Antigen recognition receptor targeting CD371, and use of the same

Abstract

To provide an antigen recognition receptor targeting CD371, and use thereof.SOLUTION: There is provided an antigen recognition receptor specifically targeting CD371, and a cell containing such a CD371 targeted antigen recognition receptor. Further there is provided use of the CD371 targeted antigen recognition receptor for treatment. There are provided a method and a composition for immunotherapy. The invention relates to an antigen recognition receptor specifically targeting CD371 (e.g., chimeric antigen receptor (CAR) or T-cell receptor (TCR)), a cell containing such a receptor, and a method for using such a cell for treatment.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 900,141, filed September 13, 2019, and U.S. Provisional Application No. 62 / 936,951, filed November 18, 2019, the contents of each of which are incorporated by reference herein in their entirety and claim priority to each of which. Sequence Listing

[0002] This application contains a Sequence Listing that was submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy was created on September 11, 2020, is named 0727341147_ST25, and is 211,921 bytes in size. 1. Introduction

[0003] The subject matter of the present disclosure provides methods and compositions for immunotherapy, which relate to antigen recognition receptors (e.g., chimeric antigen receptors (CARs) or T cell receptors (TCRs)) that specifically target CD371, cells containing such receptors, and methods of using such cells for treatment. [Background technology]

[0004] 2. Background of the invention Cell-based immunotherapy is a potentially curative therapy for the treatment of cancer. T cells and other immune cells can be modified to target tumor antigens by the introduction of genetic material that encodes an artificial or synthetic receptor for the antigen, called a chimeric antigen receptor (CAR), specific for the selected antigen. Targeted T cell therapy using CAR has recently shown clinical success in the treatment of hematological malignancies.

[0005] Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. It is characterized by the accumulation of immature myeloid cells in the bone marrow, which leads to impaired hematopoiesis. Chemotherapy and hematopoietic stem cell transplantation (HSCT) are the standard treatments for AML. However, the majority of patients eventually relapse and succumb to the disease.

[0006] AML is the most common acute leukemia in adults.Standard induction chemotherapy regimens have not changed substantially over the past 40 years, and overall survival remains very poor.Frequent recurrent abnormalities involving genes coding for epigenetic modifiers have been identified.The development of CAR therapy for AML is hindered by the lack of suitable targets.Therefore, there is a need for novel therapeutic strategies to design CARs that target antigens that are highly expressed in AML cells and have limited expression in normal tissues to treat AML, and for strategies that can induce potent cancer eradication with minimal toxicity and immunogenicity. Summary of the Invention [Means for solving the problem]

[0007] 3. Overview of the Invention The subject matter of the present disclosure provides an antigen recognition receptor that specifically targets CD371, and a cell that includes such a CD371-targeted antigen recognition receptor.The subject matter of the present disclosure further provides the use of the CD371-targeted antigen recognition receptor for treatment.

[0008] The subject matter of the present disclosure provides an antigen-recognizing receptor comprising an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to CD371. In certain embodiments, the extracellular antigen-binding domain is a single chain variable fragment (scFv). In certain embodiments, the extracellular antigen-binding domain is a human scFv. In certain embodiments, the extracellular antigen-binding domain is an optionally cross-linked Fab. In certain embodiments, the extracellular antigen-binding domain is a F(ab)2. In certain embodiments, one or more of the scFv, Fab and F(ab)2 are included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain.

[0009] In certain embodiments, the extracellular antigen binding domain comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28 or a conservative modification thereof; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29 or a conservative modification thereof; and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30 or a conservative modification thereof; (b) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34 or a conservative modification thereof; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35 or a conservative modification thereof; and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36 or a conservative modification thereof; (c) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40 or a conservative modification thereof; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42 or a conservative modification thereof; and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43 or a conservative modification thereof; (d) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 462 or a conservative modification thereof; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 47 or a conservative modification thereof; and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48 or a conservative modification thereof; (e) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52 or a conservative modification thereof; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 53 or a conservative modification thereof; and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 54 or a conservative modification thereof; or (f) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58 or a conservative modification thereof; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59 or a conservative modification thereof; and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 60 or a conservative modification thereof. Includes.

[0010] In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:28; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:29; and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:30.

[0011] In certain embodiments, the extracellular antigen binding domain comprises: (a) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31 or a conservative modification thereof; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32 or a conservative modification thereof; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33 or a conservative modification thereof; (b) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37 or a conservative modification thereof; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof; and a light chain variable region CDR3 comprising SEQ ID NO: 39 or a conservative modification thereof; (c) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43 or a conservative modification thereof; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44 or a conservative modification thereof; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45 or a conservative modification thereof; (d) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49 or a conservative modification thereof; a light chain variable region CDR2 comprising SEQ ID NO: 50 or a conservative modification thereof; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51 or a conservative modification thereof; (e) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 55 or a conservative modification thereof; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 56 or a conservative modification thereof; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof; or (f) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 61 or a conservative modification thereof; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 62 or a conservative modification thereof; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63 or a conservative modification thereof. Includes.

[0012] In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:28; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:29; and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:30.

[0013] In certain embodiments, the extracellular antigen binding domain comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29; a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33; (b) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35; a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39; (c) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41; a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42; a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45; (d) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 46; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 47; a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48; a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51; (e) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 53; a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 54; a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 55; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 56; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57; or (f) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59; a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 60; a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 61; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 62; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63 Includes.

[0014] In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:28; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:29; a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:30; a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:31; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:32; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:33.

[0015] In certain embodiments, the extracellular antigen binding domain comprises a heavy chain variable region comprising 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% homologous or identical to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11. In certain embodiments, the extracellular antigen binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1.

[0016] In certain embodiments, the extracellular antigen binding domain comprises a light chain variable region comprising 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% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12. In certain embodiments, the extracellular antigen binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2.

[0017] In certain embodiments, the extracellular antigen binding domain comprises: (a) 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% homologous or identical to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11. and (b) a light chain variable region comprising 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% homologous or identical to the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12. In certain embodiments, the extracellular antigen binding domain comprises (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11; and (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12.

[0018] In certain embodiments, the extracellular antigen binding domain comprises: (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2; (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:4; (c) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:6; (d) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8; (e) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10; or (f) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12 Includes.

[0019] In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2.

[0020] In certain embodiments, the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. In certain embodiments, the linker has the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, or SEQ ID NO: 94. In certain embodiments, the linker has the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14.

[0021] In certain embodiments, the extracellular antigen binding domain comprises a signal peptide covalently joined to the 5' end of the extracellular antigen binding domain. In certain embodiments, the extracellular antigen binding domain comprises a V H -V LThe variable regions include a heavy chain and a light chain variable region located at

[0022] In certain embodiments, the extracellular antigen-binding domain binds to CD371 with low binding affinity. In certain embodiments, the extracellular antigen-binding domain binds to CD371 with low binding affinity. -8 M or higher dissociation constant (K d ) and binds to CD371.

[0023] In certain embodiments, the transmembrane domain comprises a CD28 polypeptide. In certain embodiments, the intracellular signaling domain comprises a CD3ζ polypeptide. In certain embodiments, the intracellular signaling domain further comprises at least one costimulatory signaling region. In certain embodiments, 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. In certain embodiments, the at least one costimulatory signaling region comprises a CD28 polypeptide or a 4-1BB polypeptide.

[0024] In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR) or a T cell-like fusion protein. In certain embodiments, the antigen-recognizing receptor is a CAR.

[0025] In certain embodiments, the antigen-recognizing receptor is recombinantly expressed. In certain embodiments, the antigen-recognizing receptor is expressed from a vector. In certain embodiments, the vector is a gamma-retroviral vector.

[0026] The subject matter of the present disclosure provides a cell comprising the antigen recognition receptor of the present disclosure. In certain embodiments, the cell is transduced with the antigen recognition receptor. In certain embodiments, the antigen recognition receptor is constitutively expressed on the surface of the cell.

[0027] In certain embodiments, the cell is engineered to express a cytokine or a fragment thereof. In certain embodiments, the cell further comprises an exogenous polypeptide of a cytokine or a fragment thereof. In certain embodiments, the cell further comprises a nucleic acid molecule encoding a cytokine or a fragment thereof. In certain embodiments, the cytokine is selected from the group consisting of IL-18, IL-33, IL-36, and combinations thereof. In certain embodiments, the cytokine is IL-18.

[0028] In certain embodiments, the cell is an immune response cell. In certain embodiments, the cell is a lymphoid lineage cell or a myeloid lineage cell. In certain embodiments, the cell is selected from the group consisting of T cells, natural killer (NK) cells, and stem cells from which lymphoid cells can be differentiated. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is a cytotoxic T lymphocyte (CTL) or a regulatory T cell. In certain embodiments, the stem cell is a pluripotent stem cell. In certain embodiments, the pluripotent stem cell is an embryonic-like stem cell or an induced pluripotent stem cell.

[0029] The subject matter of the present disclosure further provides a nucleic acid molecule encoding the antigen-recognition receptor of the present disclosure. In certain embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27. In certain embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 22. The subject matter of the present disclosure further provides a vector comprising the nucleic acid molecule of the present disclosure. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a gamma-retroviral vector.

[0030] In addition, the presently disclosed subject matter provides a host cell expressing the nucleic acid molecules disclosed herein. In certain embodiments, the host cell is a T cell.

[0031] The presently disclosed subject matter further provides a composition comprising the cells disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0032] The subject matter of the present disclosure further provides a method of reducing tumor burden in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of the cells or compositions of the present disclosure. In certain embodiments, the method reduces the number of tumor cells, reduces tumor size, and / or eradicates tumors in the subject. The subject matter of the present disclosure further provides a method of increasing or extending the survival time of a subject having a tumor or neoplasm. In certain embodiments, the method comprises administering to the subject an effective amount of the cells or compositions of the present disclosure. The subject matter of the present disclosure further provides a method of treating and / or preventing a tumor or neoplasm in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of the cells or compositions of the present disclosure. In certain embodiments, the tumor or neoplasm is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), glioblastoma, myelodysplastic syndrome (MDS), and chronic myeloid leukemia (CML). In certain embodiments, the tumor or neoplasm is acute myeloid leukemia (AML).

[0033] The subject matter of the present disclosure further provides a method for generating a cell of the present disclosure comprising a CD371-targeting antigen recognition receptor. In certain embodiments, the method comprises introducing a nucleic acid molecule encoding the antigen recognition receptor into the cell.

[0034] In addition, the subject matter of the present disclosure provides a kit for reducing tumor burden in a subject, treating and / or preventing tumor or neoplasm in a subject, and / or increasing or extending the survival time of a subject with a tumor or neoplasm.In certain embodiments, the kit comprises the cells described herein.In certain embodiments, the kit further comprises instructions for using the cells for reducing tumor burden in a subject, treating and / or preventing tumor or neoplasm in a subject, and / or increasing or extending the survival time of a subject with a tumor or neoplasm. 4. Brief description of the drawings

[0035] The following detailed description is given by way of example and is not intended to limit the invention to the specific embodiments described and may be understood in conjunction with the accompanying drawings. [Brief description of the drawings]

[0036] [Figure 1] FIG. 1 depicts the structure of an antigen-recognizing receptor according to the presently disclosed subject matter.

[0037] [Figure 2-1] Figure 2 depicts detection of CD371-targeted CARs on the surface of transduced human T cells. Antibodies against EGFRt (cetuximab-APC) and Myc tag (9B11-PE) detected surface expression of EGFRt and human anti-human CARs in B10-based CAR constructs. Controls included non-transduced human T cells that do not express EGFRt or the MYC tag, as well as non-MYC tag-containing CAR constructs Etah19h28z (anti-human CD19 CAR T cells) and EtC1HVh28z (anti-CD371 CAR T cells derived from the mouse anti-human CD371 antibody 107537). [Figure 2-2]Figure 2 depicts detection of CD371-targeted CARs on the surface of transduced human T cells. Antibodies against EGFRt (cetuximab-APC) and Myc tag (9B11-PE) detected surface expression of EGFRt and human anti-human CARs in B10-based CAR constructs. Controls included non-transduced human T cells that do not express EGFRt or the MYC tag, as well as non-MYC tag-containing CAR constructs Etah19h28z (anti-human CD19 CAR T cells) and EtC1HVh28z (anti-CD371 CAR T cells derived from the mouse anti-human CD371 antibody 107537).

[0038] [Diagram 3] Figure 3 depicts the tumor cytolytic activity of CD371-targeted CAR T cells. Human CD371-targeted CAR T cells rested for 4 days (4dR) were co-cultured with CD33+ / CD371+U937 cells expressing GFP and firefly luciferase (U937gL) at different effector:tumor (E:T) ratios. Bioluminescence was measured after 24 hours and plotted as a percentage of the signal detected in the co-culture of non-functional CAR T cells (based on B10L4H but without CD28 or CD3 zeta signaling domains) and U937gL. Et.C1HVh28z represents a CD371-targeted CAR derived from the murine anti-human CD371 antibody 1075.7, and EtM195MTh28Z represents a CD33-targeted CAR T cell derived from the murine anti-CD33 antibody M195 (see Zhao et al., Haematologica (2010):95:71-78 (2010)).

[0039] [Figure 4]Figure 4 represents the tumor cell lysis activity of CD371-targeted CAR T cells in a 24-hour killing assay. CD371-targeted CAR T cells from a healthy donor (referred to as "Donor C") were co-cultured with CD33+ / CD371+U937 cells expressing GFP and firefly luciferase (U937gL) at different effector:tumor (E:T) ratios. Bioluminescence was measured after 24 hours and plotted as a percentage of the signal detected in the co-culture of non-functional CAR T cells (based on B10L4H but without CD28 or CD3 zeta signaling domains) and U937gL. Et.C1HVh28z represents CD371-targeted CAR derived from the mouse anti-human CD371 antibody 1075.7, and EtM195Mth28z represents CD33-targeted CAR T cells derived from the mouse anti-human CD33 antibody M195.

[0040] [Diagram 5] Figure 5 represents the tumor cell lysis activity of CD371-targeted CAR T cells in a 24-hour killing assay. CD371-targeted CAR T cells from a healthy donor (referred to as "Donor D") were co-cultured with CD33+ / CD371+U937 cells expressing GFP and firefly luciferase (U937gL) at different effector:tumor (E:T ratios). Bioluminescence was measured after 24 hours and plotted as a percentage of the signal detected in the co-culture of non-functional CAR T cells (based on B10L4H but without CD28 or CD3 zeta signaling domains) and U937gL. Et.C1HVh28z represents CD371-targeted CAR derived from the mouse anti-human CD371 antibody 1075.7, and EtM195Mth28z represents CD33-targeted CAR T cells derived from the mouse anti-human CD33 antibody M195.

[0041] [Figure 6]FIG. 6 depicts the activity of CD371-targeted CAR T cells in a recursive stimulation assay. CD371-targeted CAR T cells from a healthy donor (referred to as "Donor C") were co-cultured with CD33+ / CD371+U937gL cells at an E:T ratio of 1:12.5 and a concentration of 30,000 CAR positives / ml. CAR T cells were counted and characterized by flow cytometry approximately every 4-5 days, and a starting number of tumor cells were added back to the culture (indicated by arrows). Et.B10LHdel represents non-functional CAR T cells (lacking signaling domains), Et.C1HVh28z represents CD371-targeted CAR derived from the mouse anti-human CD371 antibody 1075.7, and EtM195Mth28z represents CD33-targeted CAR T cells derived from the mouse anti-human CD33 antibody M195.

[0042] [Figure 7] FIG. 7 depicts the activity of CD371-targeted CAR T cells in a recurrent stimulation assay. Healthy donor-derived (referred to as "Donor D") CD371-targeted CAR T cells were co-cultured with CD33+ / CD371+U937gL cells at an E:T ratio of 1:12.5 and a concentration of 30,000 CAR positives / ml. CAR T cells were counted and characterized by flow cytometry approximately every 4-5 days, and a starting number of tumor cells were added back to the culture (indicated by arrows). Et.B10LHdel represents non-functional CAR T cells (lacking signaling domains), Et.C1HVh28z represents CD371-targeted CAR derived from the mouse anti-human CD371 antibody 1075.7, and EtM195Mth28z represents CD33-targeted CAR T cells derived from the mouse anti-human CD33 antibody M195.

[0043] [Figure 8]Figures 8A and 8B represent the in vitro antitumor activity of CD371-targeted CAR T cells (second generation B10HL-based CAR T cells and B10LH-based CAR T cells) in a 24-hour killing assay of U937gL from different healthy donors. CD371-targeted CAR T cells from healthy human donors (referred to as "Donor A" and "Donor B") were co-cultured with CD371-positive U937 cells expressing GFP and firefly luciferase (U937gL) at different effector:tumor ratios. Bioluminescence was measured after 24 hours and plotted as a percentage of the signal detected in the co-culture of non-functional CD371-targeted CAR T cells (based on B10L4H but without CD28 or CD3 zeta signaling domains) and U937gL. Figure 8A shows the results for Donor A. Figure 8B shows the results for Donor B.

[0044] [Figure 9] Figures 9A and 9B represent the in vitro antitumor activity of CD371-targeted CAR T cells (second generation B10HL-based CAR T cells and B10LH-based CAR T cells) in a 24-hour killing assay of HL60gL from different healthy donors. CD371-targeted CAR T cells from healthy human donors (referred to as "Donor A" and "Donor B") were co-cultured with CD371-positive HL60 cells expressing GFP and firefly luciferase (HL60gL) at different effector:tumor ratios. Bioluminescence was measured after 24 hours and plotted as a percentage of the signal detected in the co-culture of non-functional CD371-targeted CAR T cells (based on B10L4H but without CD28 or CD3 zeta signaling domains) and HL60gL. Figure 9A shows the results for Donor A. Figure 9B shows the results for Donor B.

[0045] [Figure 10]Figures 10A and 10B show the generation of CRISPR knockout cell lines of human CD371 (hCD371). Human CD371 was knocked out from HL60gL (Figure 10A) and U937gL (Figure 10B) using CRISPR. Knockout was confirmed by flow cytometry using anti-human CD371 APC-conjugated antibody. Figure 10A shows the knockout of HL60gL. Figure 10B shows the knockout of U937gL.

[0046] [Figure 11] Figures 11A and 11B show the cytotoxic activity of CD371-targeted CAR T cells (second generation B10-based CAR T cells, i.e., B10-HL and B10LH-based CAR T cells) against antigen-negative U937gL in a 24-hour killing assay from different healthy donors. CD371-targeted CAR T cells from healthy human donors (referred to as "Donor A" and "Donor B") were co-cultured with CD371-negative U937 cells expressing RFP and Cypridina luciferase (U937RFPcyp.371KO) at different effector:tumor ratios. Bioluminescence was measured after 24 hours and plotted as a percentage of the signal detected in the co-culture of non-functional CD371-targeted CAR T cells (based on B10L4H but without CD28 or CD3 zeta signaling domains) and U937RFPcyp.371KO. Figure 11A shows the results for donor A. Figure 11B shows the results for donor B.

[0047] [Figure 12]Figures 12A and 12B represent the cytotoxic activity of CD371-targeted CAR T cells (second generation B10-based CAR T cells, i.e., B10-HL and B10LH-based CAR T cells) against antigen-negative HL60gL in a 24-hour killing assay from different healthy donors. CD371-targeted CAR T cells from healthy human donors (referred to as "Donor A" and "Donor B") were co-cultured with CD371-negative HL60 cells expressing GFP and firefly luciferase (HL60L.371KO) at different effector:tumor ratios. Bioluminescence was measured after 24 hours and plotted as the percentage of the signal detected in the co-culture of non-functional CD371-targeted CAR T cells (based on B10L4H but without CD28 or CD3 zeta signaling domains) and HL60gL.371KO. Figure 12A shows the results for Donor A. FIG. 12B shows the results for donor B.

[0048] [Figure 13] Figures 13A and 13B show interferon gamma (IFN-γ) secretion of CD371-targeted CAR T cells (second generation B10-based CAR T cells, i.e., B10-HL and B10LH-based CAR T cells). Human CD371-targeted CAR T cells were co-cultured alone, with CD371-negative U937 cells, or with CD371+ U937 cells at a 1:1 effector:tumor ratio (E:T) (4.0×104:4.0×104 cells in 200 μl). After 24 hours, supernatants were collected and IFN-γ was measured using a bead-based multiplex assay. Figure 13A shows the results for donor A. Figure 13B shows the results for donor B.

[0049] [Figure 14]Figures 14A and 14B show interleukin-2 (IL-2) secretion of CD371-targeted CAR T cells (second generation B10-based CAR T cells, i.e., B10-HL and B10LH-based CAR T cells). Human CD371-targeted CAR T cells were co-cultured alone, with CD371-negative U937 cells, or with CD371+ U937 cells at an effector:tumor ratio (E:T) of 1:1 (4.0x104:4.0x104 cells in 200 μl). After 24 hours, supernatants were collected and IL-2 was measured using a bead-based multiplex assay. Figure 14A shows the results for donor A. Figure 14B shows the results for donor B.

[0050] [Figure 15] Figures 15A and 15B represent tumor necrosis factor alpha (TNF-α) secretion of CD371-targeted CAR T cells (second generation B10-based CAR T cells, i.e., B10-HL and B10LH-based CAR T cells). Human CD371-targeted CAR T cells were co-cultured alone, with CD371-negative U937 cells, or with CD371+ U937 cells at an effector:tumor ratio (E:T) of 1:1 (4.0×104:4.0×104 cells in 200 μl). After 24 hours, supernatants were collected and TNF-α was measured using a bead-based multiplex assay. Figure 15A shows the results for donor A. Figure 15B shows the results for donor B.

[0051] [Figure 16]Figures 16A and 16B represent cell proliferation of B10-based CAR T cells in a return stimulation assay. CD371-targeted CAR T cells were generated from two healthy donors (Donor A and Donor B). CAR T cells were co-cultured with CD371+U937gL at an E:T ratio of 1:5 and a concentration of 50,000 CAR positive / ml. Approximately every 5 days, CAR T cells were counted and characterized by flow cytometry. The starting number of tumor cells was added back to the culture (indicated by arrows). Figure 16A shows the results for Donor A. Figure 16B shows the results for Donor B.

[0052] [Figure 17] Figure 17 illustrates a mouse xenograft model of AML. NCG mice were inoculated with 5x104 U937gL AML FAB-M5 cell lines via tail vein. CD371-targeted CAR T cells were administered to the mice 3 days later. Tumor dynamics were non-invasively measured by bioluminescence imaging approximately every 5 days, and survival was monitored.

[0053] [Figure 18] Figure 18 represents the survival of AML cell line xenografted mice (donor 1) treated with human CD371-targeted CAR T cells. NCG mice were inoculated with 5x104 U937gL tumor cells and treated 3 days later with approximately 1.25x106 human CD371-targeted CAR T cells. Mice survival was monitored.

[0054] [Figure 19] Figure 19 shows the survival of AML cell line xenografted mice (donor 2) treated with human CD371-targeted CAR T cells. NCG mice were inoculated with U937gL tumor cells and treated with approximately 1.25x106 human CD371-targeted CAR T cells 3 days later. Mice were monitored for survival.

[0055] [Figure 20]Figure 20 shows the in vivo activity of B10-based CAR T cells. NCG mice were inoculated with 5x104 U937gL tumor cells and treated 3 days later with various doses of CD371-targeted CAR T cells (1.25x105, 2.5x105, 5.0x105 and 1.0x106). Mice survival was monitored within 55 days (Figure 20A) and 60 days (Figure 20B). [Figure 21] FIG. 21 depicts binding of scFv to HEK293 cells expressing human CD371.

[0056] [Figure 22] Figure 22 shows the in vitro cytotoxicity of second generation B10HL-based CAR T cells against CD371-positive targets. B10HL-based CAR T cells have different lengths of G4S linkers. CD371-targeted CAR T cells from healthy human donors (including donors A and B) were co-cultured with antigen-positive U937 cells expressing GFP and firefly luciferase (U937gL) at different effector:tumor ratios. Bioluminescence was measured after 24 hours and plotted as a percentage of the signal detected in the co-culture of non-functional CD371-targeted CAR T cells (based on B10L4H but without CD28 or CD3 zeta signaling domains) and U937gL.

[0057] [Diagram 23] Figure 23 shows the in vivo activity of B10-based CAR T cells. NCG mice were inoculated with 5x104 U937gL tumor cells and treated with 5x105 CAR T cells. Mice were monitored for survival. B10H3L, B10H5L and IL33-secreting B10H4L human CD371-targeted CAR T cells outperformed B10H4L-based CAR T cells in vivo.

[0058] [Figure 24]Figure 24 shows the in vivo activity of B10-based CAR T cells. NCG mice were inoculated with 5x104 U937gL tumor cells and treated with 2.5x105 CAR T cells. Mice were monitored for survival. B10H4L human CD371-targeted CAR T cells secreting IL18 and IL33 outperformed B10H4L-based CAR T cells in vivo.

[0059] [Diagram 25] Figure 25 represents the in vivo activity of B10-based CAR T cells. NCG mice were inoculated with 5x104 U937gL tumor cells and treated with 1.0x105 CAR T cells. Mice were monitored for survival. At a low dose of 1.0x105, IL18-secreting B10H4L human CD371-targeted CAR T cells outperformed all other constructs in vivo. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] 5. Detailed Description of the Invention The subject matter of the present disclosure provides an antigen recognition receptor (e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR)) that specifically targets CD371. The subject matter of the present disclosure further provides a cell comprising such a receptor. The cell can be an immune response cell, e.g., a genetically modified immune response cell (e.g., a T cell or an NK cell). The subject matter of the present disclosure also provides a method of using such a cell for treatment, e.g., to treat and / or prevent a tumor or neoplasm (e.g., AML).

[0061] Non-limiting embodiments of the present disclosure are described herein and by way of examples.

[0062] For purposes of clarity of disclosure, and not for purposes of limitation, the detailed description is set forth in the following subsections: 5.1. Definition; 5.2.CD371; 5.3. Antigen recognition receptors; 5.4.Cell; 5.5. Compositions and Vectors; 5.6. Polypeptides; 5.7. Formulation and Administration; 5.8. Method of treatment; and 5.9.Kit Divide into: 5.1.Definition

[0063] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide those of ordinary skill in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Journal of Microbiology and Molecular Biology (2nd ed. 1994); 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). As used herein, the following terms have the following meanings unless otherwise specified: have the meaning ascribed to them.

[0064] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on the method by which the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, as practiced in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably 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-fold, more preferably within 2-fold of a value.

[0065] "Immune response cell" refers to a cell that functions in an immune response, or its progenitor or progeny. In certain embodiments, the immune response cell is a cell of lymphoid lineage. Non-limiting examples of cells of lymphoid lineage include T cells, natural killer (NK) cells, B cells, and stem cells from which lymphoid cells can differentiate. In certain embodiments, the immune response cell is a cell of myeloid lineage.

[0066] "Activating immune response cells" refers to the induction of signal transduction or protein expression changes in cells that result in the initiation of an immune response. For example, when CD3 chains cluster in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs), a signal transduction cascade occurs. In certain embodiments, when endogenous TCR or exogenous CAR binds to an antigen, the formation of an immune synapse occurs, which involves 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 then initiates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce the global gene expression of T cells to increase the production of IL-2 for the proliferation and expression of master regulator T cell proteins to initiate a T cell-mediated immune response.

[0067] "Stimulating immune response cells" refers to signals that result in a robust and long-lasting immune response. In various embodiments, this occurs after activation of immune cells (e.g., T cells) or is mediated simultaneously through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, and ICOS. Receiving multiple stimulatory signals may be important to boost a robust and long-lasting T cell-mediated immune response. T cells may be rapidly inhibited and become unresponsive to antigens. The effects of these costimulatory signals may vary, but they generally result in increased gene expression to generate long-lived, proliferative, anti-apoptotic T cells that robustly respond to antigens for complete and sustained eradication.

[0068] The term "antigen-recognizing receptor" as used herein refers to a receptor that can recognize a target antigen (e.g., CD371). In certain embodiments, an antigen-recognizing receptor can activate an immune cell or immune response cell (e.g., T cell) upon its binding to a target antigen.

[0069] As used herein, the term "antibody" 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 commonly 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 intact antibodies, are cleared more rapidly from circulation and may have less nonspecific tissue binding of intact antibodies (Wahl et al.,. Nucl Med (1983);24:316-325). As used herein, the term includes whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and atypical 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 C L The light chain constant region consists of the C L It consists of one domain: V H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V Lis 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.

[0070] As used herein, "CDR" is defined as the amino acid sequences of the complementarity determining regions of an antibody, which are the hypervariable regions of the immunoglobulin heavy and light chains. See, e.g., Kabat et al. 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 comprises three heavy chain CDRs and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for antibody binding to antigen or epitope. In certain embodiments, CDR regions are represented using the IMGT numbering system. In certain embodiments, CDR regions are represented using the IMGT numbering system, which can be accessed at http: / / www.imgt.org / IMGT_vquest / input.

[0071] As used herein, the term "single chain variable fragment" or "scFv" refers to a V H Heavy chains (V, VL) of immunoglobulins (e.g., mouse or human) covalently linked to form a VL heterodimer. H ) and light chain (VL ) is a fusion protein of the variable regions of the heavy chain (V H ) and light chain (V L ) are either directly bonded or V H N-terminus of V L and the C-terminus of V H The C-terminus of V L The linker is joined by a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids) that connects the N-terminus of the extracellular antigen-binding domain to the N-terminus of the extracellular antigen-binding domain. The linker is usually glycine-rich for flexibility, as well as serine or threonine-rich for solubility. The linker can link the heavy and light chain variable regions of the extracellular antigen-binding domain. Non-limiting examples of linkers are described in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014 / 087010. and the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the linker is a G4S linker.

[0072] In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 13, which is provided below. GGGGSGGGGSGGGSGGGGS [SEQ ID NO: 13]

[0073] In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 14, which is provided below. GGGGSGGGGSGGGGS [SEQ ID NO: 14]

[0074] In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:91, which is provided below. GGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 91]

[0075] In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:92, which is provided below. GGGGSGGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 92]

[0076] In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:93, which is provided below. GGGGS [SEQ ID NO: 93]

[0077] In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:94, which is provided below. GGGGSGGGGS [SEQ ID NO: 94]

[0078] 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 are described in Huston, et al. Proc. Nat. Acad. Sci. USA, (1988);85:5879-5883; U.S. Patent No. 5,091,512. 3, 5,132,405 and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754, H and V L The scFv can be expressed from a nucleic acid containing the coding sequence. Antagonistic scFvs with inhibitory activity have been described (e.g., Zhao et al., Hyrbidoma (Larchmt) (2008);27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle (2012);August 12; Shieh et al., J Imunol (2009);183(4):2277-85; Giomarrelli et al., J Immunol (2009);183(4):2277-85). 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 have been described (Peter et al., J Biol Chern (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).

[0079] The term "chimeric antigen receptor" or "CAR" as used herein refers to a molecule that comprises an extracellular antigen-binding domain and a transmembrane domain fused to an intracellular signaling domain that can activate or stimulate immune response cells. In certain embodiments, the extracellular antigen-binding domain of a CAR comprises an scFv. The scFv can be derived from the fusion of the variable heavy and light chain regions of an antibody. Alternatively, or in addition, the scFv can be derived from that of a Fab (e.g., obtained from a Fab library instead of from an antibody). In certain embodiments, the scFv is fused to a transmembrane domain and then fused to an intracellular signaling domain. "Substantially identical" or "substantially homologous" refers to 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 sequences are 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.

[0080] Sequence identity can be determined using sequence analysis software (e.g., Genetics Computer The degree of identity can be measured by using sequence analysis software packages (BLAST, BESTFIT, GAP or PILEUP / PRETTYBOX programs) from the BLAST 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. In an exemplary approach to determine the degree of identity, the BLAST program can be used with a probability score of e-3 to e-100, which indicates closely related sequences.

[0081] An "effective amount" is an amount sufficient to affect beneficial or desired clinical results upon treatment. An effective amount can be administered to a subject in one or more doses. In certain embodiments, an effective amount can be an amount sufficient to palliate, improve, stabilize, reverse, or delay the progression of a disease, or otherwise 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 skill of a person of ordinary skill in the art. Several factors are typically considered when determining the appropriate dosage to achieve an effective amount. 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 being administered.

[0082] As used herein, the term "endogenous" refers to a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue.

[0083] 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" encompasses any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous and overexpressed nucleic acid molecules and polypeptides. An "exogenous" nucleic acid refers to a nucleic acid that is not present in a native wild-type cell, for example, an exogenous nucleic acid may vary from its endogenous counterpart by sequence, by position / location, or both. For clarity, an exogenous nucleic acid may have the same or different sequence compared to its native endogenous counterpart, which may be introduced by genetic engineering into the cell itself or its precursor cell, and may be linked to alternative control sequences, such as non-native promoters or secretion sequences, as necessary.

[0084] 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 the cell or in a sample obtained from the 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.

[0085] By "modulate" is meant to alter, 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%.

[0086] By "increase" is meant a positive alteration of at least about 5%. The alteration can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100%, or more.

[0087] By "reduce" is meant to negatively alter by at least about 5%. The alteration may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0088] The terms "isolated," "purified," or "biologically pure" refer to material that is free to various degrees from components that normally accompany it as found in the material's native state. "Isolate" refers to a degree of separation from the original source or surroundings. "Purify" refers to a degree of separation that is greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not significantly affect the biological properties of the protein or cause other deleterious consequences. That is, a nucleic acid or peptide is purified if it is substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA techniques, or chemical precursors or other chemicals if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can refer to a nucleic acid or protein that gives rise to essentially one band in an electrophoretic gel. For proteins that can be subject to modifications, such as phosphorylation or glycosylation, different modifications can give rise to different isolated proteins that can be purified separately.

[0089] By "isolated cell" is meant a cell that has been separated from the molecular and / or cellular components that naturally accompany the cell.

[0090] The term "antigen-binding domain," as used herein, refers to a domain that is capable of specifically binding to a particular antigenic determinant or group of antigenic determinants present on a cell.

[0091] "Neoplasm" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion into other tissues or organs. Neoplastic growth is typically uncontrolled and progressive, occurring under conditions that would not induce or cause the cessation of normal cell proliferation. Neoplasms can affect a variety of cell types, tissues, or organs, including, but not limited to, organs selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, genitourinary tract, ureter, urethra, uterus, and vagina, or tissues or cell types thereof. Neoplasia includes cancers, such as sarcoma, carcinoma, or plasmacytoma (malignant tumor of plasma cells). Neoplasia can be a primary tumor or a primary cancer. In addition, neoplasia can be in a metastatic state.

[0092] By "receptor" is meant a polypeptide or portion thereof present on a cell membrane that selectively binds one or more ligands.

[0093] "Recognize" refers to selective binding to a target. T cells that recognize a tumor can express a receptor (e.g., TCR or CAR) that binds to a tumor antigen.

[0094] "Reference" or "control" refers to a standard for comparison. For example, the level of scFv-antigen binding by a cell expressing a CAR and an scFv can be compared to the level of scFv-antigen binding in a corresponding cell expressing a CAR alone.

[0095] By "secreted" is meant a polypeptide that is released from the cell via the secretory pathway as vesicles that pass through the endoplasmic reticulum, the Golgi apparatus, and transiently fuse with the cell plasma membrane, releasing the protein outside the cell.

[0096] By "signal sequence" or "leader sequence" is meant a peptide sequence (e.g., 5, 10, 15, 20, 25 or 30 amino acids) present at the N-terminus of a newly synthesized protein that directs entry of the protein into the secretory pathway.

[0097] By "specifically binds" or "specifically binds to" or "specifically targets" is meant a polypeptide or fragment thereof that recognizes and / or binds to a biological molecule of interest (e.g., a polypeptide, e.g., a CD371 polypeptide) but does not substantially recognize and / or bind to other molecules in a sample, e.g., a biological sample, that naturally contains a polypeptide of the present disclosure (e.g., a CD371 polypeptide).

[0098] The terms "comprises" and "comprising" are intended to have the broad meaning ascribed to them in U.S. patent law and can mean "includes," "including," etc.

[0099] As used herein, "treatment" refers to clinical intervention that seeks to change the disease course of the individual or cell being treated, and can be performed for prevention or during the course of clinical pathology. The therapeutic effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. By preventing the progression of a disease or disorder, treatment can prevent the deterioration caused by the disorder in an affected or diagnosed subject, or a subject suspected of having the disorder, and treatment can prevent the onset of the disorder or symptoms of the disorder in a subject at risk of the disorder or a subject suspected of having the disorder.

[0100] 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 pet animals. 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. The term "immunocompromised" as used herein refers to a subject with an immune deficiency. The subject is highly vulnerable to opportunistic infections, which are infections caused by organisms that do not normally cause disease in people with healthy immune systems, but can affect people with poorly functioning or suppressed immune systems.

[0101] Other aspects of the presently disclosed subject matter are described in the following disclosure and are within the scope of the presently disclosed subject matter. 5.2.CD371

[0102] CD371 (CEC12A), also known as DCAL-2, MICL or CLL-1, is a 30 kD C-type lectin transmembrane glycoprotein. It is expressed in monocytes, granulocytes, natural killer (NK) cells and basophils. CD371 is an immune inhibitory receptor that recruits the Src homology 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). CD371 is involved in autoimmune and CD371 has been implicated as a negative regulatory urate crystal (monosodium urate, MSU) receptor that controls immune and inflammatory diseases (Neumann et al., Immunity (2014);40:389-99). It 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).

[0103] In certain embodiments, the CD371 is human CD371 comprising or consisting of the amino acid sequence having NCBI reference number: NP_612210.4 (SEQ ID NO: 15), or a fragment thereof. SEQ ID NO:15 is provided below. [ka]

[0104] In certain embodiments, CD371 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 the amino acid sequence set forth in SEQ ID NO: 15, or a fragment thereof. 5.3. Antigen Recognition Receptors

[0105] The antigen-recognizing receptor of the present disclosure specifically targets or binds to CD371.In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR).In certain embodiments, the antigen-recognizing receptor is a T cell receptor (TCR).In certain embodiments, the antigen-recognizing receptor is a TCR-like fusion molecule.

[0106] The subject matter of the present disclosure also provides a nucleic acid molecule encoding an antigen-recognizing receptor of the present disclosure. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide of a CD371-targeting antigen-recognizing receptor disclosed herein. T cell receptor (TCR)

[0107] In certain embodiments, the antigen recognition receptor is a TCR. A TCR is a disulfide-linked heterodimeric protein consisting of two variable chains expressed as part of a complex with an invariant CD3 chain molecule. The TCR found on the surface of T cells is responsible for recognizing antigens as peptides that bind to major histocompatibility complex (MHC) molecules. In certain embodiments, the TCR comprises an alpha chain and a beta chain (encoded by TRA and TRB, respectively). In certain embodiments, the TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0108] Each chain of the TCR is composed of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is proximal to the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. The variable domains of both chains each have three complementarity determining regions (CDRs).

[0109] In certain embodiments, the TCR can form a receptor complex with three dimeric signaling modules CD3δ / ε, CD3γ / ε and CD247ζ / ζ or ζ / η. When the TCR complex binds to its antigen and MHC (peptide / MHC), the T cell expressing the TCR complex is activated.

[0110] In certain embodiments, the TCR is an endogenous TCR. In certain embodiments, the antigen recognition receptor is a naturally occurring TCR.

[0111] In certain embodiments, the antigen-recognizing receptor is an exogenous TCR. In certain embodiments, the antigen-recognizing receptor is a recombinant TCR. In certain embodiments, the antigen-recognizing receptor is a non-naturally occurring TCR. In certain embodiments, the non-naturally occurring TCR differs from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, the non-naturally occurring TCR differs from any naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more amino acid residues. In certain embodiments, the non-naturally occurring TCR is modified by at least one amino acid residue from a naturally occurring TCR. In certain embodiments, a non-naturally occurring TCR has at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, or more amino acid residues altered from a naturally occurring TCR. Chimeric Antigen Receptors (CARs)

[0112] In certain embodiments, the antigen recognition receptor is CAR.CAR is an engineered receptor that grafts or imparts the specificity of interest onto immune effector cells.CAR can be used to graft the specificity of monoclonal antibodies onto T cells, and the transfer of their coding sequences is facilitated by retroviral vectors.

[0113] There are three generations of CARs. "First generation" CARs are typically composed of an extracellular antigen binding domain (e.g., scFv) fused to a transmembrane domain fused to a cytoplasmic / intracellular signaling domain. "First generation" CARs can provide de novo antigen recognition and bind to CD4 through their CD3 ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. + and CD8+ The "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 the T cell. "Second generation" CARs include those that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). "Third generation" CARs include those that provide multiple costimulations (e.g., CD28 and 4-1BB) and activation (CD3ζ). In certain embodiments, the antigen recognition receptor is a first generation CAR. In certain embodiments, the antigen recognition receptor is a CAR that does not include the intracellular signaling domain of a costimulatory molecule or a fragment thereof. In certain embodiments, the antigen recognition receptor is a second generation CAR.

[0114] In certain embodiments, the CAR comprises an extracellular antigen-binding domain that specifically binds CD371, a transmembrane domain, and an intracellular signaling domain. 5.3.2.1. Extracellular Antigen-Binding Domain of the CAR

[0115] In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a mouse scFv. In certain embodiments, the scFv is identified by screening an scFv phage library with an antigen-Fc fusion protein.

[0116] In certain embodiments, the extracellular antigen-binding domain is a Fab. In certain embodiments, the Fab is cross-linked. In certain embodiments, the extracellular antigen-binding domain is a F(ab)2.

[0117] Any of the above molecules may be included in a fusion protein with a heterologous sequence to form an extracellular antigen-binding domain. In certain non-limiting embodiments, the extracellular antigen-binding domain of the CAR (e.g., embodied in an scFv or analog thereof) is about 1×10 -6 M or less, e.g., about 1×10 -7 M or less, approximately 1 x 10 -8 M or less, approximately 1 x 10 -9 M or less, approximately 1 x 10 -10 M or less, or about 1 x 10 -11 M or a dissociation constant (K d ) and binds to CD371 (e.g., human CD371). In certain embodiments, the extracellular antigen-binding domain of the CAR has a molecular weight of about 1×10 -7 M or less K d In certain embodiments, the extracellular antigen-binding domain of the CAR binds to CD371 (e.g., human CD371) at about 1×10 -8 M or less K d In certain embodiments, the extracellular antigen-binding domain of the CAR binds to CD371 (e.g., human CD371) at about 1.5×10 -8 M or approx. 1×10 -8 K of M d In certain embodiments, the extracellular antigen-binding domain of the CAR binds to CD371 (e.g., human CD371) at about 1×10 -8 M ~ approx. 1×10 -7 K of M d It binds to CD371 (e.g., human CD371) at .

[0118] In certain embodiments, the extracellular antigen-binding domain of the CAR binds to CD371 (e.g., human CD371) with low binding affinity. In certain embodiments, the extracellular antigen-binding domain of the CAR binds to CD371 (e.g., human CD371) with low binding affinity. -8 M or higher, approximately 1×10 -8 M or higher, approximately 1×10 -7 M or higher, or about 1×10 -6M or higher K d It binds to CD371 (e.g., human CD371) at .

[0119] The binding of the extracellular antigen-binding domain of 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 interest by using a labeled reagent (e.g., antibody or scFv) specific for the complex of interest. For example, scFv can be radiolabeled and used in radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated herein by reference). Radioisotopes can be detected by means such as using a gamma counter or scintillation counter, or by autoradiography.In certain embodiments, CD371-targeting extracellular antigen binding domain 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).In one embodiment, CD371-targeting human scFv is labeled with GFP.

[0120] In certain embodiments, the CDRs are identified according to the IMGT numbering system.

[0121] In certain embodiments, the extracellular antigen-binding domain of the CAR (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:1. H For example, the extracellular antigen binding domain of the CAR (e.g., scFv) can comprise a VFv 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:1. H In certain embodiments, the extracellular antigen-binding domain comprises a V H SEQ ID NO:1 is provided in Table 1 below.

[0122] In certain embodiments, the extracellular antigen-binding domain of the CAR (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:2. L For example, the extracellular antigen binding domain of the CAR (e.g., scFv) can comprise a VFv 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:2. L In certain embodiments, the extracellular antigen-binding domain comprises a V L SEQ ID NO:2 is provided in Table 1 below.

[0123] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 28, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:29 or a conservative modification thereof HCDR2 and a V H SEQ ID NOs: 28-30 are provided in Table 1.

[0124] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 31, or a conservative modification thereof. L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:32 or a conservative modification thereof L CDR2 and a V L SEQ ID NOs: 31-33 are provided in Table 1.

[0125] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 28, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:29 or a conservative modification thereof H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO: 30 or a conservative modification thereof H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:31 or a conservative modification thereof L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:32 or a conservative modification thereof L CDR2 and a V L Includes CDR3.

[0126] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO: 28. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:29 H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO:30 H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:31 L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:32 L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:33 LIncludes CDR3.

[0127] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:1. H and V comprising the amino acid sequence set forth in SEQ ID NO:2 L In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:13.

[0128] In certain embodiments, the variable region within the extracellular antigen-binding domain of the CAR comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable region is V from the N-terminus to the C-terminus. H -V L In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 107, which is provided in Table 1. In certain embodiments, the scFv is designated "B10H4L."

[0129] In certain embodiments, the variable region within the extracellular antigen-binding domain of the CAR comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable region is V from the N-terminus to the C-terminus. L -V H In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the scFv is designated "B10L4H". An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 is set forth in SEQ ID NO: 22. SEQ ID NOs: 16 and 22 are provided in Table 1 below. Table 1 [Table 1]

[0130] In certain embodiments, the extracellular antigen binding domain of the CAR (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 sequence set forth in SEQ ID NO:3. H For example, the extracellular antigen binding domain of the CAR (e.g., scFv) can comprise a VFv that comprises 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:3. H In certain embodiments, the extracellular antigen-binding domain comprises a V H SEQ ID NO:3 is provided in Table 2 below.

[0131] In certain embodiments, the extracellular antigen binding domain of the CAR (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:4. L For example, the extracellular antigen binding domain of the CAR (e.g., scFv) can comprise a VFv that comprises 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:4. L In certain embodiments, the extracellular antigen-binding domain comprises a V L SEQ ID NO:4 is provided in Table 2 below.

[0132] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) of the CAR comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO:3, as shown in Table 2. H In certain embodiments, the anti-CD371 scFv comprises a VFv comprising the amino acid sequence set forth in SEQ ID NO:4. L In certain embodiments, the anti-CD371 scFv comprises a VFv comprising the amino acid sequence set forth in SEQ ID NO:3. H and V comprising the amino acid sequence set forth in SEQ ID NO:4 L Includes.

[0133] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 34, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:35 or a conservative modification thereof H CDR2 and a V H SEQ ID NOs: 34-36 are provided in Table 2.

[0134] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 37, or a conservative modification thereof. L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:38 or a conservative modification thereof L CDR2 and a V L SEQ ID NOs: 37-39 are provided in Table 2.

[0135] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 34, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:35 or a conservative modification thereof H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO:36 or a conservative modification thereof H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:37 or a conservative modification thereof LCDR1, V comprising the amino acid sequence set forth in SEQ ID NO:38 or a conservative modification thereof L CDR2 and a V L Includes CDR3.

[0136] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO: 34. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:35 H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO:36 H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:37 L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:38 L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:39 L Includes CDR3.

[0137] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) of the CAR comprises the amino acid sequence set forth in SEQ ID NO:3. H and V comprising the amino acid sequence set forth in SEQ ID NO:4 L In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:13.

[0138] In certain embodiments, the variable region within the extracellular antigen-binding domain of the CAR comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable region is V from the N-terminus to the C-terminus. H -V L In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 108, which is provided in Table 2. In certain embodiments, the scFv is designated "C3H4L."

[0139] In certain embodiments, the variable region within the extracellular antigen-binding domain of the CAR comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable region is V from the N-terminus to the C-terminus. L -V H In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the scFv is designated "C3L4H". An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 17 is set forth in SEQ ID NO: 23. SEQ ID NOs: 17 and 23 are provided in Table 2 below. Table 2 [Table 2-1] [Table 2-2]

[0140] In certain embodiments, the extracellular antigen binding domain of the CAR (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:5. H For example, the extracellular antigen binding domain of the CAR (e.g., scFv) can comprise a VFv that comprises 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:5. H In certain embodiments, the extracellular antigen-binding domain comprises a V H SEQ ID NO:5 is provided in Table 3 below.

[0141] In certain embodiments, the extracellular antigen binding domain of the CAR (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 sequence set forth in SEQ ID NO:6. L For example, the extracellular antigen binding domain of the CAR (e.g., scFv) can comprise a VFv that comprises 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:6. L In certain embodiments, the extracellular antigen-binding domain comprises a V L SEQ ID NO:6 is provided in Table 3 below.

[0142] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 40, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 41 or a conservative modification thereof H CDR2 and a V H SEQ ID NOs: 40-42 are provided in Table 3.

[0143] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 43, or a conservative modification thereof. L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:44 or a conservative modification thereof L CDR2 and a V L SEQ ID NOs: 43-45 are provided in Table 3.

[0144] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 40, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 41 or a conservative modification thereof H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO: 42 or a conservative modification thereof H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:43 or a conservative modification thereof L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:44 or a conservative modification thereof L CDR2 and a V L Includes CDR3.

[0145] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO: 40. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:41 H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO:42 H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:43 L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:44 L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:45 L Includes CDR3.

[0146] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) of the CAR comprises the amino acid sequence set forth in SEQ ID NO:5. H and V comprising the amino acid sequence set forth in SEQ ID NO:6 L In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:13.

[0147] In certain embodiments, the variable region within the extracellular antigen-binding domain of the CAR comprises a heavy chain variable region (V HIn certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable region is V from the N-terminus to the C-terminus. H -V L In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 109, which is provided in Table 3. In certain embodiments, the scFv is designated "D6H4L."

[0148] In certain embodiments, the variable region within the extracellular antigen-binding domain of the CAR comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable region is V from the N-terminus to the C-terminus. L -V H In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the scFv is designated "D6L4H". An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 18 is set forth in SEQ ID NO: 24. SEQ ID NOs: 18 and 24 are provided in Table 3 below. Table 3 [Table 3-1] [Table 3-2]

[0149] In certain embodiments, the extracellular antigen binding domain of the CAR (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 sequence set forth in SEQ ID NO:7. HFor example, the extracellular antigen binding domain of the CAR (e.g., scFv) can comprise a VFv that comprises 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:7. H In certain embodiments, the extracellular antigen-binding domain comprises a V H SEQ ID NO:7 is provided in Table 4 below.

[0150] In certain embodiments, the extracellular antigen binding domain of the CAR (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 sequence set forth in SEQ ID NO:8. L For example, the extracellular antigen binding domain of the CAR (e.g., scFv) can comprise a VFv that comprises 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:8. L In certain embodiments, the extracellular antigen-binding domain comprises a V L SEQ ID NO:8 is provided in Table 4 below.

[0151] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 46, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:47 or a conservative modification thereof H CDR2 and a V H SEQ ID NOs: 46-48 are provided in Table 4.

[0152] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 49, or a conservative modification thereof. L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:50 or a conservative modification thereof L CDR2 and a V L SEQ ID NOs: 49-51 are provided in Table 4.

[0153] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 46, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:47 or a conservative modification thereof H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO:48 or a conservative modification thereof H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:49 or a conservative modification thereof L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:50 or a conservative modification thereof L CDR2 and a V L Includes CDR3.

[0154] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO: 46. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:47 H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO:48 H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:49 L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:50 L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:51 L Includes CDR3.

[0155] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:7. Hand V comprising the amino acid sequence set forth in SEQ ID NO:8 L In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:13.

[0156] In certain embodiments, the variable region within the extracellular antigen-binding domain of the CAR comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable region is V from the N-terminus to the C-terminus. H -V L In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 110, which is provided in Table 4. In certain embodiments, the scFv is designated "A11H4L."

[0157] In certain embodiments, the variable region within the extracellular antigen-binding domain of the CAR comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable region is V from the N-terminus to the C-terminus. L -V H In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the scFv is designated "A11L4H". An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 19 is set forth in SEQ ID NO: 25. SEQ ID NOs: 19 and 25 are provided in Table 4 below. Table 4 [Table 4-1] [Table 4-2]

[0158] In certain embodiments, the extracellular antigen binding domain of the CAR (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 sequence set forth in SEQ ID NO:9. H For example, the extracellular antigen binding domain of the CAR (e.g., scFv) can comprise a VFv that comprises 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:9. H In certain embodiments, the extracellular antigen-binding domain comprises a V H SEQ ID NO:9 is provided in Table 5 below.

[0159] In certain embodiments, the extracellular antigen binding domain of the CAR (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 sequence set forth in SEQ ID NO: 10. L For example, the extracellular antigen binding domain of the CAR (e.g., scFv) can comprise a VFv that comprises 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:10. L In certain embodiments, the extracellular antigen-binding domain comprises a V L SEQ ID NO: 10 is provided in Table 5 below.

[0160] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 52, or a conservative modification thereof. HCDR1, V comprising the amino acid sequence set forth in SEQ ID NO:53 or a conservative modification thereof H CDR2 and a V H SEQ ID NOs: 52-54 are provided in Table 5.

[0161] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 55, or a conservative modification thereof. L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:56 or a conservative modification thereof L CDR2 and a V L SEQ ID NOs: 55-57 are provided in Table 5.

[0162] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 52, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:53 or a conservative modification thereof H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO:54 or a conservative modification thereof H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:55 or a conservative modification thereof L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:56 or a conservative modification thereof L CDR2 and a V L Includes CDR3.

[0163] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:52. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:53 H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO:54 H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:55 L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:56 LCDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:57 L Includes CDR3.

[0164] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:9. H and V comprising the amino acid sequence set forth in SEQ ID NO:10 L In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:13.

[0165] In certain embodiments, the variable region within the extracellular antigen-binding domain of the CAR comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable region is V from the N-terminus to the C-terminus. H -V L In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 111, which is provided in Table 5. In certain embodiments, the scFv is designated "E4H4L."

[0166] In certain embodiments, the variable region within the extracellular antigen-binding domain of the CAR comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable region is V from the N-terminus to the C-terminus. L -V H In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the scFv is designated "E4L4H". An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 20 is set forth in SEQ ID NO: 26. SEQ ID NOs: 20 and 26 are provided in Table 5 below. Table 5 [Table 5-1] [Table 5-2]

[0167] In certain embodiments, the extracellular antigen binding domain of the CAR (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 sequence set forth in SEQ ID NO:11. H For example, the extracellular antigen binding domain of the CAR (e.g., scFv) can comprise a VFv that comprises 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:11. H In certain embodiments, the extracellular antigen-binding domain comprises a V H SEQ ID NO:11 is provided in Table 6 below.

[0168] In certain embodiments, the extracellular antigen binding domain of the CAR (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 sequence set forth in SEQ ID NO: 12. L For example, the extracellular antigen binding domain of the CAR (e.g., scFv) can comprise a VFv that comprises 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:12. L In certain embodiments, the extracellular antigen-binding domain comprises a V LSEQ ID NO:12 is provided in Table 6 below.

[0169] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 58, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:59 or a conservative modification thereof H CDR2 and a V H SEQ ID NOs: 58-60 are provided in Table 6.

[0170] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 61, or a conservative modification thereof. L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:62 or a conservative modification thereof L CDR2 and a V L SEQ ID NOs: 61-63 are provided in Table 6.

[0171] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 58, or a conservative modification thereof. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:59 or a conservative modification thereof H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO: 60 or a conservative modification thereof H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO: 61 or a conservative modification thereof L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:62 or a conservative modification thereof L CDR2 and a V L Includes CDR3.

[0172] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:58. HCDR1, V comprising the amino acid sequence set forth in SEQ ID NO:59 H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO:60 H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:61 L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:62 L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:63 L Includes CDR3.

[0173] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:11. H and V comprising the amino acid sequence set forth in SEQ ID NO:12 L In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:13.

[0174] In certain embodiments, the variable region within the extracellular antigen-binding domain of the CAR comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable region is V from the N-terminus to the C-terminus. H -V L In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 112, which is provided in Table 6. In certain embodiments, the scFv is designated "E8H4L."

[0175] In certain embodiments, the variable region within the extracellular antigen-binding domain of the CAR comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable region is V from the N-terminus to the C-terminus. L -V HIn certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO:21. In certain embodiments, the scFv is designated "E8L4H". An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:21 is set forth in SEQ ID NO:27. SEQ ID NOs:21 and 27 are provided in Table 6 below. Table 6 [Table 6-1] [Table 6-2]

[0176] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding properties of the mesothelin-targeting CAR of the present disclosure (e.g., the extracellular antigen-binding domain of the CAR) that contains 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 CAR 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 charge and polarity. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid in the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, histidine, negatively charged amino acids include aspartic acid, glutamic acid, and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Additionally, amino acids can be classified by 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 a CDR region can be replaced with other amino acid residues from the same group, and the altered antibody can be tested for retained function (i.e., the functions described in (c)-(l) above) using the functional assays described herein. In certain embodiments, no more than 1, no more than 2, no more than 3, no more than 4, no more than 5 residues in a particular sequence or in a CDR region are altered.

[0177] 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:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12). H and / or V L The amino acid sequence may contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to a particular sequence, but may retain the ability to bind to a target antigen (e.g., mesothelin). In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in a particular sequence (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12). In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs of the extracellular antigen-binding domain (e.g., in the FRs). In certain embodiments, the extracellular antigen-binding domain comprises a V-terminal amino acid sequence selected from SEQ ID NOs:1-12. H and / or V L The nucleic acid sequence includes a sequence, including post-translational modifications of that sequence (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12).

[0178] As used herein, the percentage of homology between two amino acid sequences is equal to the percentage of identity between the two sequences.The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., homology%=number of identical positions / total number of positions×100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.Comparing the sequences and determining the percentage of identity between two sequences can be achieved using mathematical algorithms.

[0179] 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)) incorporated into the ALIGN program (version 2.0) using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program in the GCG software package (available at www.gcg.com) using either a Blossum62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. The rhythm can be used to determine.

[0180] Additionally, or alternatively, the amino acid sequences of the presently disclosed subject matter can be further used as a "query sequence" to perform a search against public databases, e.g., to identify related sequences. Such searches can 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 with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the identified 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) can be used.

[0181] In certain embodiments, the extracellular antigen-binding domain of a CAR of the disclosure is selected for binding to CD371 (e.g., human CD371), e.g., from any one of the V of the scFvs of the disclosure (e.g., V10, C3, D6, A11, E4, and D8). H CDR1, CDR2 and CDR3 sequences and V L In certain embodiments, the extracellular antigen-binding domain of the CAR of the present disclosure cross-competes with a reference antibody or antigen-binding fragment thereof comprising the CDR1, CDR2 and CDR3 sequences. In certain embodiments, the extracellular antigen-binding domain of the CAR of the present disclosure cross-competes with any one of the V of any one of the scFvs of the present disclosure (e.g., V10, C3, D6, A11, E4 and D8) for binding to CD371 (e.g., human CD371). H and V L It cross-competes with a reference antibody or antigen-binding portion thereof comprising the sequence.

[0182] In certain embodiments, the extracellular antigen-binding domain of a CAR of the present disclosure comprises, for binding to CD371 (e.g., human CD371), the V H CDR1, CDR2 and CDR3 sequences and V LCross-competes with a reference antibody or an antigen-binding portion thereof comprising the CDR1, CDR2 and CDR3 sequences. For example, the extracellular antigen-binding domain of the CAR of the present disclosure comprises a V-terminal end having an amino acid sequence set forth in SEQ ID NO: 28 for binding to CD371 (e.g., human CD371). H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:29 H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO:30 H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:31 L CDR1, V comprising an amino acid sequence as set forth in SEQ ID NO:32 L CDR2 and V comprising an amino acid sequence as set forth in SEQ ID NO:33 L In certain embodiments, the extracellular antigen-binding domain of the CAR of the present disclosure cross-competes with the V of scFv B10 for binding to CD371 (e.g., human CD371). H and V L For example, the extracellular antigen-binding domain of the CAR of the present disclosure may be a V-seq. H and V comprising an amino acid having the sequence set forth in SEQ ID NO:2 L or an antigen-binding portion thereof.

[0183] In certain embodiments, the extracellular antigen-binding domain binds to the same epitope on CD371 (e.g., human CD371) as the reference antibody or antigen-binding portion thereof. For example, the extracellular antigen-binding domain of a CAR of the present disclosure can be, for example, the V of any one of the scFvs of the present disclosure (e.g., B10, C3, D6, A11, E4, and E8). H CDR1, CDR2 and CDR3 sequences and V LIt binds to the same epitope on CD371 (e.g., human CD371) as a reference antibody or antigen-binding portion thereof that comprises the CDR1, CDR2, and CDR3 sequences. In certain embodiments, the extracellular antigen-binding domain of a CAR of the present disclosure is, for example, the V of any one of the scFvs of the present disclosure (e.g., B10, C3, D6, A11, E4, and E8). H and V L It binds to the same epitope on EMR2 (eg, human EMR2) as a reference antibody or antigen-binding portion thereof that comprises the sequence.

[0184] In certain embodiments, the extracellular antigen-binding domain of the CAR of the present disclosure is the V H CDR1, CDR2 and CDR3 sequences and V L It binds to the same epitope on CD371 (e.g., human CD371) as a reference antibody or antigen-binding fragment thereof that comprises the CDR1, CDR2 and CDR3 sequences. For example, the extracellular antigen-binding domain of the CAR of the present disclosure comprises the V H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:29 H CDR2, V comprising the amino acid sequence set forth in SEQ ID NO:30 H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:31 L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:32 L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:33 L Binds to the same epitope on CD371 (e.g., human CD371) as a reference antibody or antigen-binding fragment thereof that comprises CDR3. In certain embodiments, the extracellular antigen-binding domain of the CAR of the present disclosure is the V H and V L Binds to the same or substantially the same epitope on CD371 (e.g., human CD371) as a reference antibody or antigen-binding fragment thereof comprising the sequence. For example, the extracellular antigen-binding domain of a CAR of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:1. H and V comprising the amino acid sequence set forth in SEQ ID NO:2 Lbinds to the same epitope on CD371 (e.g., human CD371) as a reference antibody or antigen-binding fragment thereof comprising:

[0185] Extracellular antigen-binding domains that cross-compete or compete with a reference antibody or antigen-binding portion thereof for binding to CD371 (e.g., human CD371) 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 herein by reference in its entirety. In certain embodiments, the antibody binding assay comprises measuring the initial binding of a reference antibody to a CD371 polypeptide, mixing the reference antibody with a test extracellular antigen binding domain, measuring the second binding of the reference antibody to a CD371 polypeptide in the presence of the test extracellular antigen binding domain, and comparing the initial binding of the reference antibody to the second binding, wherein a decrease in the second binding of the reference antibody to a CD371 polypeptide compared to the initial binding indicates that the test extracellular antigen binding domain cross-competes with the reference antibody for binding to CD371, e.g., recognizes the same or substantially the same epitope, overlapping epitope, or adjacent epitope. In certain embodiments, the reference antibody is labeled, e.g., with a fluorescent dye, biotin, or peroxidase. In certain embodiments, the CD371 polypeptide is expressed in cells, e.g., in a flow cytometry test. In certain embodiments, the CD371 polypeptide is immobilized on a surface containing a Biacore ship (e.g., in a Biacore test) or other medium suitable for surface plasmon resonance analysis. The binding of the reference antibody in the presence of a completely unrelated antibody (that does not bind to CD371) can serve as a high control value. The low control value can be obtained by incubating the labeled reference antibody with an unlabeled reference antibody, where competition and reduced binding of the labeled reference antibody will occur. In certain embodiments, a test extracellular antigen binding domain that reduces the binding of the reference antibody to the CD371 polypeptide 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 CD371. In certain embodiments, the assay is performed at room temperature.

[0186] In certain embodiments, the antibody binding assay comprises measuring the initial binding of the test extracellular antigen binding domain to a CD371 polypeptide, mixing the test extracellular antigen binding domain with a reference antibody, measuring the second binding of the test extracellular antigen binding domain to a CD371 polypeptide in the presence of the reference antibody, and comparing the initial binding of the test extracellular antigen binding domain to the second binding, where a decrease in the second binding of the test extracellular antigen binding domain to a CD371 polypeptide compared to the initial binding indicates that the test extracellular antigen binding domain cross-competes with the reference antibody for binding to CD371, e.g., recognizes the same or substantially the same epitope, an overlapping epitope, or an adjacent epitope. In certain embodiments, the test extracellular antigen binding domain is labeled, e.g., with a fluorescent dye, biotin, or peroxidase. In certain embodiments, the CD371 polypeptide is expressed in cells, e.g., in a flow cytometry test. In certain embodiments, the CD371 polypeptide is immobilized on a surface comprising a Biacore ship (e.g., in a Biacore test) 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 to CD371) can serve as a high control value. A low control value can be obtained by incubating a labeled test extracellular antigen binding domain with an unlabeled test extracellular antigen binding domain, where competition and reduced binding of the labeled test extracellular antigen binding domain will occur. In certain embodiments, a test extracellular antigen binding domain that reduces binding to the CD371 polypeptide 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% in the presence of a reference antibody is considered to be an extracellular antigen binding domain that cross-competes with the reference antibody for binding to CD371. In certain embodiments, the assay is performed at room temperature.

[0187] In certain non-limiting embodiments, the extracellular antigen binding domain of the CAR 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 certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 91. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 92. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 93. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 94.

[0188] In certain embodiments, the variable region within the extracellular antigen-binding domain of the CAR comprises a heavy chain variable region (V H In certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable region is V from the N-terminus to the C-terminus. H -V L Located at.

[0189] In certain embodiments, the variable region within the extracellular antigen-binding domain of the CAR comprises a light chain variable region (V L In certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable region is V from the N-terminus to the C-terminus. L -V H Located at.

[0190] In addition, the extracellular antigen-binding domain can include a leader or signal peptide that directs the nascent protein to the endoplasmic reticulum. The signal peptide or leader can be essential when the CAR is glycosylated and anchored to the cell membrane. The 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 the newly synthesized protein that directs the entry of the protein into the secretory pathway. In certain embodiments, the signal peptide is covalently attached to the 5' end of the extracellular antigen-binding domain. In certain embodiments, the signal peptide comprises a CD8 polypeptide, for example, the CAR comprises a truncated CD8 signal peptide. 5.3.2.2. CAR Transmembrane Domain

[0191] In certain non-limiting embodiments, 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 cluster and a signal is transmitted to the cell. According to the subject matter of the present disclosure, the transmembrane domain of the CAR can 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.

[0192] In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., the transmembrane domain of CD8 or a fragment thereof).

[0193] In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., the transmembrane domain of CD8 or a fragment thereof). In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., the transmembrane domain of human CD8 or a fragment thereof). In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence 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: 64) or a fragment thereof, and / or optionally comprises up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 64 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acids in length. Alternatively, or in addition, in various non-limiting embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1-235, 1-50, 50-100, 100-150, 150-200, 137-209, or 200-235 of SEQ ID NO: 64. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 137-209 of SEQ ID NO: 64. SEQ ID NO: 64 is provided below. [ka]

[0194] In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., the transmembrane domain of mouse CD8 or a fragment thereof). In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence 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: 65) or a fragment thereof, and / or optionally comprises up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 65 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 247 amino acids in length. Alternatively, or in addition, in various non-limiting embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1-247, 1-50, 50-100, 100-150, 150-200, 151-219, or 200-247 of SEQ ID NO: 65. 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: 65. SEQ ID NO: 65 is provided below. [ka]

[0195] In certain embodiments, 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).

[0196] In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a fragment thereof). In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence 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: 66) or a fragment thereof, and / or optionally contains up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 66 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. Alternatively, or in addition, in various non-limiting embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 150-200, 153-179, or 200-220 of SEQ ID NO: 66. 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: 66. SEQ ID NO: 66 is provided below. [ka]

[0197] An exemplary nucleotide sequence encoding amino acids 153-179 of SEQ ID NO:66 is set forth in SEQ ID NO:67, which is provided below. [ka]

[0198] In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., the transmembrane domain of mouse CD28 or a fragment thereof). In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence 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: 68) or a fragment thereof, and / or optionally contains up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 66 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 218 amino acids in length. Alternatively, or in addition, in various non-limiting embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 150-200, 151-177, or 200-218 of SEQ ID NO: 68. 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: 68. SEQ ID NO: 68 is provided below. [ka]

[0199] In certain non-limiting embodiments, the CAR further comprises a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region can be flexible enough to allow the antigen-binding domain to be oriented in different directions to facilitate antigen recognition while retaining the activating activity of the CAR.

[0200] In certain embodiments, 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, or a fragment thereof, or a modified hinge region or fragment thereof, the native or modified hinge region of CD8b or a fragment thereof, the native or modified hinge region of ICOS or a fragment thereof, the native or modified hinge region of ICAM-1 or a fragment thereof, the native or modified hinge region of CTLA-4 or a fragment thereof, the native or modified hinge region of CD27 or a fragment thereof, the native or modified hinge region of CD40 or a fragment thereof, the native 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. The hinge / spacer region can be a hinge region from IgG1, or an immunoglobulin CH2CH3 region, and a portion of CD3, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO: 66 or 68), a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO: 64 or 65), a variation of any of the foregoing 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 thereto, or a synthetic spacer sequence. 5.3.2.3. CAR Intracellular Signaling Domain

[0201] In certain embodiments, the CAR comprises an intracellular signaling domain. In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide. CD3ζ can activate or stimulate cells (e.g., lymphoid lineage cells, e.g., T cells). Wild-type ("native") CD3ζ comprises three functional basic-rich stretch (BRS) regions (BRS1, BRS2 and BRS3) of three functional immunoreceptor tyrosine-based activation motifs (ITAMs). CD3ζ transmits activation signals to cells (e.g., lymphoid lineage 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.

[0202] In certain embodiments, the intracellular signaling domain of the CAR comprises native CD3zeta. In certain embodiments, the CD3zeta polypeptide comprises or consists of an amino acid sequence 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: 69) or a fragment thereof, and / or optionally contains up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD3zeta polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 69 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acids in length. Alternatively, or in addition, in various non-limiting embodiments, the CD3ζ polypeptide comprises or consists of the amino acid sequence of amino acids 1-164, 1-50, 50-100, 52-164, 100-150, or 150-164 of SEQ ID NO: 69. 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: 69. SEQ ID NO: 69 is provided below. [ka]

[0203] In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3 polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 89. SEQ ID NO: 89 is provided below. [ka]

[0204] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:89 is set forth in SEQ ID NO:90, which is provided below. [ka]

[0205] In certain non-limiting embodiments, the intracellular signaling domain of CAR further comprises at least a costimulatory signaling region.In certain embodiments, the costimulatory signaling region comprises at least one costimulatory molecule or a fragment thereof.In certain embodiments, the costimulatory signaling region comprises at least one intracellular domain of costimulatory molecule or a fragment thereof.

[0206] As used herein, "costimulatory molecule" refers to a cell surface molecule other than antigen receptor or its ligand, which can provide an efficient response of lymphocytes to antigen. In certain embodiments, costimulatory molecule can provide optimal lymphocyte activation. Non-limiting examples of costimulatory molecule include CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKGD2, CD2, FN14, HVEM, LTBR, ​​CD28H, TNFR1, TNFR2, BAFF-R, BCMA, TACI, TROY, RANK, CD40, CD27, CD30, EDAR, XEDAR, GITR, DR6 and NGFR, and combinations thereof. Costimulatory molecule can bind to costimulatory ligand, a protein expressed on cell surface, which upon binding to its receptor, generates a costimulatory response, i.e., an intracellular response that results in the stimulation provided when antigen recognition receptor (e.g., chimeric antigen receptor (CAR)) binds to its target antigen. As an example, 4-1BB ligand (i.e., 4-1BBL) can be used in combination with a CAR signal to induce CAR + It can bind 4-1BB to provide an intracellular signal that induces effector cell function of a T cell.

[0207] In certain embodiments, 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. The CD28 polypeptide can comprise or have 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 set forth in SEQ ID NO: 66 or a fragment thereof, and / or can optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 66 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. Alternatively, or in addition, in various non-limiting embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, 180-220, or 200-220 of SEQ ID NO: 66. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide comprising or consisting of the amino acid sequence of amino acids 180-220 of SEQ ID NO: 66.

[0208] An exemplary nucleic acid sequence encoding amino acids 180-220 of SEQ ID NO:66 is set forth in SEQ ID NO:70, which is provided below. [ka]

[0209] In certain embodiments, a CD28 polypeptide 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 set forth in SEQ ID NO:68 or a fragment thereof, and / or optionally contains up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, a CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO:68 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. Alternatively, or in addition, in various non-limiting embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 150-218, 178-218, or 200-218 of SEQ ID NO: 68. In certain embodiments, a 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: 68.

[0210] In certain embodiments, the intracellular signaling domain of the CAR comprises a 4-1BB polypeptide, e.g., a costimulatory signaling region comprising the intracellular domain of 4-1BB or a fragment thereof. The 4-1BB polypeptide can 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: 71) or a fragment thereof, and / or optionally contains up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the 4-1BB polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 71 that is at least 20, or at least 30, or at least 40, or at least 50, or at least 100, or at least 150, or at least 150, and up to 255 amino acids in length. Alternatively, or in addition, in various non-limiting embodiments, the 4-1BB polypeptide comprises or consists of the amino acid sequence of amino acids 1-255, 1-50, 50-100, 100-150, 150-200, or 200-255 of SEQ ID NO: 71. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a 4-1BB polypeptide comprising or consisting of the amino acid sequence of amino acids 214-255 of SEQ ID NO: 71. SEQ ID NO: 71 is provided below. [ka]

[0211] An exemplary nucleic acid sequence encoding amino acids 214-255 of SEQ ID NO:71 is set forth in SEQ ID NO:72, which is provided below. [ka]

[0212] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region that comprises 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. 5.3.2.4. Example CAR

[0213] In certain embodiments, the CAR is a CD371-targeting CAR. In certain embodiments, the CAR comprises (a) (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a VDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a V L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a fragment thereof), and (c) 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). In certain embodiments, H and V L is linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14. H and V L is V from the N-terminus to the C-terminus L -V H In certain embodiments, the CAR is designed as "Et.B10L3H_MT_h28Z". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 73, which is provided below. [ka]

[0214] An exemplary nucleic acid sequence for the amino acid sequence of SEQ ID NO:73 is set forth in SEQ ID NO:74, which is provided below. [ka] [ka]

[0215] In certain embodiments, the CAR is a CD371-targeting CAR. In certain embodiments, the CAR comprises (a) (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a VDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a V L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a portion thereof), and (c) an intracellular signaling domain comprising (i) a CD3ζ polypeptide, and (ii) a costimulatory signaling region comprising a 4-1BB polypeptide (e.g., the intracellular domain of human 4-1BB or a portion thereof). In certain embodiments, H and V L is linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 13. H and V L is V from the N-terminus to the C-terminus L -V H In certain embodiments, the CAR is designed as "Et.B10L4H_MT_hBBZ". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 75, which is provided below. [ka]

[0216] An exemplary nucleic acid sequence for the amino acid sequence of SEQ ID NO:75 is set forth in SEQ ID NO:76, which is provided below. [ka] [ka]

[0217] In certain embodiments, the CAR is a CD371-targeting CAR. In certain embodiments, the CAR comprises (a) (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a VDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a V L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a portion thereof); and (c) 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 portion thereof). In certain embodiments, H and V L is linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 13. H and V L is V from the N-terminus to the C-terminus L -V HIn certain embodiments, the CAR is designed as "Et.B10L4H_MT_h28Z". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 77, which is provided below. [ka] [ka]

[0218] An exemplary nucleic acid sequence for the amino acid sequence of SEQ ID NO:77 is set forth in SEQ ID NO:78, which is provided below. [ka] [ka]

[0219] In certain embodiments, the CAR is a CD371-targeting CAR. In certain embodiments, the CAR comprises (a) (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a VDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a V L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a portion thereof), and (c) 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 portion thereof). In certain embodiments, H and V Lis linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14. H and V L is V from the N-terminus to the C-terminus H -V L In certain embodiments, the CAR is designed as "Et.B10H3L_MT_h28Z". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 79, which is provided below. [ka] [ka]

[0220] An exemplary nucleic acid sequence for the amino acid sequence of SEQ ID NO:79 is set forth in SEQ ID NO:80, which is provided below. [ka] [ka]

[0221] In certain embodiments, the CAR is a CD371-targeting CAR. In certain embodiments, the CAR comprises (a) (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a VDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a V L V containing CDR3 L(b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a portion thereof); and (c) 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 portion thereof). In certain embodiments, H and V L is linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 13. H and V L is V from the N-terminus to the C-terminus H -V L In certain embodiments, the CAR is designated as "Et.B10H4L_MT_h28Z". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 81, which is provided below. [ka]

[0222] An exemplary nucleic acid sequence for the amino acid sequence of SEQ ID NO:81 is set forth in SEQ ID NO:82, which is provided below. [ka] [ka]

[0223] In certain embodiments, the CAR is a CD371-targeting CAR. In certain embodiments, the CAR comprises (a) (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a VDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and a V LV containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a portion thereof); and (c) 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 portion thereof). In certain embodiments, H and V L is linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14. H and V L is V from the N-terminus to the C-terminus H -V L In certain embodiments, the CAR is designated as "Et.C3H3L_MT_h28Z". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 83, which is provided below. [ka]

[0224] An exemplary nucleic acid sequence for the amino acid sequence of SEQ ID NO:83 is set forth in SEQ ID NO:84, which is provided below. [ka] [ka]

[0225] In certain embodiments, the CAR is a CD371-targeting CAR. In certain embodiments, the CAR comprises (a) (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a VDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36. H V containing CDR3 Hand (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and a V L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a portion thereof), and (c) 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 portion thereof). In certain embodiments, H and V L is linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14. H and V L is V from the N-terminus to the C-terminus L -V H In certain embodiments, the CAR is designated as "Et.C3L3H_MT_h28Z". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 85, which is provided below. [ka]

[0226] An exemplary nucleic acid sequence for the amino acid sequence of SEQ ID NO:85 is set forth in SEQ ID NO:86, which is provided below. [ka] [ka]

[0227] In certain embodiments, the CAR is a CD371-targeting CAR. In certain embodiments, the CAR comprises (a) (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a VDR comprising the amino acid sequence set forth in SEQ ID NO: 42. HV containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44, and a V comprising the amino acid sequence set forth in SEQ ID NO: 45. L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a portion thereof), and (c) 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 portion thereof). In certain embodiments, H and V L is linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14. H and V L is V from the N-terminus to the C-terminus L -V H In certain embodiments, the CAQR is designated as "Et.D6L3H_MT_h28Z". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 87, which is provided below. [ka]

[0228] An exemplary nucleic acid sequence for the amino acid sequence of SEQ ID NO:87 is set forth in SEQ ID NO:88, which is provided below. [ka] [ka]

[0229] In certain embodiments, the CAR is a CD371-targeting CAR. In certain embodiments, the CAR comprises (a) (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a VDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a V L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a fragment thereof), and (c) 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). In certain embodiments, H and V L is linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 93. H and V L is V from the N-terminus to the C-terminus H -V L In certain embodiments, the CAR is designed as "Et.B10H1L_MT_h28Z". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 95, which is provided below. [ka] [ka]

[0230] An exemplary nucleic acid sequence for the amino acid sequence of SEQ ID NO:95 is set forth in SEQ ID NO:96, which is provided below. [ka] [ka]

[0231] In certain embodiments, the CAR is a CD371-targeting CAR. In certain embodiments, the CAR comprises (a) (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a VDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a V L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a fragment thereof), and (c) 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). In certain embodiments, H and V L is linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 94. H and V L is V from the N-terminus to the C-terminus H -V L In certain embodiments, the CAR is designed as "Et.B10H2L_MT_h28Z". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 97, which is provided below. [ka] [ka]

[0232] An exemplary nucleic acid sequence for the amino acid sequence of SEQ ID NO:97 is set forth in SEQ ID NO:98, which is provided below. [ka] [ka]

[0233] In certain embodiments, the CAR is a CD371-targeting CAR. In certain embodiments, the CAR comprises (a) (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a VDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a V L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a fragment thereof), and (c) 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). In certain embodiments, H and V L is linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 91. H and V L is V from the N-terminus to the C-terminus H -V L In certain embodiments, the CAR is designed as "Et.B10H5L_MT_h28Z". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 99, which is provided below. [ka]

[0234] An exemplary nucleic acid sequence for the amino acid sequence of SEQ ID NO:99 is set forth in SEQ ID NO:100, which is provided below. [ka] [ka]

[0235] In certain embodiments, the CAR is a CD371-targeting CAR. In certain embodiments, the CAR comprises (a) (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a VDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. H V containing CDR3 H and (ii) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a V L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a fragment thereof), and (c) 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). In certain embodiments, H and V L is linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 92. H and V L is V from the N-terminus to the C-terminus H -V L In certain embodiments, the CAR is designed as "Et.B10H6L_MT_h28Z". In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 101, which is provided below. [ka]

[0236] An exemplary nucleic acid sequence for the amino acid sequence of SEQ ID NO:101 is set forth in SEQ ID NO:102, which is provided below. [ka] [ka]

[0237] In certain embodiments, the CAR of the present disclosure further comprises an inducible promoter to express the nucleic acid sequence in human cells. The promoter for use in expressing the CAR gene can be a constitutive promoter, such as the ubiquitin C (UbiC) promoter. 5.3.3. TCR-like fusion molecules

[0238] In certain embodiments, the antigen-recognizing 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", e.g., those disclosed in International Patent Application No. PCT / US19 / 017525, which is incorporated herein by reference in its entirety), and T cell receptor fusion constructs (TRuC) (e.g., 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 herein by reference in its entirety).

[0239] In certain embodiments, the TCR-like fusion molecule comprises an antigen-binding chain comprising an extracellular antigen-binding domain and a constant domain, wherein the TCR-like fusion molecule binds to an antigen in an HLA-independent manner. In certain embodiments, the constant domain comprises a T cell receptor constant region selected from the group consisting of a native or modified TRAC peptide, a native or modified TRBC peptide, a native or modified TRDC peptide, a native or modified TRGC peptide, and any variant or functional fragment thereof. In certain embodiments, the constant domain comprises a native or modified TRAC peptide. In certain embodiments, the constant domain comprises a native or modified TRBC peptide. In certain embodiments, the constant domain can form a homodimer or a heterodimer with another constant domain. In certain embodiments, the antigen-binding chain can associate with a CD3ζ polypeptide. In certain embodiments, the antigen-binding chain can activate a CD3ζ polypeptide associated with the antigen-binding chain upon binding to an antigen. In certain embodiments, the activation of the CD3ζ polypeptide can activate an immune response cell. In certain embodiments, the TCR-like fusion molecule can combine with the CD3 complex and provide HLA-independent antigen recognition. In certain embodiments, the TCR-like fusion molecule replaces the endogenous TCR in the CD3 / TCR complex. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule can dimerize with another extracellular antigen-binding domain. In certain embodiments, 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 certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises one or two immunoglobulin variable regions. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises a heavy chain variable region (V) of an antibody. H In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule comprises the light chain variable region of an antibody (V LIn certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule is capable of dimerizing with another extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain of the TCR-like fusion molecule is capable of dimerizing with another extracellular antigen-binding domain. H where V H is the V of the antibody L In certain embodiments, 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 variable fragment (Fv). L where V L is the V of the antibody H and dimerize with another extracellular antigen-binding domain comprising the variable fragment (Fv).

[0240] The TCR-like fusion molecule can bind to a tumor antigen or a pathogen antigen. In certain embodiments, the TCR-like fusion molecule binds to a tumor antigen. 5.4.Cells

[0241] The subject matter of the present disclosure provides a cell comprising a CD371-targeting antigen-recognition receptor of the present disclosure (e.g., as disclosed in Section 5.3). In certain embodiments, the cell is selected from the group consisting of a cell of lymphoid lineage and a cell of myeloid lineage. In certain embodiments, the cell is an immune response cell. In certain embodiments, the immune response cell is a cell of lymphoid lineage.

[0242] In certain embodiments, the cell is a lymphoid lineage cell. The lymphoid lineage cell can provide antibody production, regulation of the cellular immune system, detection of foreign pathogens in the blood, detection of cells foreign to the host, etc. Non-limiting examples of lymphoid lineage cells include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphoid cells can be differentiated. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., an embryonic stem cell).

[0243] In certain embodiments, the cell is a T cell. T cells can be lymphocytes that mature in the thymus and are primarily involved in cell-mediated immunity. T cells participate 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 T cells (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 gamma delta T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. The patient's own T cells can be genetically modified to target specific antigens by the introduction of antigen-recognition receptors, e.g., CARs or TCRs. In certain embodiments, the immune response cells are T cells. T cells are CD4 + T cells or CD8 + In certain embodiments, the T cells are CD4 + In certain embodiments, the T cells are CD8 + T cells.

[0244] In certain embodiments, the cell is a NK cell. Natural killer (NK) cells are part of cell-mediated immunity and can be lymphocytes that act during natural immune response. NK cells do not require prior activation to carry out their cytotoxic effect on target cells.

[0245] Types of human lymphocytes of the presently disclosed subject matter include, but are not limited to, peripheral donor lymphocytes, e.g., 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 full-length tumor antigens, including 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 artificial antigen-presenting cells (AAPCs) , which disclose selective in vitro expanded antigen-specific peripheral blood leukocytes using dendritic cells (PCs) or pulsed dendritic cells (Hu et al., 2003).

[0246] The cells (eg, T cells) can be autologous, non-autologous (eg, allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0247] The subject cell of the present disclosure is myeloid lineage cell.Non-limiting examples of myeloid lineage cell include monocyte, macrophage, neutrophil, dendritic cell, basophil, neutrophil, eosinophil, megakaryocyte, mast cell, erythrocyte, platelet, and stem cell from which myeloid cell can differentiate.In certain embodiments, stem cell is pluripotent stem cell (for example, embryonic stem cell or induced pluripotent stem cell).

[0248] In certain embodiments, the cells of the present disclosure can modulate the tumor microenvironment. Tumors have a microenvironment hostile to the host immune response, including a series of mechanisms by malignant cells to protect themselves from immune recognition and elimination. This "hostile tumor microenvironment" includes the infiltration-regulatory CD4 +The tumor microenvironment contains a variety of 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 (CTLA-4 and PD-1) expressed by activated T cells. These mechanisms of immunosuppression play a role in maintaining tolerance and suppressing inappropriate immune responses; however, within the tumor microenvironment, these mechanisms prevent effective anti-tumor immune responses. Taken together, these immunosuppressive factors can induce either significant anergy or apoptosis of adoptively transferred CAR-modified T cells upon encounter with targeted tumor cells.

[0249] In certain embodiments, cells may be transduced with a CD371-targeted antigen recognition receptor of the present disclosure such that the cells express the antigen recognition receptor.

[0250] In certain embodiments, the cells further comprise a soluble single chain variable fragment (scFv) that binds a polypeptide having immunosuppressive or immunostimulatory activity. In certain embodiments, immunosuppressive activity refers to the induction of signal transduction or changes in protein expression in cells (e.g., activated immune response cells) that result in a decrease in immune response. Polypeptides known to suppress or decrease immune responses through their binding include CD47, PD-1, CTLA-4, and their corresponding ligands including SIRPa, PD-L1, PD-L2, B7-1 and B7-2. Such polypeptides are present in the tumor microenvironment and inhibit the immune response against neoplastic cells. In various embodiments, inhibiting, blocking or antagonizing the interaction of immunosuppressive polypeptides and / or their ligands enhances the immune response of immune response cells.

[0251] In certain embodiments, immune stimulatory activity refers to the induction of signal transduction or changes in protein expression in cells (e.g., activated immune response cells) that result in an increase in immune response. Immune stimulatory activity may include pro-inflammatory activity. Polypeptides known to stimulate or increase immune response through their binding include CD28, OX-40, 4-IBB, and their corresponding ligands including B7-1, B7-2, OX-40L, and 4-1BBL. Such polypeptides are present in the tumor microenvironment and activate immune response against neoplastic cells. In various embodiments, promoting, stimulating, or agonizing pro-inflammatory polypeptides and / or their ligands enhances the immune response of immune response cells.

[0252] Cells comprising a soluble scFv that binds an antigen recognition receptor (e.g., a CAR) and a polypeptide having immunosuppressive or immunostimulatory activity are disclosed in International Patent Publication No. WO 2014 / 134165, which is incorporated herein by reference in its entirety.

[0253] In certain embodiments, the cells further comprise exogenous CD40L. Cells comprising an antigen recognition receptor (e.g., a CAR) and exogenous CD40L are disclosed in International Patent Publication No. WO 2014 / 134165.

[0254] Furthermore, in certain embodiments, the cells are engineered to express IL-18. In certain embodiments, the cells further comprise an exogenous IL-18 polypeptide. In certain embodiments, the exogenous IL-18 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 103, which is provided below. [ka]

[0255] In certain embodiments, the cell further comprises a nucleic acid molecule encoding an IL-18 polypeptide. In certain embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 104, which is provided below. [ka]

[0256] Alternatively, in certain embodiments, the cells further comprise an altered promoter / enhancer at the IL-18 locus that can increase IL-18 gene expression, e.g., a constitutive or inducible promoter is positioned to drive IL-18 gene expression.

[0257] Cells that contain an antigen recognition receptor (e.g., CAR) and are engineered to express IL-18, e.g., cells that contain an exogenous IL-18 polypeptide or an altered promoter / enhancer at the IL-18 locus, are disclosed in International Patent Publication No. WO 2018 / 027155, which is incorporated by reference in its entirety.

[0258] Additionally or alternatively, the cells are engineered to express IL-33. In certain embodiments, the cells further comprise an exogenous IL-33 polypeptide. In certain embodiments, the exogenous IL-33 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 105, which is provided below. [ka]

[0259] In certain embodiments, the cell further comprises a nucleic acid molecule encoding an IL-33 polypeptide. In certain embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 106, which is provided below. [ka]

[0260] Alternatively, in certain embodiments, the cells further comprise an engineered promoter / enhancer at the IL-33 locus, which can increase IL-33 gene expression, e.g., a constitutive or inducible promoter is positioned to drive IL-33 gene expression. Cells that comprise an antigen recognition receptor (e.g., CAR) and are engineered to express IL-33, e.g., cells that comprise an exogenous IL-33 polypeptide or an engineered promoter / enhancer at the IL-33 locus, are disclosed in International Patent Publication No. WO 2019 / 099479, which is incorporated herein by reference in its entirety.

[0261] Additionally or alternatively, the cells are engineered to express IL-36. In certain embodiments, the cells further comprise an exogenous IL-36 polypeptide. In certain embodiments, the cells further comprise a promoter / enhancer modified in the IL-36 locus, which can increase IL-36 gene expression, for example, a constitutive promoter or an inducible promoter is arranged to drive IL-36 gene expression. Cells that comprise an antigen recognition receptor (e.g., CAR) and are engineered to express IL-36, for example, cells that comprise an exogenous IL-36 polypeptide or a promoter / enhancer modified in the IL-36 locus, are disclosed in International Patent Publication No. 2019 / 099483, which is incorporated herein by reference in its entirety. 5.5. Compositions and Vectors

[0262] The presently disclosed subject matter provides compositions comprising a CD371-targeting antigen-recognizing receptor of the present disclosure (e.g., those disclosed in Section 5.3). Cells comprising such compositions are also provided.

[0263] In certain embodiments, the CD371-targeting antigen recognition receptor of the present disclosure is operably linked to a promoter.

[0264] Furthermore, the presently disclosed subject matter provides nucleic acid compositions comprising a polynucleotide encoding a CD371-targeting antigen-recognizing receptor of the present disclosure (e.g., those disclosed in Section 5.3). Cells comprising such nucleic acid compositions are also provided.

[0265] In certain embodiments, the nucleic acid composition further comprises a promoter operably linked to the CD371-targeting antigen recognition receptor of the present disclosure.

[0266] In certain embodiments, the promoter is endogenous or exogenous.In certain embodiments, the exogenous promoter is selected from the elongation factor (EF)-1 promoter, the promoter of cytomegalovirus immediate early promoter (CMV), the promoter of simian virus 40 early promoter (SV40), the phosphoglycerate kinase (PGK) promoter and the metallothionein promoter.In certain embodiments, the promoter is an inducible promoter.In certain embodiments, the inducible promoter is selected from the NFAT transcription response element (TRE) promoter, the CD69 promoter, the CD25 promoter and the IL-2 promoter.

[0267] The compositions and nucleic acid compositions can be administered to a subject or delivered to cells by methods known in the art or as described herein. Genetic modification of cells (e.g., T cells or NK cells) can be achieved by transducing a substantially homogeneous cell composition with a recombinant DNA construct. In certain embodiments, a retroviral vector (e.g., a gamma retroviral vector or a lentiviral vector) is used to introduce a DNA construct into cells. For example, a polynucleotide encoding an antigen-recognizing receptor can be cloned into a retroviral vector, and expression can be driven from its endogenous promoter, from a retroviral long terminal repeat, or from a promoter specific to the target cell type of interest. Non-viral vectors can be used as well.

[0268] For the initial genetic modification of cells to contain the CD371-targeting antigen-recognizing receptor (e.g., CAR or TCR) of the present disclosure, a retroviral vector can be used for transduction, however, any other suitable viral vector or non-viral delivery system can be used. The antigen-recognizing receptor can be constructed in a single multicistronic expression cassette, in multiple expression cassettes in a single vector, or in multiple vectors. Examples of elements that create polycistronic expression cassettes 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-κB 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). Also suitable are combinations of retroviral vectors and appropriate packaging systems, where the capsid protein is functional to infect human cells. Examples of suitable vectors include, but are 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). Amphotropic virus-producing cell lines are known. Non-amphotropic particles, such as particles pseudotyped with VSVG, RD114 or GALV envelopes, and any others known in the art, are also suitable.

[0269] Potential methods of transduction 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., al. J Clin Invest (1992); 89:1817).

[0270] Other transducing viral vectors can be used to modify cells. In certain embodiments, the vector selected exhibits high efficiency of infection, as well as stable integration and expression (e.g., 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 virus vectors, vaccinia virus, bovine papilloma virus, or herpes viruses, such as Epstein-Barr virus (see, for example, 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; (See also vectors in Johnson, Chest (1995) 107:77S- 83S.) Retroviral vectors have been particularly well developed and are used in clinical practice (Rosenberg et al., N Engl J Med (1990);323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0271] Non-viral techniques can also be used for genetic modification of cells. For example, nucleic acid molecules can be introduced into cells by administering nucleic acids 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). It can be introduced into cells. Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation and protoplast fusion. Liposomes can also be potentially useful for delivery of DNA into cells. 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 the cells (or their progeny) are injected into targeted tissues or injected systemically. Recombinant receptors can also be induced or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases or TALE nucleases, CRISPR). Transient expression can be obtained by RNA electroporation.

[0272] Any targeted genome editing method can also be used to deliver the antigen-recognizing receptor of the present disclosure to cell or subject.In certain embodiments, the CRISPR system is used to deliver the antigen-recognizing receptor of the present disclosure disclosed herein.In certain embodiments, zinc finger nuclease is used to deliver the antigen-recognizing receptor of the present disclosure.In certain embodiments, the TALEN system is used to deliver the antigen-recognizing receptor of the present disclosure.

[0273] The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool found in prokaryotic cells. When utilized for genome editing, the system includes Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (crRNA, containing the RNA used by Cas9 to guide it to the correct section of the host DNA together with a region that binds to tracrRNA (generally in the form of a hairpin loop) that forms an active complex with Cas9), transactivating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9), and an optional portion of DNA repair template (DNA that guides the cell repair process allowing the insertion of a specific DNA sequence). CRISPR / Cas9 often uses a plasmid to transfect the target cell. The crRNA is the sequence that Cas9 uses to identify and directly bind to the target DNA in the cell, and thus needs to be designed for each application. A repair template carrying a CAR expression cassette must also be designed for each application, as it must overlap sequences on either side of the cut and encode the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single-stranded guide RNA (sgRNA). This sgRNA can be combined with the Cas9 gene into a plasmid for transfection into cells.

[0274] Zinc finger nucleases (ZFNs) are artificial restriction enzymes that are created by combining a zinc finger DNA binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific DNA sequences that allow 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 for creating new 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 II restriction endonuclease FokI. Using the endogenous homologous recombination (HR) machinery and a homologous DNA template bearing the CAR expression cassette, ZFNs can be used to insert the CAR expression cassette into the genome. When the targeting sequence is cleaved by the ZFN, the HR machinery searches for homology between the damaged chromosome and the homologous DNA template and then copies the sequence of the template between the two cut ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0275] Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences in DNA. TALEN systems operate on roughly the same principle as ZFNs. They are created by combining a transcription activator-like effector DNA binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of a repeating motif of 33-34 amino acids with two variable positions that strongly recognize specific nucleotides. By assembling a variety of these TALEs, the TALE DNA binding domain can be engineered to bind the desired DNA sequence, thereby guiding the nuclease to cleave at a specific location in the genome. cDNA expression for use in methods of polynucleotide therapy can be directed from any suitable promoter (e.g., human cytomegalovirus (CMV) promoter, simian virus 40 (SV40) promoter or metallothionein promoter) and regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of nucleic acids.The enhancers used can include, but are not limited to, those characterized as tissue- or cell-specific enhancers.Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by cognate regulatory sequences, or, if desired, by regulatory sequences derived from heterologous sources, including any of the promoters or regulatory elements described above.

[0276] The method for delivering genome editing agent / system can be changed according to need.In certain embodiments, the components of the selected genome editing method are delivered as DNA constructs in one or more plasmids.In certain embodiments, the components are delivered via viral vectors.Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, sonication, magnetofection, adeno-associated virus, envelope protein pseudotyping of viral vectors, cis- and trans-acting elements of replication-competent vectors, herpes simplex virus, and chemical mediators (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles and cell-penetrating peptides). Polypeptides

[0277] The subject matter of the present disclosure provides a method for optimizing an amino acid sequence or a nucleic acid sequence by generating changes in the sequence. Such changes may include certain mutations, deletions, insertions, or post-translational modifications. The subject matter of the present disclosure further includes analogs of any naturally occurring polypeptide disclosed herein, including but not limited to CD371, CD8, CD28, 4-1BB, and CD3zeta. Analogs may differ from naturally occurring polypeptides disclosed herein by differences in amino acid sequence, by post-translational modifications, or both. Analogs may exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more homology or identity with all or a portion of the naturally occurring amino acid sequence of the subject matter of the present disclosure. The length of sequence comparison is at least 5, 10, 15 or 20 amino acid residues, for example at least 25, 50 or 75 amino acid residues, or more than 100 amino acid residues. Again, in an exemplary procedure for determining the degree of identity, the BLAST program uses an e -3 ~e -100The probability score can be used to modify the in These include chemical derivatizations in vivo and in vitro, such as acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing, or after treatment with isolated modifying enzymes. Analogs may also differ from naturally occurring polypeptides by alterations in the primary sequence. These include both natural and induced genetic variants, such as those produced by random mutagenesis, by exposure to irradiation or ethane methyl sulfate, or by site-directed mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra. Also included are cyclized peptides, molecules and analogs that contain residues other than L-amino acids, e.g., D-amino acids, or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids.

[0278] In addition to full-length polypeptides, the subject matter of the present disclosure also provides fragments of any of the polypeptides disclosed herein. As used herein, the term "fragment" means at least 5, 10, 13, or 15 amino acids. In certain embodiments, a fragment comprises at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids. In certain embodiments, a fragment comprises at least 60-80, 100, 200, 300, or more contiguous amino acids. Fragments can be generated by methods known to those of skill in the art, or can result from normal protein processing (e.g., removal of amino acids from a nascent polypeptide that are not required for biological activity, or removal of amino acids by alternative mRNA splicing or alternative protein processing events). 5.7. Formulation and Administration

[0279] The subject matter of the present disclosure also provides a composition comprising the cells of the present disclosure. The composition comprising the cells of the present disclosure can be conveniently provided as a sterile liquid preparation, for example, an isotonic aqueous solution, suspension, emulsion, dispersion or viscous composition, which may be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, particularly by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact period with specific tissues. The liquid or viscous composition can include a carrier, which can be a solvent or dispersion medium, for example, containing water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.

[0280] Sterile injectable solutions can be prepared by incorporating the genetically modified cells in a required amount of a suitable solvent with various amounts of other ingredients, if desired. Such compositions may be in admixture with suitable carriers, diluents or excipients, such as sterile water, saline, glucose, dextrose, etc. The compositions can also be lyophilized. Depending on the desired route of administration and preparation, the compositions can contain auxiliary substances such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffers, gelling or thickening additives, preservatives, flavoring agents, coloring agents, etc. Suitable preparations can be prepared without undue experimentation by reference to standard texts such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, which is incorporated herein by reference.

[0281] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents and buffers. Prevention of microbial activity can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of injectable pharmaceutical forms can be achieved by the use of agents that delay absorption, such as aluminum monostearate and gelatin. However, according to the subject matter of the present disclosure, any vehicle, diluent or additive used must be compatible with genetically modified cells.

[0282] The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and tears.The desired isotonicity of the composition can be achieved using sodium chloride or other pharma- ceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes.Sodium chloride can be used especially for buffers containing sodium ions.

[0283] The viscosity of the composition can be maintained at a selected level by using a pharma- ceutically acceptable thickening agent if desired. For example, methylcellulose is readily available economically and easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, etc. The concentration of the thickening agent can depend on the agent selected. The important point is to use an amount that achieves the selected viscosity. Obviously, the selection of suitable carriers and other additives depends on the exact route of administration and the nature of a particular dosage form, for example, a liquid dosage form (for example, whether the composition is formulated into a solution, suspension, gel, or another liquid form, for example, a sustained release form or a liquid-filled form).

[0284] The composition comprising the cells of the present disclosure can be provided to a subject systemically or directly to induce and / or enhance immune response to an antigen and / or treat and / or prevent neoplasia. In certain embodiments, the cells of the present disclosure or the composition comprising the same are directly injected into the organ of interest (e.g., the organ affected by neoplasia). Alternatively, the cells of the present disclosure or the composition comprising the same are provided indirectly to the organ of interest, for example, by administration to the circulatory system (e.g., tumor vasculature). Expansion and differentiation agents can be provided before, during, or after administration of the cells or the composition to increase the generation of cells (e.g., T cells or NK cells) in vitro or in vivo.

[0285] The cells of the present disclosure can be administered in any physiologically acceptable vehicle, typically intravascularly, although they may also be introduced into bone or other convenient sites where the cells may find a suitable site for regeneration and differentiation (e.g., the thymus).

[0286] The amount of cells administered can vary depending on the subject being treated. In certain embodiments, about 10 4 ~about 10 10 pieces, about 10 4 ~about 10 7 pieces, about 10 5 ~about 10 7 pieces, about 10 5 ~about 10 9 Pieces, or about 10 6 ~about 10 8 of the cells of the present disclosure are administered to a subject. More effective cells may be administered in even smaller numbers. Typically, at least about 1 x 10 5 cells were administered, ultimately resulting in approximately 1 x 10 10 In certain embodiments, the number of cells in the blood reaches at least about 1×10 or more. 5 , 5×10 5 , 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1×10 8or about 5 x 10 8 The cells of the present disclosure are administered to a subject. In certain embodiments, about 1×10 6 The cells of the present disclosure are administered to the subject.The exact determination of the dosage that will be considered as an effective dosage may be based on individual factors for each subject, including the size, age, sex, weight and condition of the particular subject.The dosage can be easily ascertained by those skilled in the art from the present disclosure and knowledge in the art.

[0287] The cells of the present disclosure can include purified populations of cells. One of skill in the art can easily determine the percentage of cells of the present disclosure in a population using various well-known methods, such as fluorescence-activated cell sorting (FACS). Suitable ranges of purity in a population containing immune response cells of the present disclosure are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%. In certain embodiments, the purity is about 70% to about 75%, about 75% to about 80%, or about 80% to about 85%. In certain embodiments, the purity is about 85% to about 90%, about 90% to about 95%, and about 95% to about 100%. The dosage can be easily adjusted by one of skill in the art (e.g., a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, etc.

[0288] One of skill in the art can readily determine the amount of cells and optional additives, vehicles and / or carriers to be administered in the compositions and methods. Typically, any additives (in addition to active cells and / or agents) are present in an amount of 0.001 to 50% (by weight) of solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, e.g., about 0.0001 to about 5% by weight, about 0.0001 to about 1% by weight, about 0.0001 to about 0.05% by weight, or about 0.001 to about 20% by weight, about 0.01 to about 10% by weight, or about 0.05 to about 5% by weight. For any composition to be administered to animals or humans, the following can be determined: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model, e.g., rodents such as mice; the dosage of the composition, the concentration of components therein, and the timing of administering the composition, that elicits a suitable response. Such determinations do not require undue experimentation from the knowledge of one of ordinary skill in the art, this disclosure, and the documents cited herein, and the times for sequential administration can be ascertained without undue experimentation.

[0289] In certain embodiments, the composition is a pharmaceutical composition comprising the cells of the present disclosure and a pharma- ceutically acceptable carrier.

[0290] The administration of the composition can be autologous or heterologous. For example, cells can be obtained from one subject and administered to the same subject or to a different compatible subject. Peripheral blood-derived cells or their progeny (e.g., derived in vivo, ex vivo or in vitro) can be administered. When administering the composition of the present disclosure (e.g., the pharmaceutical composition comprising the cells of the present disclosure), it can be formulated into a unit dose injectable form (solution, suspension, emulsion).

[0291] The cells and compositions of the disclosure can be administered by any method known in the art, including, but not limited to, oral administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, intraosseous administration, intraperitoneal administration, pleural administration, and direct administration to a subject. 5.8. Treatment Method

[0292] The subject cells of the present disclosure and compositions comprising same can be used to treat and / or prevent tumors or neoplasms. Such cells can be administered to a subject (e.g., a human subject) in need thereof for the treatment and / or prevention of tumors or neoplasms (e.g., acute myeloid leukemia (AML), multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), glioblastoma). In certain embodiments, the cells are T cells. T cells are CD4 + T cells or CD8 + In certain embodiments, the T cells are CD4 + T cells.

[0293] The subject matter of the present disclosure provides a method for inducing and / or increasing an immune response in a subject in need thereof. The cells of the present disclosure and compositions comprising the same can be used in therapy or medicine. The subject matter of the present disclosure provides various methods of using the cells (e.g., T cells) or compositions comprising the same. For example, the cells of the present disclosure and compositions comprising the same can be used to reduce tumor burden in a subject. The cells of the present disclosure can reduce the number of tumor cells, reduce tumor size, and / or eradicate tumors in a subject. The cells of the present disclosure and compositions comprising the same can be used to treat and / or prevent tumors or neoplasms in a subject. The cells of the present disclosure and compositions comprising the same can be used to extend the survival of a subject suffering from a tumor or neoplasm. Such methods include administering the cells of the present disclosure or compositions comprising the same (e.g., pharmaceutical compositions) to achieve a desired effect, such as alleviating an existing condition or preventing recurrence. For treatment, the amount administered is an amount effective to produce a desired effect. An effective amount can be provided in one or a series of administrations. An effective amount can be provided in a bolus or by continuous perfusion.

[0294] The subject matter of the present disclosure provides various methods of using cells (e.g., T cells) or compositions comprising the same.For example, the subject matter of the present disclosure provides a method of reducing tumor burden in a subject.In certain embodiments, the method of reducing tumor burden comprises administering the cells of the present disclosure or compositions comprising the same to a subject.The cells of the present disclosure can reduce the number of tumor cells, reduce tumor size, and / or eradicate tumor in a subject.

[0295] The tumor or neoplasm may be a solid tumor. Non-limiting examples of solid tumors include mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, gastric cancer, and bile duct carcinoma.

[0296] The subject matter of the present disclosure also provides a method for increasing or extending the survival time of a subject having a tumor or neoplasm. In certain embodiments, the method for increasing or extending the survival time of a subject having a tumor or neoplasm comprises administering to the subject an immune response cell of the present disclosure or a composition comprising the same. The method can reduce or eradicate tumor burden in the subject. In addition, the subject matter of the present disclosure provides a method for increasing the immune response in a subject, comprising administering to the subject a cell of the present disclosure or a composition comprising the same. The subject matter of the present disclosure further provides a method for treating and / or preventing a tumor or neoplasm in a subject, comprising administering to the subject a cell of the present disclosure or a composition comprising the same.

[0297] Non-limiting examples of neoplasms or tumors include acute myeloid leukemia (AML), multiple myeloma, chronic lymphocytic leukemia (CLL), lymphoma (Hodgkin's lymphoma, non-Hodgkin's lymphoma), glioblastoma, myelodysplastic syndrome (MDS), and chronic myeloid leukemia (CML), bone cancer, intestinal cancer, liver cancer, skin cancer, head and neck cancer, melanoma (cutaneous or intraocular malignant melanoma), kidney cancer (e.g., clear cell carcinoma), throat cancer, prostate cancer (e.g., hormonal refractory prostate adenocarcinoma), blood cancers (e.g., leukemia, lymphoma, and myeloma), uterine cancer, rectal cancer, anal cancer, bladder cancer, brain cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, polycythemia vera, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, cancer of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, including those induced by asbestos Environmentally induced cancers, including Waldenstrom's hypergammaglobulinemia, heavy chain disease, and sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, spermatozoa, sarcom ... Solid tumors such as epithelioma, embryonal carcinoma, Wilms' tumor, cervical cancer, salivary gland cancer, uterine cancer, testicular cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, Schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0298] In certain embodiments, the tumor or neoplasm is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma, chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma, non-Hodgkin's lymphoma, glioblastoma, myelodysplastic syndrome (MDS) and chronic myeloid leukemia (CML). In certain embodiments, the tumor or neoplasm is AML.

[0299] The subject may have an advanced form of the disease, in which case the goal of treatment may include reducing or reversing disease progression and / or ameliorating side effects. The subject may have a history of a condition that has already been treated, in which case the goal of therapy typically includes reducing or delaying the risk of recurrence.

[0300] As a result of surface expression of the CD371-targeted antigen recognition receptor of the present disclosure, the adoptively transferred cells (e.g., immune response cells, e.g., T cells or NK cells) are endowed with enhanced selective cytolytic activity at the tumor site. Furthermore, following their localization to the tumor or viral infection and their proliferation, the cells transform the tumor or viral infection site into a more conducive environment for a broad range of immune cells (tumor-infiltrating lymphocytes, NK cells, NKT cells, dendritic cells and macrophages) that participate in the physiological anti-tumor or anti-viral response.

[0301] Further modifications can be introduced into the cells (e.g., T cells) of the present disclosure to avoid or minimize the risk of immunological complications (known as "malignant T cell transformation"), such as graft-versus-host disease (GvHD), or when healthy tissue expresses the same target antigen as tumor cells, resulting in a similar outcome to GvHD. A potential solution to this problem is to engineer a suicide gene into the cells of the present disclosure. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible caspase 9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can allow for the elimination of T cells by administering anti-EGFR monoclonal antibodies (e.g., cetuximab). The EGFRt can be covalently attached upstream of an antigen recognition receptor (e.g., CAR). The suicide gene may be included in a vector that includes a nucleic acid encoding an antigen recognition receptor (e.g., a CAR) of the present disclosure. In this method, administration of a prodrug (e.g., a prodrug (e.g., AP1903 that can activate iCasp-9) designed to activate the suicide gene during T cell transformation of a malignant tumor (e.g., GVHD) causes apoptosis in suicide gene-activated cells expressing the antigen recognition receptor (e.g., a CAR). Incorporation of a suicide gene into an antigen recognition receptor (e.g., a CAR) of the present disclosure provides an additional level of safety by its ability to eliminate the majority of cells expressing the receptor within a very short time. Cells (e.g., T cells) of the present disclosure that have been incorporated with a suicide gene may be preemptively eliminated at a given time point after cell infusion or eradicated at the earliest sign of toxicity. 5.9.Kit

[0302] The subject matter of the present disclosure provides a kit for inducing and / or enhancing immune response in a subject, treating and / or preventing tumor or neoplasm in a subject, reducing tumor burden in a subject, and / or increasing or extending the survival time of a subject having a tumor or neoplasm in a subject.In certain embodiments, the kit comprises a cell of the present disclosure or a composition comprising the same.In certain embodiments, the kit comprises a sterile container, such a container may be in the form of a box, an ampoule, a bottle, a vial, a tube, a bag, a sachet, a blister pack, or other suitable container known in the art.Such a container can be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding medicine.In certain non-limiting embodiments, the kit comprises a nucleic acid molecule encoding a CD371-targeting antigen-recognition receptor (e.g., CAR or TCR) of the present disclosure.

[0303] Optionally, the cells and / or nucleic acid molecules are provided with instructions for administering the cells or nucleic acid molecules to a subject having or at risk of developing a tumor or neoplasm. The instructions generally include information about the use of the composition for the treatment and / or prevention of tumors or neoplasms. In certain embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosing schedule and administration for the treatment or prevention of tumors or neoplasms; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if present), or as a label attached to the container, or as a separate sheet, pamphlet, card, or folder provided in or with the container. EXAMPLES

[0304] 6. Working Example The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the skill of the art. Such techniques may be found in any of the numerous publications, including those set forth in "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987);“Current Protocols in These techniques are fully described in such publications as "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides disclosed herein and may be considered, for example, in making and practicing the presently disclosed subject matter. Techniques particularly useful for certain embodiments are discussed in the sections that follow.

[0305] The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the disclosed cells and compositions and are not intended to limit the scope of what the inventors regard as their invention. Example 1 Generation of CD371-targeted CAR

[0306] Six CD371-targeting CARs were generated: "Et.B10L3H_MT_h28z", "Et.B10L4H_MT_hBBz", "Et.B10L4H_MT_h28z", "Et.B10H3L_MT_h28z", "Et.B10H4L_MT_h28z", "Et.C3H3L_MT_h28z", "Et.C3L3H_MT_h28z" and "Et.D6L3H_MT_h28z". The structure of the CAR is illustrated in FIG. 1.

[0307] As shown in Figure 2, CAR was detected by EGFR and Myc tags on the surface of transduced human T cells. Antibodies against EGFRt (cetuximab-APC) and Myc tag (9B11-PE) were used to detect surface expression of EGFRt and human anti-human CAR in B10-based constructs. Controls included non-transduced human T cells expressing neither EGFRt nor Myc tag, as well as non-Myc tag-containing CAR constructs Etah19h28Z (anti-human CD19 CAR T cells) and EtC1HVh28Z (anti-CD371 CAR T cells derived from mouse anti-human CD371 antibody 1075.7). Example 2 In vitro cytotoxic activity of CD371-targeted CAT T cells

[0308] T cells were transfected with several CD371-targeting CARs (i.e., "Et.C1HVh28Z", "Et.B10LH_MT_h28Z", "Et.B10LHg4s_MT_h28Z", "Et.B10HL_MT_h28Z", "Et.C3HL_MT_h28Z", "Et.C3LH_MT_h28Z" and "Et.D6LH_MT_h28Z"). Et.C1HVh28Z was used as a positive control, which is derived from the mouse anti-human CD371 antibody 1075.7. "Et.B10LHg4s_MT_hDEL" is a non-functional CD371-targeting CAR T cell (based on B10L4H but without the CD28 or CD3 zeta signaling domains). These CD371-targeted CAR T cells were co-cultured with CD371+U937 cells (U937gL) expressing GFP and firefly luciferase at different effector:tumor ratios. After 24 hours, bioluminescence was measured and plotted as a percentage of the signal detected in the co-culture of non-functional CD371-targeted CAR T cells (based on B10L4H but without CD28 or CD3 zeta signaling domains) and U937gL. The results are shown in Figure 3. As shown in Figure 3, CD371-targeted CAR T cells were able to lyse cells expressing CD371. Example 3 In vitro cytotoxic activity of CD371-targeted CAT T cells

[0309] T cells were transfected with several CD371-targeting CARs (i.e., "Et.C1HVh28Z", "Et.B10L4H_MT_h28Z", "Et.B10L3H_MT_h28Z", "Et.B10L4H_MT_hBBZ", "Et.B10H3L_MT_h28Z", "Et.C3HL_MT_h28Z", "Et.C3LH_MT_h28Z" and "Et.M195_MT_h28Z"). Et.C1HVh28Z was used as a positive control, which is derived from the mouse anti-human antibody 1075.7. "Et.M195MTh28Z" represents CD33-targeting CAR T cells derived from the mouse anti-human CD33 antibody M195. "Et.M195MTh28Z" was used as a positive control, since U937 cells also express CD33. Healthy human donor-derived (i.e., donor C and donor D) CD371-targeted CAR T cells were co-cultured with CD33+ / CD371+ U937 cells expressing GFP and firefly luciferase (U937gL) at different effector:tumor ratios. After 24 hours, bioluminescence was measured and plotted as a percentage of the signal detected in the co-culture of non-functional CD371-targeted CAR T cells (based on B10L4H but without CD28 or CD3 zeta signaling domains) and U937gL. The results are shown in Figure 4 (for donor C) and Figure 5 (for donor D). As shown in Figures 4 and 5, B10-based CAR (both HL / LH) T cells outperformed C3-based CAR T cells. Example 4 Antigenic stimulation of CD371-targeted CAR T cells

[0310] Healthy human donor-derived (i.e., Donor C and Donor D) CD371-targeted CAR T cells were co-cultured with CD33+ / CD371+U937gL at an E:T ratio of 1:12.5 and a concentration of 30,000 CAR+ / mL. Approximately every 4-5 days, CAR T cells were counted and characterized by flow cytometry, and the starting number of tumor cells was added back to the culture (indicated by arrows). Results are shown in Figure 6 (for donor C) and Figure 7 (for donor D). "Et.B10LHdel" represents non-functional CAR T cells (lacking signaling domains). "Et.C1HVh28Z" represents CD371-targeted CAR derived from mouse anti-human CD371 antibody 1075.7, and "EtM195MTh28Z" or "M19528" represents CD33-targeted CAR T cells derived from mouse anti-human CD33 antibody M195. As shown in Figures 6 and 7, B10-based CAR T cells outperformed C3-based CAR T cells in long-term repeated stimulation assays. Example 5 In vitro cytotoxic activity of CD371-targeted CAT T cells

[0311] Healthy human donor-derived (i.e., Donor A and Donor B) CD371-targeted CAR T cells were co-cultured with CD371-positive U937 or HL60 cells (U937gL or HL60gL) expressing GFP and firefly luciferase at different effector:tumor ratios. Bioluminescence was measured 24 hours after co-culture and plotted as a percentage of the signal detected in the co-culture of non-functional CD371-targeted CAR T cells (based on B10L4H but without CD28 or CD3 zeta signaling domains) and U937gL. The results are shown in Figures 8A and 8B (for U937gL cells) and Figures 9A and 9B (for HL60 cells). As shown in Figures 8A-8B and 9A-9B, B10HL-based CD371-targeted CAR T cells outperformed B10LH-based CD371-targeted CAR T cells. Example 6 Antigen-specific cytotoxic activity of CD371-targeted CAR T cells

[0312] Whether the cytotoxic activity of CD371-targeting CAR T cells is specific to CD371 was evaluated. Human CD371 (hCD371) CRISPR knockout cell line was generated. Human CD371 was knocked out from HL60gL and U937gL using CRISPR, and knockout was confirmed by flow cytometry using anti-human CD371 APC-conjugated antibody.See Figure 10.

[0313] Healthy human donor-derived (i.e., Donor A and Donor B) CD371-targeted CAR T cells were co-cultured with antigen-negative U937 cells expressing RFP and Cypridina luciferase or HL60 (U937RFPcyp.371KO or HLgL.371KO) at different effector:tumor ratios. After 24 hours, bioluminescence was measured and plotted as a percentage of the signal detected in co-cultures of non-functional CD371-targeted CAR T cells (based on B10L4H but without CD28 or CD3 zeta signaling domains) and U937RFPcyp.371KO. Results are shown in Figures 11A-11B (for U937 cells) and Figures 12A-12B (for HL60 cells). As shown in Figures 11A-11B and 12A-12B, both B10HL-based CD371-targeted CAR T cells and B10LH-based CD371-targeted CAR T cells showed no significant cytotoxicity against CD371-negative U937 cells and limited cytotoxicity against CD371-negative HL60 cells. Example 7 Cytokine secretion by CD371-targeted CAR T cells

[0314] The ability to produce cytokines by CD371-targeted CAR T cells was evaluated. CD371-targeted CAR T cells were co-cultured alone, with CD371-negative tumor cells, or with CD371-positive U937 cells at a 1:1 effector:tumor ratio (40,000:40,000 cells in 200 μl). After 24 hours, supernatants were collected from the cultures and secretion levels of IFN-γ, IL-2, and TNF-α were measured using a bead-based multiplex assay. The results are shown in Figures 13A-13B (for IFN-γ), Figures 14A-14B (for IL-2), and Figures 15A-15B (for TNF-α). As shown in Figures 13A-13B, Figures 14A-14B, and Figures 15A-15B, B10HL-based CAQR T cells produced more cytokines in an antigen-dependent manner compared to B10LH-based CAR T cells. Example 8 Proliferative capacity of CD371-targeted CAR T cells

[0315] CAR T cells were generated from two healthy donors (i.e., Donor A and Donor B). CD371-targeted CAR T cells were co-cultured with CD371+U937gL at an E:T ratio of 1:5 and a concentration of 50,000 CAR+ / mL. CAR T cells were counted and characterized by flow cytometry approximately every 5 days. The starting number of tumor cells was added back to the culture at the time indicated by the arrow. The results are shown in Figure 16. As shown in Figure 16, B10HL-based CD371-targeted CAR T cells and B10LH-based CD371-targeted CAR T cells showed nearly comparable expansion capacity in vitro. Example 9 In vivo antitumor activity of CD371-targeted CAR T cells

[0316] The in vivo antitumor activity of B10-based CD371-targeted CAR T cells was evaluated. Figure 17 shows a xenograft model of AML. NCG mice were injected with 5.0 x 10 4AML FAB-M5 cell line of U937gL tumor cells was inoculated via the tail vein, and approximately 1.25 × 10 6 Each donor was treated with 10 CAR T cells. The mice were monitored for survival. The results are shown in Figure 18 (for donor 1) and Figure 19 (for donor 2). As shown in Figures 18 and 19, B10HL-based CAR T cells showed superior survival in AML xenograft mice compared to B10LH-based CAR T cells.

[0317] We next assessed whether the in vivo activity was dose-dependent. NCG mice were dosed with 5.0 × 10 4 U937gL tumor cells were inoculated and 3 days later, various doses of CAR T cells (1.25×10 5 , 2.5×10 5 , 5.0×10 5 and 1.0×10 6 The results are shown in Figure 20. As shown in Figure 20, B10HL-based CAR T cells outperformed B10LH-based CAR T cells in a dose-response treatment model. Example 10 B10 scFv binding to cell lines expressing CD371

[0318] In both orientations (V H -V L or V L -V H The binding of the B10 (also referred to as "1B10") scFv of 1B10 to cells expressing CD371 was evaluated. The results are shown in FIG. 21. As shown in FIG. 21, binding to cells was evaluated using the V L -V H However, the V H -V L No binding of oriented B10 scFv was observed by flow cytometry, suggesting low affinity and therefore the requirement for bivalent binding. Example 11 B10 scFv binding to recombinant CD371 in solution

[0319] For affinity measurements of scFv, biotinylated CD371 was captured with streptavidin and soluble scFv was used as the analyte. Table 8 shows the dissociation constants (K D ), binding rate (k on ) and dissociation rate (k off ) are shown. Consistent with the flow cytometry results, V H -V L Weak binding of the oriented 1B10 scFv was observed, but the dissociation constant could not be calculated by any curve fitting method.

[0320] It is therefore surprising that CARs comprising scFvs with lower binding affinity were superior in vitro and in vivo to CARs comprising scFvs with higher binding affinity, as shown in Examples 5, 7 and 9. Table 8.V H -V L or V L -V H Binding affinity of oriented B10 scFv to soluble CD371 [Table 8]

[0321] Example 12 In vivo and in vitro activity of second-generation B10HL-based CAR T cells Second generation CAR T cells based on B10HL with truncated EGFR (Et), Myc tag (MT) and G4S linkers of various lengths were successfully generated. Et.B10H4L_MT_h28Z is described in Example 1. Et.B10H1L_MT_h28Z is V H -V LEt.B10H2L_MT_h28Z contained a B10-based scFv (designated as B10H1L) with a 5 amino acid G4S linker (GGGGS [SEQ ID NO: 93]) and a CD28Z-based signaling domain. H -V L The scFv comprised a B10-based scFv (designated as B10H2L) with a 10 amino acid long G4S linker (GGGGSGGGGS [SEQ ID NO: 94]) and a CD28Z-based signaling domain. Et.B10H5L_MT_h28Z is V H -V L The scFv comprised a B10-based scFv (designated as B10H5L) with a 24 amino acid long G4S linker (GGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 91]) and a CD28Z-based signaling domain. Et.B10H6L_MT_h28Z is V H -V L The scFv contained a B10-based scFv (designated as B10H6L) with a CD28Z-based domain and a 29 amino acid G4S linker (GGGGSGGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 92]).

[0322] Additional B10H4L-based second generation CAR T cells were generated that incorporate either constitutive IL18 (designated as "Et.B10H4Lmt28ZpIL18") or IL33 (designated as "Et.B10H4Lmt28ZpIL33") secretion. T cells containing Et.B10H4Lmt28ZpIL18 contain a CAR designated as "Et.B10H4L_MT_h28Z" and an exogenous IL-18 polypeptide (e.g., an exogenous IL-18 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 103). T cells containing Et.B10H4Lmt28ZpIL33 contain a CAR designated as "Et.B10H4L_MT_h28Z" and an exogenous IL-33 polypeptide (e.g., an exogenous IL-33 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 105).

[0323] As shown in FIG. H -V L CAR T cells bearing scFvs of the V orientation L -V H To evaluate the in vivo activity of these B10-based CAR T cells, 5 × 10 4 U937gL tumor cells were inoculated and treated with various doses of CAR T cells. The survival of the treated mice was monitored and recorded. As shown in Figure 23, 5×10 5 At a dose of 2.5×10 CAR T cells, human CD371-targeted CAR T cells based on B10H3L, B10H5L, and IL33-secreting B10H4L outperformed B10H4L-based CAR T cells. As shown in FIG. 24, at a dose of 2.5×10 5 At a dose of 1.0×10 CAR T cells, B10H4L-based human CD371-targeted CAR T cells secreting IL18 and IL33 were superior to B10H4L-based CAR T cells. As shown in Figure 25, 5 At low doses of CAR T cells, IL18-secreting B10H4L human CD371-targeted CAR T cells outperformed other CAR T cells. Embodiments of the Subject Matter of the Disclosure

[0324] From the foregoing description, it is apparent that variations and modifications may be made to the subject matter of the present disclosure to accommodate various uses and conditions. Such embodiments are also within the scope of the following claims.

[0325] The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0326] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference. The present invention provides, for example, the following items. (Item 1) An antigen recognition receptor comprising an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to CD371. (Item 2) The antigen-recognizing receptor of item 1, wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv). (Item 3) 3. The antigen recognition receptor according to item 2, wherein the extracellular antigen-binding domain is a human scFv. (Item 4) 2. The antigen recognition receptor according to item 1, wherein the extracellular antigen-binding domain is a Fab, optionally cross-linked. (Item 5) 2. The antigen recognition receptor according to item 1, wherein the extracellular antigen-binding domain is F(ab)2. (Item 6) 6. The antigen-recognizing receptor according to any one of items 2 to 5, wherein one or more of scFv, Fab and F(ab)2 are included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. (Item 7) The extracellular antigen-binding domain comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28 or a conservative modification thereof; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29 or a conservative modification thereof; and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30 or a conservative modification thereof; (b) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34 or a conservative modification thereof; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35 or a conservative modification thereof; and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36 or a conservative modification thereof; (c) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40 or a conservative modification thereof; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42 or a conservative modification thereof; and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43 or a conservative modification thereof; (d) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 462 or a conservative modification thereof; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 47 or a conservative modification thereof; and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48 or a conservative modification thereof; (e) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52 or a conservative modification thereof; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 53 or a conservative modification thereof; and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 54 or a conservative modification thereof; or (f) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58 or a conservative modification thereof; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59 or a conservative modification thereof; and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 60 or a conservative modification thereof. 7. The antigen recognition receptor according to any one of items 1 to 6, comprising: (Item 8) 8. The antigen recognition receptor according to any one of items 1 to 7, wherein the extracellular antigen-binding domain comprises a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 28; a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 29; and a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 30. (Item 9) The extracellular antigen-binding domain comprises: (a) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31 or a conservative modification thereof; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32 or a conservative modification thereof; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33 or a conservative modification thereof; (b) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37 or a conservative modification thereof; a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof CDR2; and a light chain variable region CDR3 comprising SEQ ID NO:39 or a conservative modification thereof; (c) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43 or a conservative modification thereof; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44 or a conservative modification thereof; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45 or a conservative modification thereof; (d) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49 or a conservative modification thereof; a light chain variable region CDR2 comprising SEQ ID NO: 50 or a conservative modification thereof; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51 or a conservative modification thereof; (e) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 55 or a conservative modification thereof; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 56 or a conservative modification thereof; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof; or (f) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 61 or a conservative modification thereof; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 62 or a conservative modification thereof; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63 or a conservative modification thereof. The antigen recognition receptor according to any one of items 1 to 8, comprising: (Item 10) 10. The antigen recognition receptor according to any one of items 1 to 9, wherein the extracellular antigen-binding domain comprises a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 31; a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 32; and a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 33. (Item 11) The extracellular antigen-binding domain comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29; a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33; (b) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35; a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39; (c) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41; a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42; a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45; (d) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 46; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 47; a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48; a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 51; (e) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 52; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 53; a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 54; a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 55; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 56; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57; or (f) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 60; a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 61; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 62; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63 11. The antigen recognition receptor according to any one of items 1 to 10, comprising: (Item 12) 12. The antigen recognition receptor according to any one of items 1 to 11, wherein the extracellular antigen-binding domain comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28; a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29; a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31; a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32; and a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33. (Item 13) 13. The antigen recognition receptor according to any one of items 1 to 12, wherein the extracellular antigen-binding domain comprises a heavy chain variable region comprising 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% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11. (Item 14) 14. The antigen recognition receptor according to any one of items 1 to 13, wherein the extracellular antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11. (Item 15) 15. The antigen recognition receptor according to any one of items 1 to 14, wherein the extracellular antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence having the sequence set forth in SEQ ID NO:1. (Item 16) 16. The antigen recognition receptor according to any one of items 1 to 15, wherein the extracellular antigen-binding domain comprises a light chain variable region comprising 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% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12. (Item 17) 17. The antigen recognition receptor according to any one of items 1 to 16, wherein the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. (Item 18) 18. The antigen recognition receptor according to any one of items 1 to 17, wherein the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2. (Item 19) the extracellular antigen-binding domain comprises (a) a sequence selected from the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11 and at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11; % homologous or identical to the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12; and (b) a heavy chain variable region comprising 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 19. The antigen recognition receptor according to any one of items 1 to 18, comprising a light chain variable region comprising an amino acid sequence that is 98% or about 99% homologous or identical to the antigen recognition receptor. (Item 20) 20. The antigen recognition receptor according to any one of Items 1 to 19, wherein the extracellular antigen-binding domain comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12. (Item 21) The extracellular antigen-binding domain comprises: (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2; (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:4; (c) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:6; (d) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8; (e) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10; or (f) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12 15. The antigen recognition receptor according to item 14, comprising: (Item 22) 22. The antigen recognition receptor according to any one of items 1 to 21, wherein the extracellular antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2. (Item 23) 23. The antigen recognition receptor according to any one of items 1 to 22, wherein the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. (Item 24) 24. The antigen recognition receptor according to item 23, wherein the linker has an amino acid sequence as set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93 or SEQ ID NO: 94. (Item 25) 24. The antigen recognition receptor according to claim 23, wherein the linker has the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14. (Item 26) 26. The antigen recognition receptor according to any one of items 1 to 25, wherein the extracellular antigen-binding domain comprises a signal peptide covalently joined to the 5' end of the extracellular antigen-binding domain. (Item 27) The extracellular antigen-binding domain is, from N-terminus to C-terminus, H -V L 27. The antigen recognition receptor according to any one of items 1 to 26, comprising a heavy chain variable region and a light chain variable region positioned at (Item 28) 27. The antigen recognition receptor according to any one of items 1 to 26, wherein the extracellular antigen-binding domain binds to CD371 with low binding affinity. (Item 29) The extracellular antigen-binding domain is 1×10-8 M or higher dissociation constant (K d 27. The antigen recognition receptor according to any one of items 1 to 26, which binds to CD371 at the agonist (A) or agonist (B). (Item 30) 30. The antigen recognition receptor of any one of items 1 to 29, 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, a BTLA polypeptide, or a combination thereof. (Item 31) 31. The antigen recognition receptor of item 30, wherein the transmembrane domain comprises a CD28 polypeptide. (Item 32) 32. The antigen recognition receptor according to any one of items 1 to 31, wherein the intracellular signaling domain comprises a CD3ζ polypeptide. (Item 33) 33. The antigen recognition receptor according to any one of items 1 to 32, wherein the intracellular signaling domain further comprises at least one costimulatory signaling region. (Item 34) 34. The antigen recognition receptor of item 33, 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. (Item 35) 35. The antigen recognition receptor of item 33 or 34, wherein the at least one costimulatory signaling region comprises a CD28 polypeptide or a 4-1BB polypeptide. (Item 36) 36. The antigen recognition receptor according to any one of items 1 to 35, wherein the antigen recognition receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR) or a T cell-like fusion protein. (Item 37) 37. The antigen recognition receptor according to any one of items 1 to 36, wherein the antigen recognition receptor is a CAR. (Item 38) 38. The antigen recognition receptor according to any one of items 1 to 37, wherein the antigen recognition receptor is recombinantly expressed. (Item 39) 39. The antigen recognition receptor according to any one of items 1 to 38, wherein the antigen recognition receptor is expressed from a vector. (Item 40) 40. The antigen recognition receptor according to item 39, wherein the vector is a gamma-retroviral vector. (Item 41) A cell comprising the antigen recognition receptor according to any one of items 1 to 40. (Item 42) 42. The cell of item 41, wherein the cell is transduced with the antigen recognition receptor. (Item 43) 43. The cell according to item 41 or 42, wherein the antigen recognition receptor is constitutively expressed on the surface of the cell. (Item 44) 44. The cell according to any one of items 41 to 43, wherein the cell is engineered to express a cytokine or a fragment thereof. (Item 45) Item 44. The cell comprises an exogenous polypeptide of the cytokine or a fragment thereof. The cell according to claim 1. (Item 46) 46. ​​The cell of item 44 or 45, wherein the cell comprises a nucleic acid molecule encoding the cytokine or a fragment thereof. (Item 47) 47. The cell according to any one of items 44 to 46, wherein the cytokine is selected from the group consisting of IL-18, IL-33, IL-36 and combinations thereof. (Item 48) 48. The cell according to any one of items 44 to 47, wherein the cytokine is IL-18. (Item 49) 49. The cell according to any one of items 41 to 48, wherein the cell is an immune response cell. (Item 50) 50. The cell according to any one of items 41 to 49, wherein the cell is a lymphoid lineage cell or a myeloid lineage cell. (Item 51) 51. The cell according to any one of items 41 to 50, wherein the cell is selected from the group consisting of T cells, natural killer (NK) cells, and stem cells from which lymphoid cells can be differentiated. (Item 52) 52. The cell according to any one of items 41 to 51, wherein the cell is a T cell. (Item 53) 53. The cell of item 51 or 52, wherein the T cell is a cytotoxic T lymphocyte (CTL) or a regulatory T cell. (Item 54) 54. The cell of item 53, wherein the stem cell is a pluripotent stem cell. (Item 55) 55. The cell of item 54, wherein the pluripotent stem cell is an embryonic-like stem cell or an induced pluripotent stem cell. (Item 56) 41. A nucleic acid molecule encoding the antigen-recognition receptor according to any one of items 1 to 40. (Item 57) 57. The nucleic acid molecule according to item 56, wherein the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27. (Item 58) 58. The nucleic acid molecule according to item 56 or 57, wherein the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 22. (Item 59) 59. A vector comprising the nucleic acid molecule according to any one of Items 56 to 58. (Item 60) 60. The vector according to item 59, wherein the vector is a gamma-retroviral vector. (Item 61) 59. A host cell expressing the nucleic acid molecule according to any one of items 56 to 58. (Item 62) 62. The host cell of item 61, wherein the host cell is a T cell. (Item 63) A composition comprising the cell according to any one of items 41 to 55. (Item 64) 64. The composition according to item 63, which is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier. (Item 65) 56. A method for reducing tumor burden in a subject, comprising administering to the subject an effective amount of a cell according to any one of items 41 to 55 or a composition according to item 63 or 64. (Item 66) 66. The method of claim 65, wherein the method reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject. (Item 67) A method for increasing or extending the survival time of a subject having a tumor or neoplasm, comprising administering an effective amount of a cell according to any one of items 41 to 55 or a composition according to item 63 or 64. (Item 68) A method for treating and / or preventing a tumor or neoplasm in a subject, comprising administering an effective amount of a cell according to any one of items 41 to 55 or a composition according to item 63 or 64. (Item 69) 69. The method of any one of items 65 to 68, wherein the tumor or neoplasm is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), glioblastoma, myelodysplastic syndrome (MDS) and chronic myeloid leukemia (CML). (Item 70) 70. The method of any one of items 65 to 69, wherein the tumor or neoplasm is acute myeloid leukemia (AML). (Item 71) 41. A method for generating a cell comprising the antigen recognition receptor according to any one of items 1 to 40, comprising introducing a nucleic acid molecule encoding the antigen recognition receptor into the cell. (Item 72) 56. A kit for reducing tumor burden in a subject, for treating and / or preventing a tumor or neoplasm in a subject, and / or for increasing or extending the survival time of a subject having a tumor or neoplasm, comprising the cell of any one of items 41 to 55. (Item 73) 73. The kit of claim 72, further comprising instructions for using the cells to reduce tumor burden in a subject, to treat and / or prevent a tumor or neoplasm in a subject, and / or to increase or prolong survival of a subject having a tumor or neoplasm.

Claims

[Claim 1] The invention described in the specification.

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