least one costimulatory molecule or a fragment thereof.

[0153] As used herein, a "costimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand that can provide an efficient response of lymphocytes to antigens. In certain embodiments, a costimulatory molecule can provide optimal lymphocyte activation. Non-limiting examples of costimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, and NKGD2. A costimulatory molecule can bind to a costimulatory ligand. A costimulatory ligand is a protein expressed on the cell surface that, upon binding to its receptor, generates a costimulatory response, i.e., an intracellular response that results in the stimulation provided when a chimeric receptor (e.g., a chimeric antigen receptor (CAR)) binds to a target antigen. As an example, 4-1BB ligand (i.e., 4-1BBL) is used in combination with a CAR signal to activate the CAR. + It can bind 4-1BB to provide an intracellular signal that induces effector cell function of a T cell.

[0154] In an embodiment, the intracellular signaling domain of the CAR comprises a CD28 polypeptide, e.g., a costimulatory signaling region comprising the intracellular domain of CD28 or a fragment thereof. In an embodiment, CD28 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence represented by SEQ ID NO: 10, or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In an embodiment, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 10 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 220 amino acids in length. In certain embodiments, the CD28 polypeptide comprises, or consists of, amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, 180-220, or 200-220 of SEQ ID NO: 10. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide comprising, or consisting of, amino acids 180-220 of SEQ ID NO: 10.

[0155] An exemplary nucleic acid sequence encoding amino acids 180-220 of SEQ ID NO:10 is represented as SEQ ID NO:29 and is provided below. Aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcc [SEQ ID NO: 29]

[0156] In certain embodiments, CD28 comprises or consists of an amino acid sequence or a fragment thereof that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to 218 amino acids in length of SEQ ID NO: 12. In certain embodiments, CD28 polypeptide comprises or consists of amino acids 1-218, 1-50, 50-100, 100-150, 150-218, 178-218, or 200-218 of SEQ ID NO: 12. In one embodiment, the costimulatory signaling region of a CAR of the disclosure comprises a CD28 polypeptide comprising or consisting of amino acids 178-218 of SEQ ID NO:12.

[0157] In an embodiment, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a 4-1BB polypeptide, e.g., the intracellular domain of 4-1BB or a fragment thereof (e.g., the intracellular domain of human 4-1BB or a fragment thereof). 4-1BB may comprise or consist of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence having NCBI reference number NP_001552 (SEQ ID NO:30), or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the 4-1BB polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO:30 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 100, or at least about 150, or at least about 150 and up to about 255 amino acids in length. In certain embodiments, the 4-1BB polypeptide comprises or consists of amino acids 1-255, 1-50, 50-100, 100-150, 150-200, 200-255, or 214-255 of SEQ ID NO:30. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region that comprises a 4-1BB polypeptide that comprises or consists of amino acids 214-255 of SEQ ID NO:30. SEQ ID NO:30 is provided below. 1 MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR 61 TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC 121 CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE 181 PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG 241 CSCRFPEEEE GGCEL [SEQ ID NO: 30]

[0158] An exemplary nucleic acid sequence encoding amino acids 214-255 of SEQ ID NO:30 is represented as SEQ ID NO:31 and is provided below. aaacggggcagaaagaagctcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg [SEQ ID NO: 31]

[0159] In one embodiment, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domains or portions thereof of two or more costimulatory molecules, for example, the intracellular domain of CD28 or a fragment thereof and the intracellular domain of 4-1BB or a fragment thereof, or the intracellular domain of CD28 or a fragment thereof and the intracellular domain of OX40 or a fragment thereof.

[0160] 5.2.2. Chimeric Costimulatory Receptors (CCRs) In an embodiment, the chimeric receptor is a CCR. The CCR of the present disclosure binds to an antigen (e.g., ADGRE2 or CLEC12A) and provides a costimulatory signal, but does not provide an activation signal by itself. In an embodiment, the CCR does not include a CD3ζ polypeptide. The CCR provides costimulation, e.g., a CD28-like signal, in the absence of natural costimulatory ligands on antigen-presenting cells. Combination antigen recognition, i.e., the use of CCRs in combination with CARs, can enhance T cell responsiveness to dual antigen-expressing T cells, thereby improving selective tumor targeting. Kloss et al. describe a strategy that integrates combination antigen recognition, split signaling, and critically balanced T cell activation and costimulatory strength to generate T cells that eliminate target cells expressing a combination of antigens while sparing cells that express each antigen individually (Kloss et al., Nature Biotechnology (2013);31(1):71-75, the entire contents of which are incorporated by reference). In this approach, T cell activation requires CAR-mediated recognition of one antigen, while costimulation is independently mediated by a CCR specific for a second antigen. To achieve tumor selectivity, the combinatorial antigen recognition approach reduces the efficiency of T cell activation to a level that would be ineffective without rescue by simultaneous CCR recognition of the second antigen.

[0161] In some embodiments, the CCR comprises an extracellular antigen-binding domain that binds to an antigen (e.g., ADGRE2 or CLEC12A), a transmembrane domain, and a costimulatory signaling region that comprises an intracellular domain of at least one costimulatory molecule or a fragment thereof. In some embodiments, the CCR does not alone deliver an activation signal to an immunoresponsive cell. Non-limiting examples of costimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, and NKGD2. In some embodiments, the costimulatory signaling region of the CCR comprises an intracellular domain of a costimulatory signaling molecule or a fragment thereof. In some embodiments, one costimulatory signaling molecule is CD28. In some embodiments, one costimulatory signaling molecule is 4-1BB. In some embodiments, the costimulatory signaling region of the CCR comprises an intracellular domain of a first costimulatory signaling molecule or a fragment thereof and an intracellular domain of a second costimulatory signaling molecule or a fragment thereof. In some embodiments, the first costimulatory signaling molecule and the second costimulatory signaling molecule are CD28 and 4-1BB.

[0162] Like CARs, the extracellular antigen-binding domains of CCRs are expressed as scFv, Fab, or F(ab) 2 , or a fusion protein with a heterologous sequence that forms the extracellular antigen-binding domain of the CCR.

[0163] 5.2.3. TCR-like fusion molecules In one embodiment, the chimeric receptor is a TCR-like fusion molecule. Non-limiting examples of TCR fusion molecules include HLA-independent TCR-based chimeric antigen receptors (also known as "HIT-CARs" and disclosed, for example, in International Patent Application No. PCT / US19 / 017525, which is incorporated by reference in its entirety), T cell receptor fusion constructs (TRuCs) (for example, those disclosed in Baeuerle et al., "Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response," Nature Communications volume 10, Article number: 2087 (2019), which is incorporated by reference in its entirety), synthetic T cell receptors and antigen receptors (STARs) (for example, those disclosed in Liu et al. Science Translational Medicine (2021);13(586):eabb5191, which is incorporated by reference in its entirety), antibody-T cell receptors (AbTCRs) (for example, those disclosed in Xu et al. Cell Discovery (2018), which is incorporated by reference in its entirety). 4:62), as well as T cell antigen couplers (TACs) (e.g., those disclosed in Helsen et al. Nature Communications (2018);9:3049, incorporated by reference in its entirety).

[0164] In some embodiments, the TCR-like fusion molecule comprises an antigen-binding chain comprising an extracellular antigen-binding domain and a constant domain, and the TCR-like fusion molecule binds to an antigen in an HLA-independent manner. In some embodiments, the constant domain comprises a T cell receptor constant region selected from the group consisting of a natural or modified TRAC polypeptide, a natural or modified TRBC polypeptide, a natural or modified TRDC polypeptide, a natural or modified TRGC polypeptide, and any variant or functional fragment thereof. In some embodiments, the constant domain comprises a natural or modified TRAC polypeptide. In some embodiments, the constant domain comprises a natural or modified TRBC polypeptide. In some embodiments, the constant domain can form a homodimer or heterodimer with another constant domain. In some embodiments, the antigen-binding chain can bind to a CD3ζ polypeptide. In some embodiments, the antigen-binding chain can activate a CD3ζ polypeptide that binds to the antigen-binding chain upon binding to an antigen (e.g., ADGRE2 or CLEC12A). In some embodiments, activation of the CD3ζ polypeptide can activate an immunoresponsive cell. In an embodiment, the TCR-like fusion molecule can integrate with the CD3 complex to provide HLA-independent antigen recognition. In an embodiment, the TCR-like fusion molecule replaces the endogenous TCR in the CD3 / TCR complex. In an embodiment, the extracellular antigen-binding domain of the TCR-like fusion molecule can dimerize with another extracellular antigen-binding domain. In an embodiment, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises a ligand for a cell surface receptor, a receptor for a cell surface ligand, an antigen-binding portion of an antibody or fragment thereof, or an antigen-binding portion of a TCR. In an embodiment, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises one or two immunoglobulin variable region(s). In an embodiment, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises a heavy chain variable region (V) of an antibody. H In one embodiment, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises the light chain variable region (V LIn some embodiments, the extracellular antigen binding domain of the TCR-like fusion molecule is capable of dimerizing with another extracellular antigen binding domain. In some embodiments, the extracellular antigen binding domain of the TCR-like fusion molecule is capable of dimerizing with another extracellular antigen binding domain. H Including V H is the V of the antibody L In one embodiment, the extracellular antigen-binding domain of the TCR-like fusion molecule can be dimerized with another extracellular antigen-binding domain comprising the V of an antibody to form a fragment variable (Fv). L Including V L is the V of the antibody H It can dimerize with another extracellular antigen-binding domain comprising the fragment variable (Fv).

[0165] 5.3. Extracellular Antigen-Binding Domains of ADGRE2-Targeted Chimeric Receptors In certain embodiments, the chimeric receptor of the present disclosure targets ADGRE2. In certain embodiments, the chimeric receptor of the present disclosure comprises an extracellular antigen-binding domain that binds to ADGRE2.

[0166] Adhesion G protein-coupled receptor E2 (ADGRE2), also known as EMR2, CD312, VBU, or CD97, is a member of the adhesion GPCR family. It is expressed by monocytes / macrophages, dendritic cells, and all types of granulocytes. ADGRE2 is a cell surface receptor that binds to chondroitin sulfate moieties of glycosaminoglycan chains and promotes cell adhesion. It promotes chemotaxis, degranulation, and adhesion of granulocytes. In macrophages, ADGRE2 promotes the release of inflammatory cytokines, including IL8 and TNF. The signal is transmitted mostly through G proteins.

[0167] In some embodiments, the chimeric receptor of the present disclosure targets human ADGRE2. In some embodiments, the chimeric receptor of the present disclosure comprises an extracellular antigen-binding domain that binds to human ADGRE2. In some embodiments, human ADGRE2 comprises or consists of the amino acid sequence having Uniprot reference number Q9UHX3-1 (SEQ ID NO: 32), or a fragment thereof. SEQ ID NO: 32 is provided below. MGGRVFLVFL AFCVWLTLPG AETQDSRGCA RWCPQDSSCV NATACRCNPG FSSFSEIITT PMETCDDINE CATLSKVSCG KFSDCWNTEG SYDCVCSPGY EPVSGAKTFK NESENTCQDV DECQQNPRLC KSYGTCVNTL GSYTCQCLPG FKLKPEDPKL CTDVNECTSG QNPCHSSTHC LNNVGSYQCR CRPGWQPIPG SPNGPNNTVC EDVDECSSGQ HQCDSSTVCF NTVGSYSCRC RPGWKPRHGI PNNQKDTVCE DMTFSTWTPP PGVHSQTLSR FFDKVQDLGR DYKPGLANNT IQSILQALDE LLEAPGDLET LPRLQQHCVA SHLLDGLEDV LRGLSKNLSN GLLNFSYPAG TELSLEVQKQ VDRSVTLRQN QAVMQLDWNQ AQKSGDPGPS VVGLVSIPGM GKLLAEAPLV LEPEKQMLLH ETHQGLLQDG SPILLSDVIS AFLSNNDTQN LSSPVTFTFS HRSVIPRQKV LCVFWEHGQN GCGHWATTGC STIGTRDTST ICRCTHLSSF AVLMAHYDVQ EEDPVLTVIT YMGLSVSLLC LLLAALTFLL CKAIQNTSTS LHLQLSLCLF LAHLLFLVAI DQTGHKVLCS IIAGTLHYLY LATLTWMLLE ALYLFLTARN LTVVNYSSIN RFMKKLMFPV GYGVPAVTVA ISAASRPHLY GTPSRCWLQP EKGFIWGFLG PVCAIFSVNL VLFLVTLWIL KNRLSSLNSE VSTLRNTRML AFKATAQLFI LGCTWCLGIL QVGPAARVMA YLFTIINSLQ GVFIFLVYCL LSQQVREQYG KWSKGIRKLK TESEMHTLSS SAKADTSKPS TVN [SEQ ID NO:32]

[0168] Human ADGRE2 comprises an EGF-like 1 domain, an EGF-like 2 domain, an EGF-like 3 domain, an EGF-like 4 domain, an EGF-like 5 domain, and a GPS domain. In one embodiment, the EGF-like 1 domain comprises or consists of amino acids 25-66 of SEQ ID NO: 32. In one embodiment, the EGF-like 2 domain comprises or consists of amino acids 67-118 of SEQ ID NO: 32. In one embodiment, the EGF-like 3 domain comprises or consists of amino acids 119-162 of SEQ ID NO: 32. In one embodiment, the EGF-like 4 domain comprises or consists of amino acids 163-211 of SEQ ID NO: 32. In one embodiment, the EGF-like 5 domain comprises or consists of amino acids 212-260 of SEQ ID NO: 32. In one embodiment, the GPS domain comprises or consists of amino acids 479-529 of SEQ ID NO: 32.

[0169] In one embodiment, the chimeric receptor of the present disclosure targets an ADGRE2 polypeptide comprising or consisting of an amino acid sequence or a fragment thereof that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical to the amino acid sequence represented by SEQ ID NO:32.

[0170] In some embodiments, the extracellular antigen binding domain of the ADGRE2-targeted chimeric receptor of the present disclosure binds to the stalk region of ADGRE2. In some embodiments, the extracellular antigen binding domain of the ADGRE2-targeted chimeric receptor of the present disclosure binds to the GPS domain of ADGRE2. In some embodiments, the extracellular antigen binding domain of the ADGRE2-targeted chimeric receptor of the present disclosure binds to the EGF-like 5 domain of ADGRE2.

[0171] In some embodiments, the ADGRE2-targeted chimeric receptor is a chimeric antigen receptor (CAR). In some embodiments, the ADGRE2-targeted CAR has a structure disclosed in Section 5.2.1. In some embodiments, the ADGRE2-targeted CAR comprises an extracellular antigen-binding domain that binds to ADGRE2, a transmembrane domain, and an intracellular signaling domain.

[0172] In some embodiments, the ADGRE2-targeted chimeric receptor is a chimeric costimulatory receptor (CCR). In some embodiments, the ADGRE2-targeted CCR has a structure disclosed in Section 5.2.2. In some embodiments, the ADGRE2-targeted CCR comprises an extracellular antigen-binding domain that binds to ADGRE2, a transmembrane domain, and an intracellular signaling domain that does not provide an activation signal to an immunoresponsive cell, e.g., the intracellular signaling domain does not comprise a CD3 zeta polypeptide.

[0173] In one embodiment, the ADGRE2 targeted chimeric receptor is a TCR-like fusion molecule. In one embodiment, the ADGRE2 targeted TCR-like fusion molecule has a structure disclosed in Section 5.2.3.

[0174] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises at least about 1×10 -6 M, at least about 1 × 10 -7 M, at least about 1 × 10 -8 M, at least about 1 × 10 -9 M, or at least about 1 × 10 -10 Dissociation constant of M (KD ) to ADGRE2 (e.g., human ADGRE2). In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) has a molecular weight of about 2×10 -8 Dissociation constant of M (K D In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) has a molecular weight of about 2×10 -8 M ~ approx. 8×10 -9 Dissociation constant of M (K D ) binds to ADGRE2 (e.g., human ADGRE2).

[0175] In certain embodiments, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) has a dissociation constant (K D In certain embodiments, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) binds to ADGRE2 (e.g., human ADGRE2) with a dissociation constant (K) of at least about 50 nM, at least about 40 nM, at least about 35 nM, at least about 30 nM, at least about 25 nM, at least about 20 nM, at least about 19 nM, at least about 18 nM, at least about 17 nM, at least about 16 nM, at least about 15 nM, at least about 14 nM, at least about 13 nM, at least about 12 nM, at least about 11 nM, at least about 10 nM, at least about 9 nM, at least about 8 nM, at least about 7 nM, at least about 6 nM, or at least about 5 nM. D ) binds to ADGRE2 (e.g., human ADGRE2).

[0176] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 33, or a conservative modification thereof. HCDR1, a V having the amino acid sequence represented by SEQ ID NO:34 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 35 or a conservative modification thereof H SEQ ID NOs: 33 to 35 are shown in Table 1.

[0177] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFG comprising the amino acid sequence set forth in SEQ ID NO: 36, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO:37 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof L SEQ ID NOs: 36 to 38 are shown in Table 1.

[0178] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 33, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO:34 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO:35 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO:36 or a conservative modification thereof L CDR1, a V having the amino acid sequence represented by SEQ ID NO:37 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof L Includes CDR3.

[0179] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence represented by SEQ ID NO:33. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:34 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:35 HCDR3, V comprising the amino acid sequence represented by SEQ ID NO:36 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:37 L CDR2 and V comprising the amino acid sequence represented by SEQ ID NO:38 L Includes CDR3.

[0180] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:39. H For example, the extracellular antigen binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) can comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:39. H In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a V H SEQ ID NO:39 is shown in Table 1 below.

[0181] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:40. LFor example, the extracellular antigen binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) can include a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:40. L In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a V L SEQ ID NO: 40 is shown in Table 1 below.

[0182] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence represented by SEQ ID NO:39. H and V comprising the amino acid sequence represented by SEQ ID NO:40. L In one embodiment, V H and V L are linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149.

[0183] In one embodiment, the variable region within the extracellular antigen binding domain of the ADGRE2-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V LIn one embodiment, the ADGRE2-targeting scFv comprises or consists of the amino acid sequence represented by SEQ ID NO:41. In one embodiment, the ADGRE2-targeting scFv is represented as "ADGRE2-A." An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:41 is represented by SEQ ID NO:42. SEQ ID NOs:41 and 42 are set forth in Table 1. The CDRs set forth in Table 1 are identified according to the IMGT numbering system. [Table 1-1] [Table 1-2] [Table 1-3]

[0184] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 33, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO:34 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 35 or a conservative modification thereof H SEQ ID NOs: 33 to 35 are shown in Tables 1 and 2.

[0185] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFG comprising the amino acid sequence set forth in SEQ ID NO: 36, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO:37 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof L SEQ ID NOs: 36 to 38 are shown in Tables 1 and 2.

[0186] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 33, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO:34 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO:35 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO:36 or a conservative modification thereof L CDR1, a V having the amino acid sequence represented by SEQ ID NO:37 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof L Includes CDR3.

[0187] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence represented by SEQ ID NO:33. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:34 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:35 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:36 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:37 L CDR2 and V comprising the amino acid sequence represented by SEQ ID NO:38 L Includes CDR3.

[0188] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:43. HFor example, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) can include a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:43. H In one embodiment, the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor comprises a V H SEQ ID NO:43 is shown in Table 2 below.

[0189] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:44. L For example, the extracellular antigen-binding domain of a chimeric receptor (e.g., scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:44. L In one embodiment, the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor comprises a V L SEQ ID NO:44 is shown in Table 2 below.

[0190] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence represented by SEQ ID NO:43. H and V comprising the amino acid sequence represented by SEQ ID NO:44. L In one embodiment, V H and V Lare linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149.

[0191] In one embodiment, the variable region within the extracellular antigen binding domain of the ADGRE2-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L In one embodiment, the ADGRE2-targeting scFv comprises or consists of the amino acid sequence represented by SEQ ID NO:45, as provided in Table 2. In one embodiment, the ADGRE2-targeting scFv is designated "ADGRE2-B." The CDRs shown in Table 2 are identified according to the IMGT numbering system. [Table 2]

[0192] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 33, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO: 345 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 35 or a conservative modification thereof H SEQ ID NOs: 33 to 35 are shown in Tables 1 to 3.

[0193] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFG comprising the amino acid sequence set forth in SEQ ID NO: 36, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO:37 or a conservative modification thereof LCDR2, and a V having the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof L SEQ ID NOs: 36 to 38 are shown in Tables 1 to 3.

[0194] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 33, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO:34 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO:35 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO:36 or a conservative modification thereof L CDR1, a V having the amino acid sequence represented by SEQ ID NO:37 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof L Includes CDR3.

[0195] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence represented by SEQ ID NO:33. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:34 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:35 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:36 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:37 L CDR2 and V comprising the amino acid sequence represented by SEQ ID NO:38 L Includes CDR3.

[0196] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:46. HFor example, the extracellular antigen-binding domain of a chimeric receptor (e.g., scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:46. H In one embodiment, the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor comprises a V H SEQ ID NO: 46 is shown in Table 3 below.

[0197] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:47. L For example, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) can include a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:47. L In one embodiment, the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor comprises a V L SEQ ID NO:47 is shown in Table 3 below.

[0198] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence represented by SEQ ID NO:46. H and V comprising the amino acid sequence represented by SEQ ID NO:47. L In one embodiment, V H and V Lare linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149.

[0199] In one embodiment, the variable region within the extracellular antigen binding domain of the ADGRE2-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L In one embodiment, the ADGRE2-targeting scFv comprises or consists of the amino acid sequence represented by SEQ ID NO:48 as provided in Table 3. In one embodiment, the ADGRE2-targeting scFv is represented as "ADGRE2-C." The CDRs shown in Table 3 are identified according to the IMGT numbering system. [Table 3]

[0200] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 33, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO:34 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 35 or a conservative modification thereof H SEQ ID NOs: 33 to 35 are shown in Tables 1 to 4.

[0201] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFG comprising the amino acid sequence set forth in SEQ ID NO: 36, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO:37 or a conservative modification thereof LCDR2, and a V having the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof L SEQ ID NOs: 36 to 38 are shown in Tables 1 to 4.

[0202] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 33, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO:34 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO:35 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO:36 or a conservative modification thereof L CDR1, a V having the amino acid sequence represented by SEQ ID NO:37 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof L Includes CDR3.

[0203] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence represented by SEQ ID NO:33. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:34 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:35 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:36 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:37 L CDR2 and V comprising the amino acid sequence represented by SEQ ID NO:38 L Includes CDR3.

[0204] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:49. HFor example, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) can include a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:49. H In one embodiment, the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor comprises a V H SEQ ID NO:49 is shown in Table 4 below.

[0205] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:50. L For example, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) can comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:50. L In one embodiment, the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor comprises a V L SEQ ID NO:50 is shown in Table 4 below.

[0206] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence represented by SEQ ID NO:49. H and V comprising the amino acid sequence represented by SEQ ID NO:50. L In one embodiment, V H and VL are linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2.

[0207] In one embodiment, the variable region within the extracellular antigen binding domain of the ADGRE2-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L In one embodiment, the ADGRE2-targeting scFv comprises or consists of the amino acid sequence represented by SEQ ID NO:51, as provided in Table 4. In one embodiment, the ADGRE2-targeting scFv is designated "ADGRE2-D." The CDRs shown in Table 4 are identified according to the IMGT numbering system. [Table 4]

[0208] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 33, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO:34 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 35 or a conservative modification thereof H SEQ ID NOs: 34 to 36 are shown in Tables 1 to 5.

[0209] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFG comprising the amino acid sequence set forth in SEQ ID NO: 36, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO:37 or a conservative modification thereof LCDR2, and a V having the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof L SEQ ID NOs: 36 to 38 are shown in Tables 1 to 5.

[0210] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 33, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO:34 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO:5 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO:36 or a conservative modification thereof L CDR1, a V having the amino acid sequence represented by SEQ ID NO:37 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof L Includes CDR3.

[0211] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence represented by SEQ ID NO:33. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:34 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:35 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:36 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:37 L CDR2 and V comprising the amino acid sequence represented by SEQ ID NO:38 L Includes CDR3.

[0212] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:52. HFor example, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) can include a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:52. H In one embodiment, the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor comprises a V H SEQ ID NO:52 is shown in Table 5 below.

[0213] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:53. L For example, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) can include a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:53. L In one embodiment, the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor comprises a V L SEQ ID NO:53 is shown in Table 5 below.

[0214] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence represented by SEQ ID NO:52. H and V comprising the amino acid sequence represented by SEQ ID NO:53. L In one embodiment, V H and VL are linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2.

[0215] In one embodiment, the variable region within the extracellular antigen binding domain of the ADGRE2-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L In one embodiment, the ADGRE2-targeting scFv comprises or consists of the amino acid sequence represented by SEQ ID NO:54, as provided in Table 5. In one embodiment, the ADGRE2-targeting scFv is represented as "ADGRE2-E." The CDRs shown in Table 5 are identified according to the IMGT numbering system. [Table 5]

[0216] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 33, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO:34 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 35 or a conservative modification thereof H SEQ ID NOs: 33 to 35 are shown in Tables 1 to 6.

[0217] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFG comprising the amino acid sequence set forth in SEQ ID NO: 36, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO:37 or a conservative modification thereof LCDR2, and a V having the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof L SEQ ID NOs: 36 to 38 are shown in Tables 1 to 6.

[0218] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 33, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO:34 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO:35 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO:36 or a conservative modification thereof L CDR1, a V having the amino acid sequence represented by SEQ ID NO:37 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof L Includes CDR3.

[0219] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence represented by SEQ ID NO:33. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:34 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:35 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:36 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:37 L CDR2 and V comprising the amino acid sequence represented by SEQ ID NO:38 L Includes CDR3.

[0220] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:55. HFor example, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) can include a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:55. H In one embodiment, the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor comprises a V H SEQ ID NO:55 is shown in Table 6 below.

[0221] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:56. L For example, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) can include a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:56. L In one embodiment, the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor comprises a V L SEQ ID NO:56 is shown in Table 6 below.

[0222] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence represented by SEQ ID NO:55. H and V comprising the amino acid sequence represented by SEQ ID NO:56. L In one embodiment, V H and VL are linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149.

[0223] In one embodiment, the variable region within the extracellular antigen binding domain of the ADGRE2-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L In one embodiment, the ADGRE2-targeting scFv comprises or consists of the amino acid sequence represented by SEQ ID NO:57, as provided in Table 6. In one embodiment, the ADGRE2-targeting scFv is designated "ADGRE2-F." The CDRs shown in Table 6 are identified according to the IMGT numbering system. [Table 6]

[0224] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 33, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO:34 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 35 or a conservative modification thereof H SEQ ID NOs: 33 to 35 are shown in Tables 1 to 7.

[0225] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFG comprising the amino acid sequence set forth in SEQ ID NO: 36, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO:37 or a conservative modification thereof LCDR2, and a V having the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof L SEQ ID NOs: 36 to 38 are shown in Tables 1 to 7.

[0226] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 33, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO:34 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO:35 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO:36 or a conservative modification thereof L CDR1, a V having the amino acid sequence represented by SEQ ID NO:37 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 38 or a conservative modification thereof L Includes CDR3.

[0227] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence represented by SEQ ID NO:33. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:34 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:35 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:36 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:37 L CDR2 and V comprising the amino acid sequence represented by SEQ ID NO:38 L Includes CDR3.

[0228] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:146. HFor example, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) can include a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:146. H In one embodiment, the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor comprises a V H SEQ ID NO: 146 is shown in Table 7 below.

[0229] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:147. L For example, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) can include a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:147. L In one embodiment, the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor comprises a V L SEQ ID NO: 147 is shown in Table 7 below.

[0230] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor (e.g., an ADGRE2-targeted scFv) comprises an amino acid sequence represented by SEQ ID NO:146. H and V comprising the amino acid sequence represented by SEQ ID NO: 147. L In one embodiment, VH and V L are linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149.

[0231] In one embodiment, the variable region within the extracellular antigen binding domain of the ADGRE2-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L In one embodiment, the ADGRE2 targeting scFv comprises or consists of the amino acid sequence represented by SEQ ID NO: 148 as provided in Table 7. In one embodiment, the ADGRE2 targeting scFv is represented as "ADGRE2-G". The CDRs shown in Table 7 are identified according to the IMGT numbering system. [Table 7]

[0232] A V sequence that comprises or consists of at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to a particular sequence (e.g., SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:146, or SEQ ID NO:147). H and / or V LThe amino acid sequence may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the particular sequence(s), but retain the ability to bind to the target antigen (e.g., ADGRE2). In some embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in the particular sequence (e.g., SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:146, or SEQ ID NO:147). In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs of the extracellular antigen-binding domain (e.g., within the FRs). In certain embodiments, the extracellular antigen binding domain is a V selected from SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:146, or SEQ ID NO:147, including post-translational modifications of the sequence (SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:146, or SEQ ID NO:147). H and / or V L Contains arrays.

[0233] In one embodiment, the extracellular antigen binding domain of an ADGRE2-targeted chimeric receptor of the present disclosure has, for binding to ADGRE2, a V H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:34 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:35 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:36 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:37 L CDR2 and V comprising the amino acid sequence represented by SEQ ID NO:38 LIn one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor of the present disclosure cross-competes with a reference antibody or an antigen-binding portion thereof comprising the CDR3. H Sequence and V L It cross-competes with a reference antibody or antigen-binding portion thereof comprising the sequence.

[0234] In one embodiment, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor of the present disclosure comprises a V H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:34 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:35 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:36 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:37 L CDR2 and V comprising the amino acid sequence represented by SEQ ID NO:38 L It binds to the same or substantially the same epitope region on ADGRE2 as a reference antibody or antigen-binding portion thereof, including CDR3. In some embodiments, the extracellular antigen-binding domain of an ADGRE2-targeted chimeric receptor of the present disclosure is, for example, the V of any one of the scFvs of the present disclosure (e.g., ADGRE2-A, ADGRE2-B, ADGRE2-C, ADGRE2-D, ADGRE2-E, ADGRE2-F, and ADGRE2-G). H Sequence and V L It binds to the same or substantially the same epitope region on ADGRE2 as a reference antibody or antigen-binding portion thereof comprising the sequence.

[0235] The extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor of the present disclosure that cross-competes or competes with the reference antibody or antigen-binding portion thereof for binding to ADGRE2 can be identified by using routine methods known in the art, including, but not limited to, ELISA, radioimmunoassay (RIA), Biacore, flow cytometry, Western blotting, and any other suitable quantitative or qualitative antibody binding assay. Competitive ELISA is described in Morris, "Epitope Mapping of Protein Antigens by Competition ELISA", The Protein Protocols Handbook (1996), pp 595-600, edited by J. Walker, which is incorporated by reference in its entirety. In an embodiment, the antibody binding assay includes measuring the initial binding of the reference antibody to ADGRE2, mixing the reference antibody with the test extracellular antigen-binding domain, measuring the secondary binding of the reference antibody to ADGRE2 in the presence of the test extracellular antigen-binding domain, and comparing the initial binding and secondary binding of the reference antibody. A decrease in secondary binding of the reference antibody to ADGRE2 compared to the initial binding indicates that the test extracellular antigen-binding domain cross-competes with the reference antibody, e.g., an antibody that recognizes the same or substantially the same epitope, an overlapping epitope, or an adjacent epitope, for binding to ADGRE2. In some embodiments, the reference antibody is labeled, e.g., with a fluorescent dye, biotin, or peroxidase. In some embodiments, ADGRE2 is expressed intracellularly, e.g., in a flow cytometry test. In some embodiments, ADGRE2 is immobilized on a surface, including a Biacore ship (e.g., in a Biacore test) or other medium suitable for surface plasmon resonance analysis. Binding of the reference antibody in the presence of a completely unrelated antibody (that does not bind to ADGRE2) can serve as a high control. A low control can be obtained by incubating the labeled reference antibody with an unlabeled reference antibody, which will result in competition and decreased binding of the labeled reference antibody.In certain embodiments, a test extracellular antigen-binding domain that reduces binding of the reference antibody to ADGRE2 by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% is considered to be an extracellular antigen-binding domain that cross-competes with the reference antibody for binding to ADGRE2. In certain embodiments, the assay is performed at room temperature.

[0236] In some embodiments, the antibody binding assay includes measuring the initial binding of the test extracellular antigen binding domain to ADGRE2, mixing the test extracellular antigen binding domain with a reference antibody, measuring the secondary binding of the test extracellular antigen binding domain to ADGRE2 in the presence of the reference antibody, and comparing the initial binding and secondary binding of the test extracellular antigen binding domain. Wherein, a decrease in the secondary binding of the test extracellular antigen binding domain to ADGRE2 compared to the initial binding indicates that the test extracellular antigen binding domain cross-competes with the reference antibody, e.g., an antibody that recognizes the same or substantially the same epitope, an overlapping epitope, or an adjacent epitope, for binding to ADGRE2. In some embodiments, the test extracellular antigen binding domain is labeled, e.g., with a fluorescent dye, biotin, or peroxidase. In some embodiments, ADGRE2 is expressed intracellularly, e.g., in a flow cytometry assay. In some embodiments, ADGRE2 is immobilized on a surface that includes a Biacore ship (e.g., in a Biacore assay) or other medium suitable for surface plasmon resonance analysis. Binding of the test extracellular antigen binding domain in the presence of a completely unrelated antibody (that does not bind ADGRE2) can serve as a high control. A low control can be obtained by incubating a labeled test extracellular antigen binding domain with an unlabeled test extracellular antigen binding domain, which will result in competition and reduced binding of the labeled test extracellular antigen binding domain. In some embodiments, a test extracellular antigen binding domain whose binding to ADGRE2 in the presence of the reference antibody is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 95% is considered to be an extracellular antigen binding domain that cross-competes with the reference antibody for binding to ADGRE2. In some embodiments, the assay is performed at room temperature.

[0237] In one embodiment, the extracellular antigen binding domain of the ADGRE2-targeted chimeric receptor of the present disclosure comprises a linker connecting the heavy chain variable region and the light chain variable region of the extracellular antigen binding domain. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO:1. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO:2. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO:3. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO:4. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO:5. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO:6. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO:149.

[0238] In one embodiment, the variable region within the extracellular antigen binding domain of the ADGRE2-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L will be placed in.

[0239] In one embodiment, the variable region within the extracellular antigen binding domain of the ADGRE2-targeted chimeric receptor comprises a light chain variable region (V L In one embodiment, when the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor is an scFv, the variable regions are arranged in the order of V L -V H will be placed in.

[0240] Additionally, the ADGRE2-targeted chimeric receptor can include a leader or signal peptide that directs the nascent protein to the endoplasmic reticulum. In some embodiments, the leader or signal peptide is located (e.g., covalently linked) to the N-terminus of the extracellular antigen-binding domain of the ADGRE2-targeted chimeric receptor. A signal peptide or leader may be essential if the chimeric receptor is glycosylated and anchored to the cell membrane. A signal sequence or leader can be a peptide sequence (about 5, about 10, about 15, about 20, about 25, or about 30 amino acids long) present at the N-terminus of a newly synthesized protein that directs entry into the secretory pathway. Non-limiting examples of signal peptides or leader sequences include the IL-2 signal sequence, the CD8 leader sequence, the kappa leader sequence, the albumin leader sequence, and the prolactin leader sequence. In some embodiments, the IL-2 signal sequence comprises or consists of the amino acid sequence represented by SEQ ID NO:58 or SEQ ID NO:59. In some embodiments, the kappa leader sequence comprises or consists of the amino acid sequence represented by SEQ ID NO:60 or SEQ ID NO:61. In one embodiment, the CD8 signal sequence comprises or consists of the amino acid sequence represented by SEQ ID NO:62 or SEQ ID NO:63. In one embodiment, the albumin leader sequence comprises or consists of the amino acid sequence represented by SEQ ID NO:64. In one embodiment, the prolactin leader sequence comprises or consists of the amino acid sequence represented by SEQ ID NO:65. In one embodiment, the ADGRE2 targeted chimeric receptor comprises a signal peptide comprising a CD8 polypeptide. In one embodiment, the ADGRE2 targeted chimeric receptor comprises a signal peptide comprising a CD8 polypeptide comprising or consisting of the amino acid sequence represented by SEQ ID NO:63. MYRMQLLSCIALSLALVTNS [SEQ ID NO:58] MYSMQLASCVTLTLVLLVNS [SEQ ID NO:59] METPAQLLFLLLLWLPDTTG [SEQ ID NO: 60] METDTLLLWVLLLWVPGSTG [SEQ ID NO: 61] MALPVTALLLPLALLLHAARP [SEQ ID NO: 62] MALPVTALLLPLALLLHA [SEQ ID NO: 63] MKWVTFISLLFSSAYS [SEQ ID NO: 64] MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS [SEQ ID NO: 65]

[0241] 5.4. Examples of ADGRE2-targeted chimeric receptors In one embodiment, the ADGRE2-targeted chimeric receptor is an ADGRE2-targeted CAR. In one embodiment, the ADGRE2-targeted CAR comprises: (a)(i) a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 33, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 34, and a VDR comprising the amino acid sequence represented by SEQ ID NO: 35. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 36, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 38. L (b) an extracellular antigen-binding domain comprising a CD28 polypeptide; (c) a transmembrane domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a fragment thereof); and (d) an intracellular signaling domain comprising (i) a CD3ζ polypeptide and (ii) a costimulatory signaling region comprising a CD28 polypeptide (e.g., the intracellular domain of human CD28 or a fragment thereof). H and V L is linked via a linker comprising or consisting of the amino acid sequence represented by SEQ ID NO:2. H and V L is V from N-terminus to C-terminus H -V LIn one embodiment, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 10. In one embodiment, the costimulatory signaling region comprises a CD28 polypeptide comprising or consisting of amino acids 180-220 of SEQ ID NO: 10. In one embodiment, the hinge / spacer region comprises a CD28 polypeptide comprising or consisting of amino acids 114-152 of SEQ ID NO: 10. In one embodiment, the extracellular antigen binding domain and the transmembrane domain are linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 150. SEQ ID NO: 150 is provided below. In one embodiment, the ADGRE2-targeted CAR comprises or consists of the amino acid sequence represented by SEQ ID NO: 66, provided below. QVQLQQSGAEVAKPGASVKLSCKASGYTFTNYWMQWIKQAPGQGLEWIGAVYPGDGDTRHTQKFKGKATLTADKSTSTAYMEVSSLRSEDTAVYYCARGFTAYGMDYWGQGTTVTVS SGGGGSGGGGSGGGGSEIVLTQSPATMSASPGERVTMSCSASSSVSYMHWYQQKSGQSPKRWIYDTSKLASGVPARFSGSGSGTDYTFTISSMEPEDFATYYCQQWSSNPLTFGGGTK LEIKRAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR [SEQ ID NO: 66] RAAA [SEQ ID NO: 150]

[0242] The amino acid sequence of an exemplary nucleic acid sequence, SEQ ID NO:66, is represented in SEQ ID NO:67, provided below.

[0243] In one embodiment, the ADGRE2-targeted chimeric receptor comprises a signal peptide at the N-terminus of the extracellular antigen-binding domain, hi one embodiment, the signal peptide comprises a CD8 polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:63.

[0244] 5.5. Extracellular Antigen-Binding Domain of CLEC12A-Targeted Chimeric Receptors In certain embodiments, the chimeric receptor of the present disclosure targets CLEC12A. In certain embodiments, the chimeric receptor of the present disclosure comprises an extracellular antigen-binding domain that binds to CLEC12A.

[0245] C-type lectin domain family 12 member A (CLEC12A), also known as CLL-1, CLL1, DCAL-2, MICL, and CD371, is a 30 kD C-type lectin transmembrane glycoprotein. It is expressed in monocytes, granulocytes, natural killer (NK) cells, and basophils. CLEC12A is an immunoinhibitory receptor that recruits Src homologous phosphatases SHP-1 and SHP-2 to its phosphorylated cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) (Sancho et al., Annu Rev. Immunol (2012);30:491-529; Yan et al., Front Immunol (2015);6:408; Lahoud et al., J Immunol (2011);187:842). CLEC12A has been implicated as a negative regulatory urate crystal (monosodium urate, MSU) receptor that controls autoimmune and inflammatory diseases (Neumann et al., Immunity (2014);40:389-99). CLEC12A is a negative regulator of granulocyte and monocyte function (Marshall et al., J Biol Chem (2004);279(15):14792-802; Pyz et al., Eur J Immunol (2008);38(4):1157-63).

[0246] In an embodiment, the chimeric receptor of the present disclosure targets human CLEC12A. In an embodiment, the chimeric receptor of the present disclosure comprises an extracellular antigen-binding domain that binds to human CLEC12A. In an embodiment, the human CLEC12A comprises or consists of an amino acid sequence having Uniprot reference number Q5QGZ9-2 (SEQ ID NO: 68), or a fragment thereof. SEQ ID NO: 68 is provided below. MSEEVTYADLQFQNSSEMEKIPEIGKFGEKAPPAPSHVWRPAALFLTLLCLLLLIGLGVLASMFHVTLKIEMKKMNKLQNISEELQRNISLQLMSNMNISNKIRNLSTTLQTIATKLCRELYSKEQEHKCKP CPRRWIWHKDSCYFLSDDVQTWQESKMACAQNASLLKINNKNALEFIKSQSRSYDYWLGLSPEEDSTRGGMRVDNIINSSAWVIRNAPDLNNMYCGYINRLYVQYYHCTYKKRMICEKMANPVQLGSTYFREA [SEQ ID NO:68]

[0247] Human CLEC12A comprises a cytoplasmic domain, a transmembrane domain, and an extracellular domain. In certain embodiments, the cytoplasmic domain comprises or consists of amino acids 1 to 43 of SEQ ID NO: 68. In certain embodiments, the transmembrane domain comprises or consists of amino acids 44 to 64 of SEQ ID NO: 68. In certain embodiments, the extracellular domain comprises or consists of amino acids 65 to 265 of SEQ ID NO: 68.

[0248] In one embodiment, a chimeric receptor of the present disclosure targets a CLECL12A polypeptide comprising or consisting of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical to the amino acid sequence set forth in SEQ ID NO:68, or a fragment thereof.

[0249] In certain embodiments, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor of the present disclosure binds to the extracellular domain of CLEC12A.

[0250] In some embodiments, the CLEC12A-targeted chimeric receptor is a chimeric antigen receptor (CAR). In some embodiments, the CLEC12A-targeted CAR has a structure as disclosed in Section 5.2.1. In some embodiments, the CLEC12A-targeted CAR comprises an extracellular antigen-binding domain that binds to CLEC12A, a transmembrane domain, and an intracellular signaling domain.

[0251] In one embodiment, the CLEC12A-targeting chimeric receptor is a chimeric costimulatory receptor (CCR). In one embodiment, the CLEC12A-targeting CCR has a structure disclosed in Section 5.2.2. In one embodiment, the CLEC12A-targeting CCR comprises an extracellular antigen-binding domain that binds to CLEC12A, a transmembrane domain, and an intracellular signaling domain that does not provide an activation signal to an immunoresponsive cell, e.g., the intracellular signaling domain does not comprise a CD3ζ polypeptide.

[0252] In one embodiment, the CLEC12A targeted chimeric receptor is a TCR-like fusion molecule. In one embodiment, the CLEC12A targeted TCR-like fusion molecule has a structure disclosed in Section 5.2.3.

[0253] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises at least about 1×10 -6 M, at least about 1 × 10 -7 M, at least about 1 × 10 -8 M, at least about 1 × 10 -9 M, or at least about 1 × 10 -10 Dissociation constant of M (K D In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeting chimeric receptor (e.g., a CLEC12A-targeting scFv) binds to CLEC12A (e.g., human CLEC12A) at a concentration of at least about 2×10 -8Dissociation constant of M (K D In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeting chimeric receptor (e.g., a CLEC12A-targeting scFv) has a molecular weight of about 2×10 -8 M ~ approx. 8×10 -9 Dissociation constant of M (K D ) binds to CLEC12A (e.g., human CLEC12A).

[0254] In certain embodiments, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) has a dissociation constant (K D In certain embodiments, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) binds to CLEC12A (e.g., human CLEC12A) with a dissociation constant (K) of at least about 50 nM, at least about 40 nM, at least about 35 nM, at least about 30 nM, at least about 25 nM, at least about 20 nM, at least about 19 nM, at least about 18 nM, at least about 17 nM, at least about 16 nM, at least about 15 nM, at least about 14 nM, at least about 13 nM, at least about 12 nM, at least about 11 nM, at least about 10 nM, at least about 9 nM, at least about 8 nM, at least about 7 nM, at least about 6 nM, at least about 5 nM. D ) binds to CLEC12A (e.g., human CLEC12A).

[0255] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO:69, or a conservative modification thereof. H CDR1, a V having an amino acid sequence represented by SEQ ID NO: 70 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 71 or a conservative modification thereof H SEQ ID NOs: 69 to 71 are shown in Table 8.

[0256] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 72, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO: 73 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 74 or a conservative modification thereof L SEQ ID NOs: 72 to 74 are shown in Table 8.

[0257] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO:69, or a conservative modification thereof. H CDR1, a V having an amino acid sequence represented by SEQ ID NO: 70 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO: 71 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO: 72 or a conservative modification thereof L CDR1, V having the amino acid sequence represented by SEQ ID NO: 73 or conservative modifications L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 74 or a conservative modification thereof L Includes CDR3.

[0258] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO:69. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:70 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:71 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:72 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:73 L CDR2 and V comprising the amino acid sequence represented by SEQ ID NO:74 L Includes CDR3.

[0259] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:75. H For example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:108. H In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V H SEQ ID NO:75 is shown in Table 8 below.

[0260] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:76. L For example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:109. L In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V L SEQ ID NO:76 is shown in Table 8 below.

[0261] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO:75. H and V comprising the amino acid sequence represented by SEQ ID NO:76. L In one embodiment, V H and V L are linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2.

[0262] In one embodiment, the variable region within the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L In one embodiment, the CLEC12A-targeting scFv comprises or consists of the amino acid sequence set forth in SEQ ID NO: 79. In one embodiment, the CLEC12A-targeting scFv is designated "CLEC12A-A." An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 79 is designated SEQ ID NO: 80. SEQ ID NOs: 79 and 80 are set forth in Table 8. The CDRs set forth in Table 8 are identified according to the IMGT numbering system. [Table 8-1] [Table 8-2]

[0263] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 81, or a conservative modification thereof. HCDR1, a V having the amino acid sequence represented by SEQ ID NO: 82 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 83 or a conservative modification thereof H SEQ ID NOs: 81 to 83 are shown in Table 9.

[0264] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 84, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO: 73 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 85 or a conservative modification thereof L SEQ ID NOs: 73, 84, and 85 are shown in Table 9.

[0265] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 81, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO: 82 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO: 83 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO: 84 or a conservative modification thereof L CDR1, V having the amino acid sequence represented by SEQ ID NO: 73 or conservative modifications L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 85 or a conservative modification thereof L Includes CDR3.

[0266] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO:81. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:82 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:83 HCDR3, V comprising the amino acid sequence represented by SEQ ID NO:84 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:73 L CDR2 and V comprising the amino acid sequence represented by SEQ ID NO: 85 L Includes CDR3.

[0267] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:86. H For example, the extracellular antigen-binding domain of a CLEC12A-targeting chimeric receptor (e.g., scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:86. H In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V H SEQ ID NO:86 is shown in Table 9 below.

[0268] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:87. L For example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:87.L In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V L SEQ ID NO:87 is shown in Table 9 below.

[0269] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO:86. H and V comprising the amino acid sequence represented by SEQ ID NO:87. L In one embodiment, V H and V L are linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2.

[0270] In one embodiment, the variable region within the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L In one embodiment, the CLEC12A-targeting scFv comprises or consists of the amino acid sequence represented by SEQ ID NO: 88 as provided in Table 9. In one embodiment, the CLEC12A-targeting scFv is designated "CLEC12A-B". The CDRs shown in Table 9 are identified according to the IMGT numbering system. [Table 9]

[0271] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 89, or a conservative modification thereof. HCDR1, a V having an amino acid sequence represented by SEQ ID NO: 90 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 91 or a conservative modification thereof H SEQ ID NOs: 89 to 91 are shown in Table 10.

[0272] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 92, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO: 93 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 94 or a conservative modification thereof L SEQ ID NOs: 92 to 94 are shown in Table 10.

[0273] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeting chimeric receptor (e.g., a targeting scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 89, or a conservative modification thereof. H CDR1, a V having an amino acid sequence represented by SEQ ID NO: 90 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO: 91 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO: 92 or a conservative modification thereof L CDR1, V having the amino acid sequence represented by SEQ ID NO: 93 or conservative modifications L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 94 or a conservative modification thereof L Includes CDR3.

[0274] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO:89. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:90 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:91 HCDR3, V comprising the amino acid sequence represented by SEQ ID NO:92 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:93 L CDR2 and V comprising the amino acid sequence represented by SEQ ID NO:94 L Includes CDR3.

[0275] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:95. H For example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:95. H In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V H SEQ ID NO:95 is shown in Table 10 below.

[0276] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:96. LFor example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:96. L In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V L SEQ ID NO: 96 is shown in Table 10 below.

[0277] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO:96. H and V comprising the amino acid sequence represented by SEQ ID NO:96. L In one embodiment, V H and V L are linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2.

[0278] In one embodiment, the variable region within the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L In one embodiment, the CLEC12A-targeting scFv comprises the amino acid sequence represented by SEQ ID NO:97 as provided in Table 10. In one embodiment, the CLEC12A-targeting scFv is represented as "CLEC12A-C". The CDRs shown in Table 10 are identified according to the IMGT numbering system. [Table 10]

[0279] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 89, or a conservative modification thereof. H CDR1, a V having an amino acid sequence represented by SEQ ID NO: 90 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 98 or a conservative modification thereof H SEQ ID NOs: 89, 90, and 98 are shown in Table 11.

[0280] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 99, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO: 93 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 151 or a conservative modification thereof L SEQ ID NOs: 93, 99, and 151 are shown in Table 11.

[0281] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 89, or a conservative modification thereof. H CDR1, a V having an amino acid sequence represented by SEQ ID NO: 90 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO: 98 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO: 99 or a conservative modification thereof L CDR1, V having the amino acid sequence represented by SEQ ID NO: 93 or conservative modifications L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 151 or a conservative modification thereof L Includes CDR3.

[0282] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO:89. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:90 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:98 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:99 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:93 L CDR2, and a V L Includes CDR3.

[0283] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:100. H For example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:100. H In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V H SEQ ID NO: 100 is shown in Table 11 below.

[0284] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:101. LFor example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:101. L In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V L SEQ ID NO: 101 is shown in Table 11 below.

[0285] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO: 100. H and V comprising the amino acid sequence represented by SEQ ID NO: 101. L In one embodiment, V H and V L are linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2.

[0286] In one embodiment, the variable region within the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L In one embodiment, the CLEC12A-targeting scFv comprises the amino acid sequence represented by SEQ ID NO: 102 as provided in Table 11. In one embodiment, the CLEC12A-targeting scFv is represented as "CLEC12A-D". The CDRs shown in Table 11 are identified according to the IMGT numbering system. [Table 11]

[0287] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 81, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO: 103 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 83 or a conservative modification thereof H SEQ ID NOs: 81, 83, and 103 are shown in Table 12.

[0288] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 104, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO: 73 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 105 or a conservative modification thereof L SEQ ID NOs: 73, 104, and 105 are shown in Table 12.

[0289] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 81, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO: 103 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO: 83 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO: 104 or a conservative modification thereof L CDR1, V having the amino acid sequence represented by SEQ ID NO: 73 or conservative modifications L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 105 or a conservative modification thereof L Includes CDR3.

[0290] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO:81. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO: 103 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:83 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO: 104 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:73 L CDR2, and a V L Includes CDR3.

[0291] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:106. H For example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:106. H In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V H SEQ ID NO: 106 is shown in Table 12 below.

[0292] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence represented by SEQ ID NO:107. L For example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:107. L In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V L SEQ ID NO: 107 is shown in Table 12 below.

[0293] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO: 106. H and V comprising the amino acid sequence represented by SEQ ID NO: 107. L In one embodiment, V H and V L are linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2.

[0294] In one embodiment, the variable region within the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V LIn one embodiment, the CLEC12A-targeting scFv comprises the amino acid sequence represented by SEQ ID NO: 108 as provided in Table 12. In one embodiment, the CLEC12A-targeting scFv is represented as "CLEC12A-E". The CDRs shown in Table 12 are identified according to the IMGT numbering system. [Table 12]

[0295] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 109, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO: 103 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 83 or a conservative modification thereof H SEQ ID NOs: 83, 103, and 109 are shown in Table 13.

[0296] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 110, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO: 73 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 85 or a conservative modification thereof L SEQ ID NOs: 73, 85, and 110 are shown in Table 13.

[0297] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 109, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO: 103 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO: 83 or a conservative modification thereof HCDR3, a V having the amino acid sequence represented by SEQ ID NO: 110 or a conservative modification thereof L CDR1, V having the amino acid sequence represented by SEQ ID NO: 73 or conservative modifications L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 85 or a conservative modification thereof L Includes CDR3.

[0298] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO: 109. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO: 103 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:83 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:110 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:73 L CDR2 and V comprising the amino acid sequence represented by SEQ ID NO: 85 L Includes CDR3.

[0299] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:111. H For example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:111. H In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V H SEQ ID NO: 111 is shown in Table 13 below.

[0300] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence represented by SEQ ID NO:112. L For example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:112. L In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V L SEQ ID NO: 112 is shown in Table 13 below.

[0301] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO:111. H and V comprising the amino acid sequence represented by SEQ ID NO: 112. L In one embodiment, V H and V L are linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2.

[0302] In one embodiment, the variable region within the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor comprises a heavy chain variable region (V HIn one embodiment, when the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L In one embodiment, the CLEC12A-targeting scFv comprises the amino acid sequence represented by SEQ ID NO: 113 as provided in Table 13. In one embodiment, the CLEC12A-targeting scFv is represented as "CLEC12A-F". The CDRs shown in Table 13 are identified according to the IMGT numbering system. [Table 13]

[0303] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 89, or a conservative modification thereof. H CDR1, a V having an amino acid sequence represented by SEQ ID NO: 90 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 114 or a conservative modification thereof H SEQ ID NOs: 89, 90, and 114 are shown in Table 14.

[0304] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 115, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO: 93 or a conservative modification thereof L CDR2 and a V having the amino acid sequence represented by SEQ ID NO: 116 or a conservative modification thereof L SEQ ID NOs: 93, 115, and 116 are shown in Table 14.

[0305] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 89, or a conservative modification thereof.H CDR1, a V having an amino acid sequence represented by SEQ ID NO: 90 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO: 114 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO: 115 or a conservative modification thereof L CDR1, V having the amino acid sequence represented by SEQ ID NO: 93 or conservative modifications L CDR2 and a V having the amino acid sequence represented by SEQ ID NO: 116 or a conservative modification thereof L Includes CDR3.

[0306] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO:89. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:90 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:114 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:115 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:93 L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 116 L Includes CDR3.

[0307] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:117. H For example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:117.H In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V H SEQ ID NO: 117 is shown in Table 14 below.

[0308] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:118. L For example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:118. L In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V L SEQ ID NO: 118 is shown in Table 14 below.

[0309] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO:117. H and V comprising the amino acid sequence represented by SEQ ID NO: 118. L In one embodiment, V H and V L are linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2.

[0310] In one embodiment, the variable region within the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L In one embodiment, the CLEC12A-targeting scFv comprises the amino acid sequence represented by SEQ ID NO: 119 as provided in Table 14. In one embodiment, the CLEC12A-targeting scFv is represented as "CLEC12A-G". The CDRs shown in Table 14 are identified according to the IMGT numbering system. [Table 14]

[0311] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 120, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO: 121 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 122 or a conservative modification thereof H SEQ ID NOs: 120 to 122 are shown in Table 15.

[0312] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 123, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO: 124 or a conservative modification thereof L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 125 or a conservative modification thereof L SEQ ID NOs: 123 to 125 are shown in Table 15.

[0313] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 120, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO: 121 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO: 122 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO: 123 or a conservative modification thereof L CDR1, V having the amino acid sequence represented by SEQ ID NO: 124 or conservative modifications L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 125 or a conservative modification thereof L Includes CDR3.

[0314] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO: 120. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:121 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:122 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:123 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:124 L CDR2 and a V having the amino acid sequence represented by SEQ ID NO: 125 L Includes CDR3.

[0315] In one embodiment, the extracellular antigen-binding domain of the chimeric receptor (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:126. HFor example, the extracellular antigen-binding domain of the chimeric receptor (e.g., scFv) can comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:126. H In one embodiment, the extracellular antigen-binding domain comprises the V H SEQ ID NO: 126 is shown in Table 15 below.

[0316] In one embodiment, the extracellular antigen-binding domain of the chimeric receptor (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:127. L For example, the extracellular antigen-binding domain of the chimeric receptor (e.g., scFv) can comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:127. L In one embodiment, the extracellular antigen-binding domain comprises the V L SEQ ID NO: 127 is shown in Table 15 below.

[0317] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO: 126. H and V comprising the amino acid sequence represented by SEQ ID NO: 127. L In one embodiment, V H and V Lare linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2.

[0318] In one embodiment, the variable region within the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L In one embodiment, the CLEC12A-targeting scFv comprises the amino acid sequence represented by SEQ ID NO: 128 as provided in Table 15. In one embodiment, the CLEC12A-targeting scFv is represented as "CLEC12A-H". The CDRs shown in Table 15 are identified according to the IMGT numbering system. [Table 15]

[0319] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 129, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO: 130 or a conservative modification thereof H CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 131 or a conservative modification thereof H SEQ ID NOs: 129 to 131 are shown in Table 16.

[0320] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 132, or a conservative modification thereof. L CDR1, a V having the amino acid sequence represented by SEQ ID NO: 133 or a conservative modification thereof LCDR2, and a V having the amino acid sequence represented by SEQ ID NO: 134 or a conservative modification thereof L SEQ ID NOs: 132 to 134 are shown in Table 16.

[0321] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 129, or a conservative modification thereof. H CDR1, a V having the amino acid sequence represented by SEQ ID NO: 130 or a conservative modification thereof H CDR2, a V having the amino acid sequence represented by SEQ ID NO: 131 or a conservative modification thereof H CDR3, a V having the amino acid sequence represented by SEQ ID NO: 132 or a conservative modification thereof L CDR1, V having the amino acid sequence represented by SEQ ID NO: 133 or conservative modifications L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 134 or a conservative modification thereof L Includes CDR3.

[0322] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO: 129. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO: 130 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:131 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO: 132 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO: 133 L CDR2, and a V having the amino acid sequence represented by SEQ ID NO: 134 L Includes CDR3.

[0323] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence represented by SEQ ID NO: 135.H For example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:135. H In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V H SEQ ID NO: 135 is shown in Table 16 below.

[0324] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence represented by SEQ ID NO: 136. L For example, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) may comprise a VF comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO:136. L In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor comprises a V L SEQ ID NO: 136 is shown in Table 16 below.

[0325] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor (e.g., a CLEC12A-targeted scFv) comprises the amino acid sequence represented by SEQ ID NO: 135. H and V comprising the amino acid sequence represented by SEQ ID NO: 136. L In one embodiment, VH and V L are linked via a linker. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 149. In one embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO: 2.

[0326] In one embodiment, the variable region within the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L In one embodiment, the scFv comprises the amino acid sequence represented by SEQ ID NO: 137 as provided in Table 16. In one embodiment, the CLEC12A-targeting scFv is represented as "CLEC12A-J". The CDRs shown in Table 16 are identified according to the IMGT numbering system. [Table 16]

[0327] A V that has at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to a particular sequence (e.g., SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:135, or SEQ ID NO:136). H and / or V LThe amino acid sequence may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the particular sequence(s), but retain the ability to bind to the target antigen (e.g., CLEC12A). In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the particular sequence (e.g., SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:135, or SEQ ID NO:136). In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs of the extracellular antigen-binding domain (e.g., within the FRs). In certain embodiments, the extracellular antigen binding domain is a V selected from SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:135, or SEQ ID NO:136, including post-translational modifications of the sequence (SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:135, or SEQ ID NO:136). H and / or V L Contains arrays.

[0328] In certain embodiments, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor of the present disclosure has, for binding to CLEC12A, a V domain of, for example, any one of the scFvs of the present disclosure (e.g., CLEC12A-A, CLEC12A-B, CLEC12A-C, CLEC12A-D, CLEC12A-E, CLEC12A-F, CLEC12A-G, CLEC12A-H, and CLEC12A-J). H CDR1, CDR2, and CDR3 sequences, and V LIn one embodiment, the extracellular antigen-binding domain of a chimeric receptor of the present disclosure cross-competes with a reference antibody or antigen-binding portion thereof comprising the CDR1, CDR2, and CDR3 sequences. In one embodiment, the extracellular antigen-binding domain of a chimeric receptor of the present disclosure cross-competes with a reference antibody or antigen-binding portion thereof comprising the CDR1, CDR2, and CDR3 sequences for binding to CLEC12A, e.g., the V of any one of the scFvs of the present disclosure (e.g., CLEC12A-A, CLEC12A-B, CLEC12A-C, CLEC12A-D, CLEC12A-E, CLEC12A-F, CLEC12A-G, CLEC12A-H, and CLEC12A-J). H Sequence and V L It cross-competes with a reference antibody or antigen-binding portion thereof comprising the sequence.

[0329] In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor of the present disclosure has, with respect to binding to CLEC12A, the V H CDR1, CDR2, and CDR3 sequences, and V L Cross-competes with a reference antibody or antigen-binding portion thereof comprising the CDR1, CDR2, and CDR3 sequences. For example, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor of the present disclosure comprises a V H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:70 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:71 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:72 L CDR1, V comprising an amino acid sequence represented by SEQ ID NO:73 L CDR2 and V comprising an amino acid sequence represented by SEQ ID NO:74 L In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeting chimeric receptor of the present disclosure cross-competes with a reference antibody or an antigen-binding portion thereof comprising CDR3. H and V LFor example, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor of the present disclosure may comprise a V-seq. H and V comprising an amino acid sequence having the sequence represented by SEQ ID NO:76. L or an antigen-binding portion thereof.

[0330] In certain embodiments, the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor of the present disclosure binds to the same epitope on CLEC12A as a reference antibody or antigen-binding portion thereof. For example ... V of any one of the scFvs of the present disclosure (e.g., CLEC12A-A, CLEC12A-B, CLEC12A-C, CLEC12A-D, CLEC12A-E, CLEC12A-F, CLEC12A-G, CLEC12A-H, and CLEC12A-J). H CDR1, CDR2, and CDR3 sequences, and V L Binds to the same epitope on CLEC12A as a reference antibody or antigen-binding portion thereof comprising CDR1, CDR2, and CDR3 sequences. In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeting chimeric receptor of the present disclosure comprises, for example, the V of any one of the scFvs of the present disclosure (e.g., CLEC12A-A, CLEC12A-B, CLEC12A-C, CLEC12A-D, CLEC12A-E, CLEC12A-F, CLEC12A-G, CLEC12A-H, and CLEC12A-J). H Sequence and V L binds to the same epitope on CLEC12A as a reference antibody or antigen-binding portion thereof that comprises the sequence.

[0331] In one embodiment, the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor of the present disclosure is V H CDR1, CDR2, and CDR3 sequences, and V LBinds to the same epitope on CLEC12A as a reference antibody or antigen-binding fragment thereof comprising CDR1, CDR2, and CDR3 sequences. For example, the extracellular antigen-binding domain of a CLEC12A-targeting chimeric receptor of the present disclosure comprises the amino acid sequence represented by SEQ ID NO:69. H CDR1, V comprising the amino acid sequence represented by SEQ ID NO:70 H CDR2, V comprising the amino acid sequence represented by SEQ ID NO:71 H CDR3, V comprising the amino acid sequence represented by SEQ ID NO:72 L CDR1, V comprising the amino acid sequence represented by SEQ ID NO:73 L CDR2 and V comprising the amino acid sequence represented by SEQ ID NO:74 L Binds to the same epitope on CLEC12A as a reference antibody or antigen-binding fragment thereof comprising CDR3. In one embodiment, the extracellular antigen-binding domain of a CLEC12A-targeting chimeric receptor of the present disclosure is V H Sequence and V L The extracellular antigen-binding domain of a CLEC12A-targeting chimeric receptor of the present disclosure binds to the same or substantially the same epitope on CLEC12A as a reference antibody or antigen-binding fragment thereof comprising the sequence VV ... H and V comprising the amino acid sequence represented by SEQ ID NO:76. L binds to the same epitope on CLEC12A as a reference antibody or antigen-binding fragment thereof comprising:

[0332] Extracellular antigen-binding domains of the CLEC12A-targeted chimeric receptors of the present disclosure that cross-compete or compete with a reference antibody or antigen-binding portion thereof for binding to CLEC12A can be identified by using routine methods known in the art, such as those disclosed in Section 5.3.

[0333] In an embodiment, the extracellular antigen-binding domain of a CLEC12A-targeting chimera of the present disclosure comprises a linker connecting the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. In an embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO:1. In an embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO:2. In an embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO:3. In an embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO:4. In an embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO:5. In an embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO:6. In an embodiment, the linker comprises the amino acid sequence represented by SEQ ID NO:149.

[0334] In one embodiment, the variable region within the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor comprises a heavy chain variable region (V H In one embodiment, when the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor is an scFv, the variable regions are arranged in the order of V H -V L will be placed in.

[0335] In one embodiment, the variable region within the extracellular antigen-binding domain of a CLEC12A-targeted chimeric receptor comprises a light chain variable region (V L In one embodiment, when the extracellular antigen-binding domain of the CLEC12A-targeting chimeric receptor is an scFv, the variable regions are arranged in the order of V L -V H will be placed in.

[0336] Additionally, the CLEC12A-targeted chimeric receptor may comprise a leader or signal peptide that directs the nascent protein to the endoplasmic reticulum. In certain embodiments, the leader or signal peptide is located (e.g., covalently linked) to the N-terminus of the extracellular antigen-binding domain of the CLEC12A-targeted chimeric receptor. In certain embodiments, the CLEC12A-targeted chimeric receptor comprises a leader or signal peptide as disclosed in Section 5.3. In certain embodiments, the CLEC12A-targeted chimeric receptor comprises a signal peptide that comprises a CD8 polypeptide. In certain embodiments, the CLEC12A-targeted chimeric receptor comprises a signal peptide that comprises a CD8 polypeptide that comprises or consists of the amino acid sequence set forth in SEQ ID NO:63.

[0337] 5.6. Examples of CLEC12A-targeted chimeric receptors In one embodiment, the CLEC12A-targeted chimeric receptor is a CCR. In one embodiment, the CLEC12A-targeted CCR comprises (a)(i) a CDR1 comprising the amino acid sequence represented by SEQ ID NO:69, a CDR2 comprising the amino acid sequence represented by SEQ ID NO:70, and a VDR3 comprising the amino acid sequence represented by SEQ ID NO:71. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 72, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and a V comprising the amino acid sequence represented by SEQ ID NO: 74. L V containing CDR3 Land (b) an intracellular domain comprising a 4-1BB polypeptide (e.g., an intracellular domain of a human 4-1BB polypeptide, e.g., a portion thereof, of 4-1BB (e.g., human 4-1BB)). In certain embodiments, the CLEC12A target CCR further comprises a transmembrane domain comprising a CD8 polypeptide (e.g., a human CD8 polypeptide, e.g., a transmembrane domain of CD8 (e.g., human CD8) or a portion thereof). In certain embodiments, the intracellular domain comprises a 4-1BB polypeptide comprising or consisting of amino acids 214-255 of SEQ ID NO:30. In certain embodiments, the transmembrane domain comprises a CD8 polypeptide comprising or consisting of amino acids 137-207 of SEQ ID NO:7. In certain embodiments, the V H and V L is linked via a linker comprising or consisting of the amino acid sequence represented by SEQ ID NO: 149. H and V L is V from N-terminus to C-terminus H -V L In one embodiment, the extracellular antigen-binding domain and the transmembrane domain are linked via a linker. In one embodiment, the linker consists of the amino acid sequence represented by SEQ ID NO: 150. In one embodiment, the CLEC12A-targeting CCR comprises the amino acid sequence represented by SEQ ID NO: 138, as shown below. QLQLQESGPGLVKPSETLSLTCTVSGGSISSSTYYWGWIRQPPRKGLEWIGSTHYRGSTYYNPSLKSRVTISVDTSKNQFSLKVSSVTAADTAVYYCARELTGEVFDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIKRAAAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL [SEQ ID NO: 138]

[0338] The amino acid sequence of an exemplary nucleic acid sequence, SEQ ID NO:138, is represented below as SEQ ID NO:139.

[0339] In one embodiment, the CLEC12A-targeted chimeric receptor comprises a signal peptide at the N-terminus of the extracellular antigen-binding domain, hi one embodiment, the signal peptide comprises a CD8 polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:63.

[0340] 5.7. Cells The presently disclosed subject matter provides cells comprising a chimeric ADGRE2-targeted receptor of the present disclosure (e.g., those disclosed in Sections 5.3 and 5.4). Additionally, the presently disclosed subject matter provides cells comprising a chimeric CLEC12A-targeted receptor of the present disclosure (e.g., those disclosed in Sections 5.5 and 5.6).

[0341] In some embodiments, the cell is selected from the group consisting of a lymphoid cell and a myeloid cell. In some embodiments, the cell is an immunoresponsive cell. In some embodiments, the immunoresponsive cell is a lymphoid cell.

[0342] In some embodiments, the cell is a lymphoid cell. The lymphoid cell can provide antibody production, regulation of the cellular immune system, detection of foreign factors in blood, detection of cells foreign to the host, etc. Non-limiting examples of lymphoid cells include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphoid cells can be differentiated. In some embodiments, the stem cell is a pluripotent stem cell. In some embodiments, the pluripotent stem cell is an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC).

[0343] In an embodiment, the cell is a T cell. T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the subject matter of the present disclosure can be any type of T cell, including but not limited to helper T cells, cytotoxic T cells, memory T cells (including central memory T cells), stem cell-like memory (or stem-like memory T cells), and two types of effector memory T cells (e.g., TEM cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), tumor infiltrating lymphocytes (TILs), natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. A patient's own T cells may be genetically modified to target specific antigens through the introduction of chimeric receptors, such as CARs or CCRs. In an embodiment, the immunoresponsive cells are T cells. T cells are CD4 + T cells or CD8 + In one embodiment, the T cell is a CD4 + In one embodiment, the T cells are CD8 + T cells.

[0344] In some embodiments, the cell is a NK cell. Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during innate immune responses. NK cells do not require prior activation to exert a cytotoxic effect on target cells. In some embodiments, the cell is a genetically modified NK cell. In some embodiments, the cell is an edited NK cell. In some embodiments, the cell is a stem cell-derived NK cell. In some embodiments, the cell is a pluripotent stem cell-derived NK cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC)-derived NK cell.

[0345] Types of human lymphocytes of the presently disclosed subject matter include peripheral donor lymphocytes, e.g., those described in Sadelain et al., Nat Rev Cancer (2003);3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CARs), Morgan, RA, et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express full-length tumor antigen-recognizing T cell receptor complexes containing α and β heterodimers), Panelli et al., J Immunol (2000);164:495-504; Panelli et al., J Immunol (2000);164:4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont et al., Cancer Res (2005); 65:5417-5427; Papanicolaou et al., Blood (2003); 102:2498-2505 (disclosing antigen-specific peripheral blood leukocytes selectively expanded in vitro using artificial antigen presenting cells (AAPCs) or pulsed dendritic cells).

[0346] The cells (eg, T cells or NK cells) can be autologous, non-autologous (eg, allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0347] The subject cell of the present disclosure can be a myeloid cell.Non-limiting examples of myeloid cells include monocytes, macrophages, neutrophils, dendritic cells, basophils, neutrophils, eosinophils, megakaryocytes, mast cells, erythrocytes, platelets, and stem cells from which myeloid cells can be differentiated.In some embodiments, the stem cells are pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells).

[0348] In an embodiment, the cells of the present disclosure can regulate the tumor microenvironment. Tumors have a hostile microenvironment for the host immune response, which involves a series of mechanisms by malignant cells to protect themselves from immune recognition and elimination. This "hostile tumor microenvironment" is characterized by the infiltrating regulatory CD4 + These include various immunosuppressive factors including T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), immunosuppressive cytokines including TGF-β, and expression of ligands that target immunosuppressive receptors expressed by activated T cells (CTLA-4 and PD-1). Although these immunosuppressive mechanisms play a role in maintaining tolerance and suppressing inappropriate immune responses, within the tumor microenvironment these mechanisms prevent effective antitumor immune responses. Collectively, these immunosuppressive factors can induce significant anergy or apoptosis of matched, transferred engineered T cells upon encounter with target tumor cells.

[0349] In certain embodiments, cells can be transduced with an ADGRE2-targeted chimeric receptor of the present disclosure and / or a CLEC12A-targeted chimeric receptor of the present disclosure such that the cells express the chimeric receptor(s).

[0350] Further, the presently disclosed subject matter provides cells comprising an ADGRE2-targeted chimeric receptor of the present disclosure (e.g., those disclosed in Section 5.3) and a CLEC12A-targeted chimeric receptor of the present disclosure (e.g., those disclosed in Section 5.4). In an embodiment, the ADGRE2-targeted chimeric receptor is a CAR. In an embodiment, the CLEC12-targeted chimeric receptor is a CCR. Thus, in an embodiment, the presently disclosed subject matter provides cells comprising a CLEC12A-targeted CAR of the present disclosure and a CLEC12A-targeted CCR of the present disclosure.

[0351] In some embodiments, the cells disclosed herein exhibit a higher degree of cytolytic activity against cells positive for both ADGRE2 and CLEC12A compared to cells positive for ADGRE2 alone. In some embodiments, the ADGRE2-targeted CAR binds to ADGRE2 with low binding affinity or low binding avidity. In some embodiments, the ADGRE2-targeted CAR binds to ADGRE2 at a less accessible epitope. In some embodiments, the ADGRE2-targeted CAR binds to ADGRE2 with lower binding affinity compared to the binding affinity of the CLEC12A-targeted CCR to CLEC12A. In some embodiments, the ADGRE2-targeted CAR binds to ADGRE2 with at least 5-fold lower binding affinity compared to the binding affinity of the CLEC12A-targeted CCR to CLEC12A. In one embodiment, the ADGRE2-targeted CAR binds to ADGRE2 with a binding affinity that is 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 10,000-fold lower than the binding affinity with which a CLEC12A-targeted CCR binds to CLEC12A.

[0352] 5.5.1. Cell example In one embodiment, the cells comprise an ADGRE2-targeted CAR and a CLEC12A-targeted CCR.

[0353] In one embodiment, the ADGRE2-targeted CAR comprises (a) (i) a CDR1 comprising an amino acid sequence represented by SEQ ID NO: 33, a CDR2 comprising an amino acid sequence represented by SEQ ID NO: 34, and a VDR comprising an amino acid sequence represented by SEQ ID NO: 35. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 36, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 38. L(b) an extracellular antigen-binding domain comprising a CD28 polypeptide; (c) a transmembrane domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a fragment thereof); and (d) an intracellular signaling domain comprising (i) a CD3ζ polypeptide and (ii) a costimulatory signaling region comprising a CD28 polypeptide (e.g., the intracellular domain of human CD28 or a fragment thereof). H and V L is linked via a linker comprising or consisting of the amino acid sequence represented by SEQ ID NO:2. H and V L is V from N-terminus to C-terminus H -V L In one embodiment, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 10. In one embodiment, the costimulatory signaling region comprises a CD28 polypeptide comprising or consisting of amino acids 180-220 of SEQ ID NO: 10. In one embodiment, the hinge / spacer region comprises a CD28 polypeptide comprising or consisting of amino acids 114-152 of SEQ ID NO: 10. In one embodiment, the ADGRE2-targeted CAR comprises or consists of the amino acid sequence represented by SEQ ID NO:66.

[0354] In one embodiment, the CLEC12A-targeting CAR comprises (a) (i) a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 69, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 70, and a VDR comprising the amino acid sequence represented by SEQ ID NO: 71. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 72, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and a V comprising the amino acid sequence represented by SEQ ID NO: 74. L V containing CDR3 Land (b) an intracellular domain comprising a 4-1BB polypeptide (e.g., an intracellular domain of a human 4-1BB polypeptide, e.g., a portion thereof, of 4-1BB (e.g., human 4-1BB)). In certain embodiments, the CLEC12A target CCR further comprises a transmembrane domain comprising a CD8 polypeptide (e.g., a human CD8 polypeptide, e.g., a transmembrane domain of CD8 (e.g., human CD8) or a portion thereof). In certain embodiments, the intracellular domain comprises a 4-1BB polypeptide comprising or consisting of amino acids 214-255 of SEQ ID NO:30. In certain embodiments, the transmembrane domain comprises a CD8 polypeptide comprising or consisting of amino acids 137-207 of SEQ ID NO:7. In certain embodiments, the V H and V L is linked via a linker comprising or consisting of the amino acid sequence represented by SEQ ID NO:2. H and V L is V from N-terminus to C-terminus H -V L In one embodiment, the CLEC12A target CCR comprises the amino acid sequence represented by SEQ ID NO:136.

[0355] 5.8. Nucleic Acid Molecules, Vectors and Genetic Modifications The subject matter of the present disclosure provides a nucleic acid molecule encoding an ADGRE2-targeted chimeric receptor of the present disclosure (e.g., those disclosed in Sections 5.3 and 5.4). In some embodiments, the nucleic acid molecule further comprises a promoter operably linked to the ADGRE2-targeted CAR of the present disclosure. Cells comprising such nucleic acid molecules are also provided.

[0356] Further, the subject matter of the present disclosure provides a nucleic acid molecule encoding a CLEC12A-targeted chimeric receptor of the present disclosure (e.g., those disclosed in Sections 5.5 and 5.6). In an embodiment, the nucleic acid molecule further comprises a promoter operably linked to the CLEC12A-targeted CAR of the present disclosure. Cells comprising such nucleic acid molecules are also provided.

[0357] The presently disclosed subject matter further provides nucleic acid compositions comprising a nucleic acid molecule encoding an ADGRE2-targeted chimeric receptor and a nucleic acid molecule encoding a CLEC12A-targeted chimeric receptor. Cells comprising such nucleic acid compositions are also provided.

[0358] In some embodiments, the promoter is endogenous or exogenous. In some embodiments, the exogenous promoter is selected from the group consisting of elongation factor (EF)-1 promoter, cytomegalovirus immediate early promoter (CMV) promoter, simian virus 40 early promoter (SV40) promoter, phosphoglycerate kinase (PGK) promoter, metallothionein promoter, and ubiquitin C promoter. In some embodiments, the endogenous promoter is selected from the group consisting of TCR alpha promoter, TCR beta promoter, and beta2-microglobulin promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is selected from the group consisting of NFAT transcription response element (TRE) promoter, CD69 promoter, CD25 promoter, IL-2 promoter, 4-1BB promoter, PD1 promoter, and LAG3 promoter.

[0359] The subject matter of the present disclosure also provides a vector comprising the nucleic acid molecule of the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the retroviral vector is a gamma retroviral vector or a lentiviral vector.

[0360] In one embodiment, the vector comprises a nucleic acid molecule encoding an ADGRE2-targeted CAR of the present disclosure and a CLEC12A-targeted CCR of the present disclosure. In one embodiment, the nucleic acid molecule encodes a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 140. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence represented by SEQ ID NO: 141. SEQ ID NOs: 140 and 141 are shown below. [SEQ ID NO:140]

[0361] The nucleic acid molecule can be delivered into cells by methods known in the art or as described herein. Genetic modification of cells can be achieved by transducing recombinant DNA constructs into substantially homogeneous cell compositions. In some embodiments, retroviral vectors (e.g., gamma retroviral vectors or lentiviral vectors) are used to introduce DNA constructs into cells. For example, the polynucleotide encoding the chimeric receptor of the present disclosure can be cloned into a retroviral vector and driven to express from its endogenous promoter, retroviral long terminal repeat, or a promoter specific to the target cell type of interest. Non-viral vectors can also be used.

[0362] For the initial genetic modification of a cell containing at least one chimeric receptor of the present disclosure (e.g., two chimeric receptors of the present disclosure, e.g., an ADGRE2-targeted chimeric receptor of the present disclosure and a CLEC12A-targeted chimeric receptor of the present disclosure), a retroviral vector can be used for transduction, although any other suitable viral vector or non-viral delivery system can be used. The chimeric receptor(s) can be constructed in a single multicistronic expression cassette, in multiple expression cassettes on a single vector, or in multiple vectors. Exemplary elements making up a polycistronic expression cassette include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κΒ IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Aphthovirus IRES, Picornavirus IRES, Poliovirus IRES, and Encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). In one embodiment, the P2A peptide comprises or consists of the amino acid sequence represented by SEQ ID NO: 142, as provided below. GSGATNFSLLKQAGDVEENPGP [SEQ ID NO: 142]

[0363] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:142 is represented as SEQ ID NO:143 and is provided below. ggaagcggagctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggaccc [SEQ ID NO: 143]

[0364] Also suitable are combinations of retroviral vectors with appropriate packaging lines, in which the capsid proteins will function to infect human cells. A variety of amphiphilic virus-producing cell lines are known, including, but not limited to, PA12 (Miller et al., (1985) Mol Cell Biol (1985);5:431-437); PA317 (Miller., et al., Mol Cell Biol (1986);6:2895-2902); and CRIP (Danos et al., Proc Natl Acad Sci USA (1988);85:6460-6464). Non-amphophilic particles are also suitable, such as particles pseudotyped with VSVG, RD114 or GALV envelopes, and any other particles known in the art.

[0365] Possible transduction methods include direct co-culture of cells with producer cells (Bregni et al., Blood (1992);80:1418-1422), or culture with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations (Xu et al., Exp Hemat (1994);22:223-230; and Hughes et al. J Clin Invest (1992);89:1817).

[0366] Other transducing viral vectors can also be used to modify cells. In some embodiments, the selected vector shows high efficiency of infection and stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, or herpes viruses such as Epstein-Barr virus (see, for example, the vectors of Miller, Human Gene Thera (1990);15-14; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques (1988);6:608-614; Tolstoshev et al., Cur Opin Biotechnol (1990);1:55-61; Sharp, The Lancet (1991);337:1277-78; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-22, 1987; Anderson, Science (1984);226:401-409; Moen, Blood Cells 17:407-16, 1991; Miller et al., Biotechnol (1989);7:980-90; LeGal La Salle et al., Science (1993);259:988-90; and Johnson, Chest (1995)107:77S- 83S).Retroviral vectors have been particularly well developed and used in clinical settings (Rosenberg et al., N Engl J Med (1990);323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0367] Non-viral approaches can also be used for genetic modification of cells.For example, nucleic acid molecules can be introduced into cells by administering nucleic acid in the presence of lipofection (Feigner et al., Proc Natl Acad Sci USA (1987);84:7413;Ono et al., Neurosci Lett (1990);17:259;Brigham et al., Am J Med Sci (1989);298:278;Staubinger et al., Methods in Enzymol (1983);101:512, Wu et al., J Biol Chem (1988);263:14621;Wu et al., J Biol Chem (1989);264:16985) or by microinjection under surgical conditions (Wolff et al., Science (1990);247:1465). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also potentially be useful for the delivery of DNA to cells. The transplantation of normal genes into diseased tissues of subjects can also be achieved by transferring normal nucleic acids ex vivo into culturable cell types (e.g., autologous or heterologous primary cells or their progeny), and then injecting the cells (or their progeny) into target tissues or by systemic injection. Recombinant receptors can also be induced or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases or TALENs, CRISPR). Transient expression can be obtained by RNA electroporation.

[0368] Any targeted genome editing method can also be used to deliver at least one chimeric receptor of the present disclosure (e.g., two chimeric receptors of the present disclosure, e.g., a chimeric receptor targeted by ADGRE2 of the present disclosure and a chimeric receptor targeted by CLEC12A of the present disclosure) to a cell. In an embodiment, a CRISPR system is used to deliver at least one chimeric receptor of the present disclosure (e.g., two chimeric receptors of the present disclosure, e.g., a chimeric receptor targeted by ADGRE2 of the present disclosure and a chimeric receptor targeted by CLEC12A of the present disclosure). In an embodiment, a zinc finger nuclease is used to deliver at least one chimeric receptor of the present disclosure (e.g., two chimeric receptors of the present disclosure, e.g., a chimeric receptor targeted by ADGRE2 of the present disclosure and a chimeric receptor targeted by CLEC12A of the present disclosure). In one embodiment, the TALEN system is used to deliver at least one chimeric receptor of the present disclosure (e.g., two chimeric receptors of the present disclosure, e.g., an ADGRE2-targeted chimeric receptor of the present disclosure and a CLEC12A-targeted chimeric receptor of the present disclosure).

[0369] The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When used for genome editing, the system includes Cas9 (a protein that can modify DNA using the crRNA as its guide), CRISPR RNA (crRNA, which contains the RNA that Cas9 uses to guide Cas9 to the correct section of the host DNA along with a region that binds to the tracrRNA (usually in the form of a hairpin loop) that forms an active complex with Cas9), transactivating crRNA (tracrRNA, which binds to the crRNA and forms an active complex with Cas9), and an optional section of DNA repair template (DNA that guides the cellular repair process allowing the insertion of a specific DNA sequence). CRISPR / Cas9 often uses a plasmid to transform the target cell. The crRNA must be designed for each application, as this is the sequence that Cas9 uses to identify and directly bind to the target DNA in the cell. The repair template carrying the CAR expression cassette must also be designed for each application, as it must overlap on either side of the cut and code for the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA), which can be combined with a Cas9 gene into a plasmid and transfected into cells.

[0370] Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by combining a zinc finger DNA binding domain with a DNA cleavage domain. The zinc finger domain can be designed to target specific DNA sequences allowing the zinc finger nuclease to target desired sequences in the genome. The DNA binding domain of an individual ZFN typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method of generating novel zinc finger domains is to combine smaller zinc finger "modules" of known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the type IIs restriction endonuclease FokI. Using the endogenous homologous recombination (HR) machinery and a homologous DNA template carrying a CAR expression cassette, ZFNs can be used to insert the CAR expression cassette into the genome. Once the target sequence is cleaved by the ZFN, the HR machinery searches for homology between the damaged chromosome and a homologous DNA template and then integrates the homologous DNA template into the genome by copying the template sequence between the two cut ends of the chromosome.

[0371] Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences in DNA. TALEN systems work on roughly the same principle as ZFNs. They are generated by combining a transcription activator-like effector DNA binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of a 33-34 amino acid repeat motif with two variable positions that stro...

Claims

1. A chimeric receptor comprising an extracellular antigen-binding domain that binds to ADGRE2, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain is a) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 33, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 34, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 35; and b) a light chain variable region comprising: CDR1 comprising the amino acid sequence represented by SEQ ID NO: 36; CDR2 comprising the amino acid sequence represented by SEQ ID NO: 37; and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 38; The chimeric receptor comprising:

2. The extracellular antigen-binding domain is a single chain variable fragment (scFv), a Fab, or a F(ab). ２ 2. The chimeric receptor of claim 1, wherein optionally the scFv is a humanized scFv.

3. a) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 39, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 40; b) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 43, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 44; c) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 46, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 47; d) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 49, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 50; e) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO:52, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:53; f) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO:55 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:56; or g) The chimeric receptor of claim 1, wherein the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 146 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:

147.

4. The chimeric receptor according to any one of claims 1 to 3, wherein the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region, and optionally the linker consists of an amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:

149.

5. The heavy chain variable region and the light chain variable region are, from N-terminus to C-terminus, Ｈ -V Ｌ The chimeric receptor according to any one of claims 1 to 3, wherein the chimeric receptor is located in

6. The chimeric receptor of any one of claims 1 to 3, wherein the extracellular antigen-binding domain comprises or is an scFv, and the scFv comprises or consists of the amino acid sequence represented by SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, or SEQ ID NO:

148. (a) the extracellular antigen-binding domain comprises about 10 －８ Less than M, about 10 －９ Less than M, about 10 －１０ Less than M, about 10 －１１ Less than M, about 10 －１２ Less than M or about 10 －１３ Dissociation constant (K Ｄ ) binds to ADGRE2; and / or (b) the chimeric receptor of any one of claims 1 to 3, wherein the extracellular antigen-binding domain binds to ADGRE2 with an EC50 of about 1 nM to about 100 nM, about 10 nM to about 95 nM, or about 25 nM to about 75 nM.

8. 4. The chimeric receptor of any one of claims 1 to 3, wherein the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ 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, or a BTLA polypeptide.

9. The chimeric receptor of claim 1, wherein the intracellular domain comprises a native CD3ζ polypeptide or a modified CD3ζ polypeptide.

10. The modified CD3ζ polypeptide comprises a natural ITAM1, an ITAM2 mutant consisting of two loss-of-function mutations, and an ITAM3 mutant consisting of two loss-of-function mutations, and optionally the modified CD3ζ polypeptide comprises or consists of the amino acid sequence represented by SEQ ID NO:

27. A chimeric receptor as described in claim 9.

11. 10. The chimeric receptor of claim 9, wherein the intracellular domain further comprises at least one costimulatory signaling region, optionally wherein the at least one costimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof.

12. The chimeric receptor of any one of claims 1 to 3, wherein the chimeric receptor is a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), or a TCR-like fusion molecule.

13. A chimeric receptor comprising an extracellular antigen-binding domain that binds to CLEC12A, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain is a) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO:69, CDR2 comprising the amino acid sequence represented by SEQ ID NO:70, and CDR3 comprising the amino acid sequence represented by SEQ ID NO:71; and a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO:72, CDR2 comprising the amino acid sequence represented by SEQ ID NO:73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO:74; b) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 81, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 82, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83; and a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 84, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 85; c) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO:89, CDR2 comprising the amino acid sequence represented by SEQ ID NO:90, and CDR3 comprising the amino acid sequence represented by SEQ ID NO:91; and a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO:92, CDR2 comprising the amino acid sequence represented by SEQ ID NO:93, and CDR3 comprising the amino acid sequence represented by SEQ ID NO:94; d) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence represented by SEQ ID NO:89, a CDR2 comprising the amino acid sequence represented by SEQ ID NO:90, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO:98; and a light chain variable region comprising a CDR1 comprising the amino acid sequence represented by SEQ ID NO:99, a CDR2 comprising the amino acid sequence represented by SEQ ID NO:93, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO:151; e) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence represented by SEQ ID NO:81, a CDR2 comprising the amino acid sequence represented by SEQ ID NO:103, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO:83; and a light chain variable region comprising a CDR1 comprising the amino acid sequence represented by SEQ ID NO:104, a CDR2 comprising the amino acid sequence represented by SEQ ID NO:73, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO:105; f) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 109, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 103, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 83; and a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 110, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 73, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 85; g) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO:89, CDR2 comprising the amino acid sequence represented by SEQ ID NO:90, and CDR3 comprising the amino acid sequence represented by SEQ ID NO:114; and a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO:115, CDR2 comprising the amino acid sequence represented by SEQ ID NO:93, and CDR3 comprising the amino acid sequence represented by SEQ ID NO:116; h) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 120, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 121, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 122; and a light chain variable region comprising a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 123, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 124, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 125; or i) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 129, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 130, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 131; and a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 132, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 133, and CDR3 comprising the amino acid sequence represented by SEQ ID NO:

134. The chimeric receptor comprising:

14. The extracellular antigen-binding domain is a single chain variable fragment (scFv), a Fab, or a F(ab). ２ 14. The chimeric receptor of claim 13, comprising:

15. a) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 75, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 76; b) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 86, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 87; c) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 95, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 96; d) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 100, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 101; e) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 106, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 107; f) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 111, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 112; g) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 117, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 118; h) the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 126 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 127; or i) The chimeric receptor of claim 13, wherein the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 135 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO:

136.

16. The chimeric receptor according to any one of claims 13 to 15, wherein the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region, and optionally the linker consists of the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:

149.

17. The heavy chain variable region and the light chain variable region are, from N-terminus to C-terminus, Ｈ -V Ｌ The chimeric receptor according to any one of claims 13 to 15, wherein the chimeric receptor is located in

18. The chimeric receptor of any one of claims 13 to 15, wherein the extracellular antigen-binding domain comprises or is an scFv, and the scFv comprises or consists of the amino acid sequence represented by SEQ ID NO:79, SEQ ID NO:88, SEQ ID NO:97, SEQ ID NO:102, SEQ ID NO:108, SEQ ID NO:113, SEQ ID NO:119, SEQ ID NO:128, or SEQ ID NO:

137. (a) the extracellular antigen-binding domain comprises about 10 －８ Less than M, about 10 －９ Less than M, about 10 －１０ Less than M, about 10 －１１ Less than M, about 10 －１２ Less than M or about 10 －１３ Dissociation constant (K Ｄ and / or is an scFv that binds to CLEC12A at K D , optionally with a K D of about 0.1 pM or less, about 0.05 pM to about 0.5 pM, about 0.1 nM to about 5.0 nM, or about 0.3 nM to about 3.5 nM; and / or (b) the chimeric receptor of any one of claims 13 to 15, wherein the extracellular antigen-binding domain comprises or is an scFv that binds to CLEC12A with an EC50 of about 1 nM to about 100 nM.

20. 16. The chimeric receptor of any one of claims 13-15, wherein the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ 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, or a BTLA polypeptide.

21. The chimeric receptor of any one of claims 13 to 15, wherein the chimeric receptor is a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), or a TCR-like fusion molecule.

22. The chimeric receptor of any one of claims 13 to 15, wherein the intracellular domain does not include a CD3ζ polypeptide.

23. 16. The chimeric receptor of any one of claims 13-15, wherein the intracellular domain comprises at least one costimulatory signaling region, optionally wherein the at least one costimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof.

24. A cell comprising the chimeric receptor of claim 1 or 13.

25. A cell comprising: a) a first chimeric receptor, the first chimeric receptor being the chimeric receptor of claim 1; and b) a second chimeric receptor, the second chimeric receptor being the chimeric receptor of claim 13.

26. 26. The cell of claim 25, wherein a) the first chimeric receptor is a chimeric antigen receptor (CAR) and b) the second chimeric receptor is a chimeric costimulatory receptor (CCR).

27. The cell of claim 25 , wherein the chimeric receptor is constitutively expressed on the surface of the cell.

28. (a) the cell is an immunoresponsive cell; (b) the cell is a lymphoid or myeloid cell; (c) the cells are selected from the group consisting of T cells, natural killer (NK) cells, stem cells from which lymphoid cells can differentiate, and stem cells from which myeloid cells can differentiate; (d) the cell is a T cell; (e) the cell is a T cell selected from the group consisting of a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a tumor-infiltrating lymphocyte (TIL), a natural killer T cell, a mucosal-associated invariant T cell, and a γδ T cell; (f) the cell is a natural killer (NK) cell; (g) the cell is a natural killer (NK) cell derived from a stem cell; (h) the cell is a pluripotent stem cell; 26. The cell of claim 25, wherein (i) the cell is an embryonic-like stem cell or an induced pluripotent stem cell.

29. A nucleic acid molecule comprising: (a) a polynucleotide encoding the chimeric receptor of claim 1 or 13; (b) a first polynucleotide encoding the chimeric receptor of claim 1, and a second polynucleotide encoding the chimeric receptor of claim 13. A nucleic acid molecule comprising:

30. 30. The nucleic acid molecule of claim 29, further comprising a promoter operably linked to said chimeric receptor, optionally wherein said promoter is inducible, endogenous or exogenous.

31. A vector comprising the nucleic acid molecule of claim 29.

32. The vector of claim 31 , wherein the vector is a viral vector or a retroviral vector.

33. 30. A cell comprising a nucleic acid molecule according to claim 29, or a vector comprising the nucleic acid molecule according to claim 29, optionally wherein the cell is a T cell or a NK cell.

34. A pharmaceutical composition comprising the cells of claim 24 and a pharma- ceutically acceptable carrier.

35. A pharmaceutical composition comprising the cells of claim 25 and a pharma- ceutically acceptable carrier.

36. Approximately 25×10 ６ ~Approx. 150×10 ６ 36. The composition of claim 34 or 35, comprising about 25x10 6 to about 50x10 6 cells, or about 2.5x10 6 cells.

37. 36. The composition of claim 34 or 35, for use in reducing tumor burden, increasing or prolonging survival of a tumor-bearing subject, treating and / or preventing tumors in a subject.

38. The composition of claim 34 or 35, wherein the tumor expresses ADGRE2 and / or CLEC12A.

39. 36. The composition of claim 34 or 35, wherein the tumor is a cancer or hematological cancer, optionally the tumor is selected from the group consisting of multiple myeloma, leukemia, lymphoma, and myeloid malignancies.

40. (a) the tumor is a leukemia selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, and B-cell prolymphocytic leukemia; (b) the tumor is acute myeloid leukemia (AML); (c) the tumor is relapsed / refractory acute myeloid leukemia (R / R AML); (d) the tumor is a myeloid malignancy selected from the group consisting of myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), myeloid / lymphoid neoplasms (e.g., myeloid / lymphoid neoplasms with eosinophilia and platelet-derived growth factor receptor alpha (PDGFRA), platelet-derived growth factor receptor beta (PDGFRB), or fibroblast growth factor receptor 1 (FGFR1) rearrangements, or PCM1-JAK2), acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm, B-lymphoblastic leukemia / lymphoma, and T-lymphoblastic leukemia / lymphoma; (e) the tumor is a myelodysplastic syndrome (MDS).

41. A method for producing a cell, comprising: (a) comprising the chimeric receptor of claim 1 or 13, the method comprising introducing into the cell a nucleic acid molecule encoding the chimeric receptor; or (b) a chimeric receptor of claim 1 and a chimeric receptor of claim 13, the method comprising introducing into the cell a nucleic acid molecule encoding each of the chimeric receptors.

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