Antigen-recognition receptor targeting CD371 and uses thereof

Abstract

The subject matter of the present disclosure provides an antigen-recognition receptor that specifically targets CD371, and cells comprising such a CD371-targeting antigen-recognition receptor.The subject matter of the present disclosure further provides the use of a CD371-targeting antigen-recognition receptor for treatment.The subject matter of the present disclosure provides methods and compositions for immunotherapy.It relates to antigen-recognition receptors that specifically target CD371 (e.g., chimeric antigen receptors (CARs) or T cell receptors (TCRs)), cells comprising such receptors, and methods of using such cells for treatment.

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 herein by reference in their entirety and to which priority is claimed. Sequence Listing

[0002] This application contains a Sequence Listing that has been 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 designated 0727341147_ST25, and is 211,921 bytes in size. 1. Introduction

[0003] The presently disclosed subject matter 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 engineered to target tumor antigens by the introduction of genetic material encoding an artificial or synthetic receptor for the antigen, called a chimeric antigen receptor (CAR), specific for the selected antigen. Targeted T cell therapy using CARs 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 regimen has not changed substantially in the past 40 years, and overall survival rate remains very poor.Frequent recurring abnormalities involving genes that code for epigenetic modifiers have been identified.The development of CAR therapy for AML is hindered by the lack of suitable target.Therefore, there is a need for a new therapeutic strategy for designing CAR that targets the antigen that is highly expressed in AML cells and has limited expression in normal tissues for treating AML, and for a strategy that can induce potent cancer eradication with minimal toxicity and immunogenicity. Summary of the Invention [Means for solving the problem]

[0007] 3. Summary of the Invention The presently disclosed subject matter provides an antigen-recognition receptor that specifically targets CD371, and cells comprising such a CD371-targeting antigen-recognition receptor. The presently disclosed subject matter further provides uses of the CD371-targeting 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 the amino acid sequence set forth in 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, from N- to C-terminus, a V H -V LThe heavy chain variable region and the light chain variable region are 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 presently disclosed subject matter provides a cell comprising the antigen-recognizing receptor of the present disclosure. In certain embodiments, the cell is transduced with the antigen-recognizing receptor. In certain embodiments, the antigen-recognizing receptor is constitutively expressed on the surface of the cell.

[0027] In certain embodiments, the cells are engineered to express a cytokine or a fragment thereof. In certain embodiments, the cells further comprise an exogenous polypeptide of a cytokine or a fragment thereof. In certain embodiments, the cells further comprise 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 a T cell, a natural killer (NK) cell, and a stem cell from which a lymphoid cell 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 presently disclosed subject matter further provides a nucleic acid molecule encoding an antigen-recognizing 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 presently disclosed subject matter 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] Additionally, the presently disclosed subject matter provides host cells that express 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 pharmaceutically acceptable carrier.

[0032] The presently disclosed subject matter further provides a method for reducing tumor burden in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of a cell or composition of the present disclosure. In certain embodiments, the method reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject. The presently disclosed subject matter further provides a method for 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 a cell or composition of the present disclosure. The presently disclosed subject matter further provides a method for 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 a cell or composition 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 presently disclosed subject matter further provides methods for generating a cell of the present disclosure comprising a CD371-targeting antigen-recognition receptor. In certain embodiments, the method comprises introducing into the cell a nucleic acid molecule encoding the antigen-recognition receptor.

[0034] In addition, the presently disclosed subject matter provides kits for reducing tumor burden in a subject, treating and / or preventing a tumor or neoplasm in a subject, and / or increasing or extending the survival time of a subject having a tumor or neoplasm. In certain embodiments, the kits include the cells described herein. In certain embodiments, the kits further include instructions for using the cells to reduce tumor burden in a subject, treat and / or prevent a tumor or neoplasm in a subject, and / or increase or extend the survival time of a subject having a tumor or neoplasm. 4. Brief description of the drawings

[0035] The following detailed description, given by way of example and not intended to limit the invention to the specific embodiments described, may be understood in conjunction with the accompanying drawings, in which: [Brief explanation 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 shows the 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 murine anti-human CD371 antibody 107537). [Figure 2-2]Figure 2 shows the 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 murine anti-human CD371 antibody 107537).

[0038] [Figure 3] Figure 3 shows the tumor cytolytic activity of CD371-targeted CAR T cells. Four-day rested (4dR) human CD371-targeted CAR T cells were co-cultured with CD33 / CD371 U937 cells (U937gL) expressing GFP and firefly luciferase at different effector:tumor (E:T) ratios. Bioluminescence was measured after 24 hours and plotted as the percentage of signal detected in co-cultures of non-functional CAR T cells (based on B10L4H but lacking the CD28 or CD3 zeta signaling domain) 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 shows the tumor cell lytic 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 (U937gL) expressing GFP and firefly luciferase at different effector:tumor (E:T) ratios. Bioluminescence was measured after 24 hours and plotted as the percentage of signal detected in co-cultures of non-functional CAR T cells (based on B10L4H but lacking the CD28 or CD3 zeta signaling domain) and U937gL. Et.C1HVh28z represents a CD371-targeted CAR derived from the mouse anti-human CD371 antibody 1075.7, and EtM195Mth28z represents a CD33-targeted CAR T cell derived from the mouse anti-human CD33 antibody M195.

[0040] [Figure 5] Figure 5 shows the tumor cell lytic 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 (U937gL) expressing GFP and firefly luciferase at different effector:tumor (E:T) ratios. Bioluminescence was measured after 24 hours and plotted as the percentage of signal detected in co-cultures of non-functional CAR T cells (based on B10L4H but lacking the CD28 or CD3 zeta signaling domain) and U937gL. Et.C1HVh28z represents a CD371-targeted CAR derived from the mouse anti-human CD371 antibody 1075.7, and EtM195Mth28z represents a CD33-targeted CAR T cell derived from the mouse anti-human CD33 antibody M195.

[0041] [Figure 6]Figure 6 shows 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-positive cells / ml. CAR T cells were counted and characterized by flow cytometry approximately every 4-5 days, and the starting number of tumor cells was added back to the culture (indicated by arrows). Et.B10LHdel represents non-functional CAR T cells (lacking the signaling domain), 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-human CD33 antibody M195.

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

[0043] [Figure 8]Figures 8A and 8B show 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 (designated "Donor A" and "Donor B") were co-cultured with CD371-positive U937 cells (U937gL) expressing GFP and firefly luciferase at different effector:tumor ratios. Bioluminescence was measured after 24 hours and plotted as the percentage of signal detected in the co-culture of non-functional CD371-targeted CAR T cells (based on B10L4H but lacking the CD28 or CD3 zeta signaling domain) 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 show 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 (designated "Donor A" and "Donor B") were co-cultured with CD371-positive HL60 cells (HL60gL) expressing GFP and firefly luciferase at different effector:tumor ratios. Bioluminescence was measured after 24 hours and plotted as the percentage of signal detected in the co-culture of non-functional CD371-targeted CAR T cells (based on B10L4H but lacking the CD28 or CD3 zeta signaling domain) 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. The knockout was confirmed by flow cytometry using an 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 cells from different healthy donors in a 24-hour killing assay. CD371-targeted CAR T cells from healthy human donors (designated "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 the percentage of signal detected in co-cultures of non-functional CD371-targeted CAR T cells (based on B10L4H but lacking the CD28 or CD3 zeta signaling domain) 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 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 HL60gL cells from different healthy donors in a 24-hour killing assay. 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 signal detected in the co-culture of non-functional CD371-targeted CAR T cells (based on B10L4H but lacking the CD28 or CD3 zeta signaling domain) 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 a 1:1 effector:tumor ratio (E:T) (4.0 x 10:4.0 x 10 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 show 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 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 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 show cell proliferation of B10-based CAR T cells in a recurrent 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 cells / 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 5x10 U937gL AML FAB-M5 cell line via the tail vein. CD371-targeted CAR T cells were administered to the mice three days later. Tumor dynamics were measured noninvasively by bioluminescence imaging approximately every five days, and survival was monitored.

[0053] [Figure 18] Figure 18 depicts 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 were monitored for survival.

[0054] [Figure 19] Figure 19 depicts 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 3 days later with approximately 1.25 x 106 human CD371-targeted CAR T cells. 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 5x10 U937gL tumor cells and treated 3 days later with various doses of CD371-targeted CAR T cells (1.25x10, 2.5x10, 5.0x10, and 1.0x10). 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 G4S linkers of various lengths. CD371-targeted CAR T cells from healthy human donors (including donors A and B) were co-cultured with antigen-positive U937 cells (U937gL) expressing GFP and firefly luciferase 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 lacking the CD28 or CD3 zeta signaling domain) and U937gL.

[0057] [Figure 23] Figure 23 depicts 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 depicts 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] [Figure 25] Figure 25 depicts 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 INVENTION

[0060] 5. Detailed Description of the Invention The presently disclosed subject matter provides antigen-recognition receptors (e.g., chimeric antigen receptors (CARs) or T cell receptors (TCRs)) that specifically target CD371. The presently disclosed subject matter further provides cells comprising such receptors. The cells can be immune response cells, e.g., genetically modified immune response cells (e.g., T cells or NK cells). The presently disclosed subject matter also provides methods of using such cells for treatment, e.g., to treat and / or prevent tumors or neoplasms (e.g., AML).

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

[0062] For purposes of clarity of disclosure, and not by way 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 meanings commonly understood by those skilled in the art to which this invention belongs. The following references provide those skilled 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 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 HarperCollins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified.

[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 how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more standard deviations, as practiced in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still 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, and 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, an immune response cell is a cell of the lymphoid lineage. Non-limiting examples of cells of the lymphoid lineage include T cells, natural killer (NK) cells, B cells, and stem cells from which lymphoid cells can differentiate. In certain embodiments, an immune response cell is a cell of the 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 TCRs or exogenous CARs bind to antigens, an immune synapse is formed near the bound receptors (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.), involving the clustering of many molecules. This clustering of membrane-bound signaling 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 global gene expression in T cells, increasing the production of IL-2 for the proliferation and expression of master regulatory T cell proteins to initiate T cell-mediated immune responses.

[0067] "Stimulating immune response cells" refers to signals that result in a robust and sustained immune response. In various embodiments, this occurs after activation of immune cells (e.g., T cells) or is simultaneously mediated through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, and ICOS. Receiving multiple stimulatory signals can be important for enhancing a robust and long-lasting T cell-mediated immune response. T cells can be rapidly inhibited and become unresponsive to antigens. While the effects of these costimulatory signals vary, 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, the antigen-recognizing receptor can activate an immune cell or immune response cell (e.g., a T cell) upon binding to the 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 from the circulation more rapidly and may exhibit less nonspecific tissue binding than 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, antibodies are glycoproteins 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 (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 contains a light chain variable region (referred to herein as V L ) and light chain constant C L The light chain constant region consists of C L It consists of one domain: V H and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with regions that are more conserved called framework regions (FRs). H and V Lis composed of three CDRs and four FRs, arranged from the amino terminus to the 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 can 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 sequence of the complementarity determining region of an antibody, which is the hypervariable region of the heavy and light chains of immunoglobulins.See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th US Department of Health and Human Services, National Institutes of Health (1987), or the IMGT numbering system (Lefranc, The Immunologist (1999); 7:132-136; Lefranc et al., Dev. Comp. Immunol. (2003); 27:55-77). Generally, an antibody comprises three heavy chain CDRs and three light chain CDRs or CDR regions in variable regions. CDRs provide the majority of contact residues for antibody binding to antigen or epitope. In certain embodiments, the CDR region is represented using the IMGT numbering system. In certain embodiments, the CDR regions are represented using the IMGT numbering system, accessible 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., murine or human) covalently linked to form a VL heterodimer. H ) and light chain (V L) is a fusion protein of the variable region 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 extracellular antigen-binding domain is joined by a peptide-encoding linker (e.g., 10, 15, 20, or 25 amino acids) that connects the N-terminus of the extracellular antigen-binding domain. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can connect the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014 / 087010, 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 can be synthesized using the VFv polypeptide as described by Huston, et al. Proc. Nat. Acad. Sci. USA, (1988); 85:5879-5883; U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754. H and V LIt can be expressed from a nucleic acid containing the coding sequence. Antagonistic scFvs with inhibitory activity have been described (see, e.g., Zhao et al., 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; Giomarelli et al., Thromb Haemost (2007);97(6):955-63; Fife et al., J Clin Invst (2006);116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40)). Agonistic scFvs with stimulatory activity have been described (Peter et al., J Biol 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 comprising an extracellular antigen-binding domain and a transmembrane domain fused to an intracellular signaling domain capable of activating or stimulating 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, which is 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 a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous or identical to the amino acid or nucleic acid sequence used for comparison.

[0080] Sequence identity can be measured using sequence analysis software (e.g., the BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs, sequence analysis software packages from the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining 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 alleviate, improve, stabilize, reverse, or slow 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 skilled 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 differ from its endogenous counterpart by sequence, position / location, or both. For clarity, an exogenous nucleic acid may have the same or a different sequence from its native, endogenous counterpart, and may be introduced by genetic engineering into the cell itself or its precursor cell, and may, if necessary, be linked to an alternative regulatory sequence, such as a non-native promoter or secretion sequence.

[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] "Modulate" means to change, either positively or negatively. Exemplary modulations include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0086] By "increase" is meant a positive alteration of at least about 5%. The alteration may 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 materials that are free, to varying degrees, from components normally associated with the material as found in its native state. "Isolated" refers to a degree of separation from the original source or surroundings. "Purified" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free from other materials so that any impurities do not significantly affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide is purified when it is substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA 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 yields essentially one band in an electrophoretic gel. For proteins that can be subject to modifications, such as phosphorylation or glycosylation, different modifications can result in 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] "Neoplasia" 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 do not induce normal cell proliferation or would otherwise cause its cessation. Neoplasia can affect various cell types, tissues, or organs, including, but not limited to, organs selected from the group consisting of the 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 (a malignant tumor of plasma cells). Neoplasia can be a primary tumor or 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 tumors can express receptors (e.g., TCRs or CARs) that bind to tumor antigens.

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

[0099] As used herein, "treatment" refers to a clinical intervention that seeks to alter the disease course of the treated individual or cell, and can be performed for prophylaxis or during the course of clinical pathology. The therapeutic effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. By preventing the progression of a disease or disorder, treatment can prevent deterioration due to the disorder in an affected or diagnosed subject, or a subject suspected of having the disorder. Treatment can also 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] As used herein, an "individual" or "subject" refers to a vertebrate, such as a human or non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, hamsters, and guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, and non-human primates, such as apes and monkeys. The term "immunocompromised" as used herein refers to a subject with an immunodeficiency. Subjects are highly susceptible 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 under-functioning or suppressed immune systems.

[0101] Other aspects of the presently disclosed subject matter are described in the disclosure that follows and are within the scope of the presently disclosed subject matter. 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 motif (ITIM) (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 has been implicated as a negative regulatory urate crystal (monosodium urate, MSU) receptor that controls autoimmune and inflammatory diseases (Neumann et al., Immunity (2014);40:389-99). CD371 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 presently disclosed subject matter also provides nucleic acid molecules encoding the antigen-recognizing receptors of the present disclosure. In certain embodiments, the nucleic acid molecules comprise a nucleotide sequence encoding a CD371-targeting antigen-recognizing receptor polypeptide 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 bound 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, a TCR can form a receptor complex with three dimeric signaling modules, CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or ζ / η. Binding of the TCR complex with its antigen and MHC (peptide / MHC) activates a T cell expressing the TCR complex.

[0110] In certain embodiments, the TCR is an endogenous TCR. In certain embodiments, the antigen-recognizing 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 has been 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 gives the specificity of interest onto immune effector cells.CAR can be used to graft the specificity of monoclonal antibody onto T cells, and the transfer of their coding sequence is promoted by retroviral vector.

[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, binding to CD4 through their CD3 ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. + and CD8 + These CARs can induce both activation and activation of T cells. "Second-generation" CARs add intracellular signaling domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide additional signals to T cells. "Second-generation" CARs include 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-recognizing receptor is a first-generation CAR. In certain embodiments, the antigen-recognizing 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-recognizing receptor is a second-generation CAR.

[0114] In certain embodiments, the CAR comprises an extracellular antigen-binding domain that specifically binds to 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 using 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 x 10 -6 M or less, e.g., about 1 x 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 less 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 binding domain of about 1 x 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 x 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 x 10 -8 m or approximately 1 x 10 -8 K of M dIn certain embodiments, the extracellular antigen-binding domain of the CAR binds to CD371 (e.g., human CD371) at about 1 x 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 x 10 -6 M 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, the 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, the 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 (e.g., scFv) of the CAR comprises a V that 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. HFor example, the extracellular antigen-binding domain (e.g., scFv) of the CAR can comprise a V 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 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 (e.g., scFv) of the CAR 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 (e.g., scFv) of the CAR can comprise a V 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 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 (e.g., scFv) of the CAR 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 and a V comprising the amino acid sequence set forth in SEQ ID NO: 30 or a conservative modification thereof H SEQ ID NOs: 28 to 30 are provided in Table 1.

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

[0125] 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: 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 comprising the amino acid sequence set forth in SEQ ID NO: 33 or a conservative modification thereof L Includes CDR3.

[0126] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) of the CAR comprises a V comprising 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 L Includes CDR3.

[0127] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 1. Hand 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 regions are arranged in a V-terminal sequence 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 regions are arranged in a V-terminal sequence 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 (e.g., scFv) of the CAR comprises a V that 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:3. H For example, the extracellular antigen-binding domain (e.g., scFv) of the CAR can comprise a V 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 comprising the amino acid sequence set forth in SEQ ID NO:3. H SEQ ID NO: 3 is provided in Table 2 below.

[0131] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V that 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 (e.g., scFv) of the CAR can comprise a V 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 comprising the amino acid sequence set forth in SEQ ID NO:4. 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 V comprising the amino acid sequence set forth in SEQ ID NO: 3, as shown in Table 2. HIn certain embodiments, the anti-CD371 scFv comprises a V comprising the amino acid sequence set forth in SEQ ID NO:4. L In certain embodiments, the anti-CD371 scFv comprises a V 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 (e.g., scFv) of the CAR comprises a V 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 comprising the amino acid sequence set forth in SEQ ID NO: 36 or a conservative modification thereof H SEQ ID NOs: 34-36 are provided in Table 2.

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

[0135] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V 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 conservative modifications 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 comprising the amino acid sequence set forth in SEQ ID NO: 39 or a conservative modification thereofL Includes CDR3.

[0136] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V comprising 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 a V 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 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 regions are arranged in a V-terminal sequence 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 LIn certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable regions are arranged in a V-terminal sequence 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 (e.g., scFv) of the CAR 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 (e.g., scFv) of the CAR can comprise a V 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 comprising the amino acid sequence set forth in SEQ ID NO:5. H SEQ ID NO:5 is provided in Table 3 below.

[0141] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V that 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:6.L For example, the extracellular antigen-binding domain (e.g., scFv) of the CAR can comprise a V 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 (e.g., scFv) of the CAR comprises a V 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 comprising the amino acid sequence set forth in SEQ ID NO: 42 or a conservative modification thereof H SEQ ID NOs: 40-42 are provided in Table 3.

[0143] 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: 43, or a conservative modification thereof. L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 44 or conservative modifications thereof L CDR2 and a V comprising the amino acid sequence set forth in SEQ ID NO: 45 or a conservative modification thereof L SEQ ID NOs: 43-45 are provided in Table 3.

[0144] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V 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 HCDR3, V comprising the amino acid sequence set forth in SEQ ID NO: 43 or conservative modifications thereof L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 44 or conservative modifications thereof L CDR2 and a V comprising the amino acid sequence set forth in SEQ ID NO: 45 or a conservative modification thereof L Includes CDR3.

[0145] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V comprising 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 a V comprising 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 H In certain embodiments, when the extracellular antigen-binding domain of the CAR is an scFv, the variable regions are arranged in a V-terminal sequence from the N-terminus to the C-terminus. H -V LIn 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 regions are arranged in a V-terminal sequence 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 (e.g., scFv) of the CAR 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:7. H For example, the extracellular antigen-binding domain (e.g., scFv) of the CAR can comprise a V 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. HIn certain embodiments, the extracellular antigen-binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO:7. H SEQ ID NO: 7 is provided in Table 4 below.

[0150] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR 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:8. L For example, the extracellular antigen-binding domain (e.g., scFv) of the CAR can comprise a V 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 (e.g., scFv) of the CAR comprises a V 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 conservative modifications thereof H CDR2 and a V comprising the amino acid sequence set forth in SEQ ID NO: 48 or a conservative modification thereof H SEQ ID NOs: 46-48 are provided in Table 4.

[0152] 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: 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 comprising the amino acid sequence set forth in SEQ ID NO: 51 or a conservative modification thereof LSEQ ID NOs: 49-51 are provided in Table 4.

[0153] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V 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 conservative modifications 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 conservative modifications 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 comprising the amino acid sequence set forth in SEQ ID NO: 51 or a conservative modification thereof L Includes CDR3.

[0154] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) of the CAR comprises a V comprising 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 (e.g., scFv) of the CAR comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 7. H and 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 regions are arranged in a V-terminal sequence 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 regions are arranged in a V-terminal sequence 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 (e.g., scFv) of the CAR comprises a V that 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:9. HFor example, the extracellular antigen-binding domain (e.g., scFv) of the CAR can comprise a V 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 (e.g., scFv) of the CAR comprises a V that 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: 10. L For example, the extracellular antigen-binding domain (e.g., scFv) of the CAR can comprise a V 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 comprising the amino acid sequence set forth in SEQ ID NO: 10. L SEQ ID NO: 10 is provided in Table 5 below.

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

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

[0162] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V 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 conservative modifications 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 comprising the amino acid sequence set forth in SEQ ID NO: 57 or a conservative modification thereof L Includes CDR3.

[0163] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises a V comprising 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 L CDR2 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 (e.g., scFv) of the CAR comprises a V comprising 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 regions are arranged in a V-terminal sequence 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 regions are arranged in a V-terminal sequence 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 (e.g., scFv) of the CAR comprises a V that 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: 11. H For example, the extracellular antigen-binding domain (e.g., scFv) of the CAR can comprise a V 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 comprising the amino acid sequence set forth in SEQ ID NO: 11. H SEQ ID NO: 11 is provided in Table 6 below.

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

[0169] 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: 58, or a conservative modification thereof. HCDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 59 or a conservative modification thereof H CDR2 and a V comprising the amino acid sequence set forth in SEQ ID NO: 60 or a conservative modification thereof H SEQ ID NOs: 58-60 are provided in Table 6.

[0170] 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: 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 V comprising the amino acid sequence set forth in SEQ ID NO: 63 or a conservative modification thereof L SEQ ID NOs: 61-63 are provided in Table 6.

[0171] 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: 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 conservative modifications thereof L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO: 62 or a conservative modification thereof L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO: 63 or a conservative modification thereof L Includes CDR3.

[0172] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) of the CAR comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 58. H CDR1, 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 LCDR2 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 (e.g., scFv) of the CAR comprises a V comprising 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 regions are arranged in a V-terminal sequence 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 regions are arranged in a V-terminal sequence 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: 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) comprising 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 within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; 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; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within 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, or no more than 5 residues within a particular sequence or 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 to 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 selected from SEQ ID NOs: 1-12. H and / or V L The present invention relates to a method for producing a nucleic acid sequence comprising the steps of:

[0178] As used herein, the percent homology between two amino acid sequences is equal to the percent identity between the two sequences.The percent 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 percent identity between two sequences can be achieved using a mathematical algorithm.

[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. Additionally, 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, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0180] Additionally or alternatively, the amino acid sequences of the presently disclosed subject matter can be further used as a "query sequence" to search public databases, for example, 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 using 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 (eg, 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 a 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 a 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 cross-compete 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 the V of scFv B10 for binding to CD371 (e.g., human CD371). H CDR1, CDR2 and CDR3 sequences and V L It cross-competes with a reference antibody or antigen-binding portion thereof comprising the CDR1, CDR2, and CDR3 sequences. For example, the extracellular antigen-binding domain of the CAR of the present disclosure comprises a V comprising the 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 set forth in SEQ ID NO: 32 L CDR2 and V comprising amino acids having the sequence set forth in SEQ ID NO: 33 L In certain embodiments, the extracellular antigen-binding domain of a CAR of the present disclosure cross-competes with a reference antibody or antigen-binding portion thereof comprising CDR3 of scFv B10 for binding to CD371 (e.g., human CD371).H and V L For example, the extracellular antigen-binding domain of a CAR of the present disclosure may be a V comprising an amino acid sequence having the sequence set forth in SEQ ID NO: 1 for binding to CD371 (e.g., human CD371). 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 L It binds to the same epitope on CD371 (e.g., human CD371) as a reference antibody or antigen-binding portion thereof comprising the CDR1, CDR2, and CDR3 sequences. In certain embodiments, the extracellular antigen-binding domain of a CAR of the present disclosure comprises, 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 (e.g., 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 a CAR of the present disclosure is the V of scFv B10. 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 comprising CDR1, CDR2, and CDR3 sequences. For example, the extracellular antigen-binding domain of a CAR of the present disclosure comprises a V comprising 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 HCDR2, 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 In certain embodiments, the extracellular antigen-binding domain of a CAR of the present disclosure binds to the same epitope on CD371 (e.g., human CD371) as the reference antibody or antigen-binding fragment thereof comprising CDR3. H and V L For example, the extracellular antigen-binding domain of a CAR of the present disclosure may be a V comprising 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 binds 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 (ed. J. Walker), which is incorporated herein by reference in its entirety. In certain embodiments, the antibody binding assay includes 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 the 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 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 reference antibody is labeled, for example, with a fluorescent dye, biotin, or peroxidase. In certain embodiments, the CD371 polypeptide is expressed in cells, for example, in a flow cytometry assay. In certain embodiments, the CD371 polypeptide is immobilized on a surface containing a Biacore™ (e.g., in a Biacore™ assay) or other medium suitable for surface plasmon resonance analysis. Binding of a reference antibody in the presence of a completely unrelated antibody (that does not bind to CD371) can serve as a high control value.A control low value can be obtained by incubating a labeled reference antibody with an unlabeled reference antibody, which will result in competition and reduced binding of the labeled reference antibody. In certain embodiments, a test extracellular antigen-binding domain that reduces binding of the reference antibody to a 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 a 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, wherein 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 assay. In certain embodiments, the CD371 polypeptide is immobilized on a surface containing a Biacore™ (e.g., in a Biacore™ assay) or other medium suitable for surface plasmon resonance analysis. Binding of the test extracellular antigen-binding domain in the presence of a completely unrelated antibody (that does not bind 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, resulting in competition and reduced binding of the labeled test extracellular antigen-binding domain. 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 the 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 a 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 regions are arranged in a V-terminal sequence 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 regions are arranged in a V-terminal sequence 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 may 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 linked 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 contains 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 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 a 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 murine 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 contains 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 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 a 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 murine 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 at least 20, or at least 30, or at least 40, or at least 50, and up to 218 amino acids in length, of SEQ ID NO: 66. 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 linking the extracellular antigen-binding domain to the transmembrane domain. The spacer region may 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 modified hinge region or fragment thereof, a native or modified hinge region of CD8b or fragment thereof, a native or modified hinge region of ICOS or fragment thereof, a native or modified hinge region of ICAM-1 or fragment thereof, a native or modified hinge region of CTLA-4 or fragment thereof, a native or modified hinge region of CD27 or fragment thereof, a native or modified hinge region of CD40 or fragment thereof, a native or modified hinge region of NKGD2 or 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 cells, e.g., T cells). Wild-type ("native") CD3ζ contains 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 cells, e.g., T cells) after antigen binding. The intracellular signaling domain of the CD3ζ chain is the main transmitter of signals from endogenous TCRs.

[0202] In certain embodiments, the intracellular signaling domain of the CAR comprises native CD3ζ. In certain embodiments, the CD3ζ 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, up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD3ζ 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 the intracellular domain of at least one costimulatory molecule or a fragment thereof.

[0206] As used herein, "costimulatory molecule" refers to a cell surface molecule other than antigen receptors or their ligands, which can provide lymphocytes with an efficient response to antigens. In certain embodiments, costimulatory molecules can provide optimal lymphocyte activation. Non-limiting examples of costimulatory molecules 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 molecules can bind to costimulatory ligands, which are proteins expressed on the cell surface, which, upon binding to their receptors, generate a costimulatory response, i.e., an intracellular response that results in the stimulation provided when an antigen-recognizing 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 to 4-1BB to provide an intracellular signal that induces effector cell function in T cells.

[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%, or 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 optionally contains up to one, 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 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%, or 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 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, the costimulatory signaling region of a CAR of the present 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%, or 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, 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 comprising the intracellular domains of two or more costimulatory molecules or portions thereof, 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 V 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 comprising the amino acid sequence set forth in SEQ ID NO: 33. L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD3ζ 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 are linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, V 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 V 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 comprising the amino acid sequence set forth in SEQ ID NO: 33. L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD3ζ 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, the V H and V L are linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, V 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.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 V 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 comprising the amino acid sequence set forth in SEQ ID NO: 33. 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 are linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, V 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 V 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 comprising the amino acid sequence set forth in SEQ ID NO: 33. 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 Lare linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, V 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 V 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 comprising the amino acid sequence set forth in SEQ ID NO: 33. 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 are linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, V 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 V 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 comprising the amino acid sequence set forth in SEQ ID NO: 39. 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 are linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, V 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 V 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 comprising the amino acid sequence set forth in SEQ ID NO: 39. 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 are linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, V 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 V 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 are linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, V 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 V 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 comprising the amino acid sequence set forth in SEQ ID NO: 33. L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD3ζ 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 are linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 93. In certain embodiments, V 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 V 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 comprising the amino acid sequence set forth in SEQ ID NO: 33. L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD3ζ 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 are linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 94. In certain embodiments, V 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.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 V 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 comprising the amino acid sequence set forth in SEQ ID NO: 33. L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD3ζ 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 are linked via a linker comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 91. In certain embodiments, V 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 V 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 comprising the amino acid sequence set forth in SEQ ID NO: 33. L V containing CDR3 L (b) an extracellular antigen-binding domain comprising a CD3ζ 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 are 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, antigen-recognizing receptor is TCR-like fusion molecule.Non-limiting examples of TCR fusion molecule include HLA-independent TCR-based chimeric antigen receptor (also known as " HIT-CAR ", for example, as disclosed in International Patent Application No. PCT / US19 / 017525, which is incorporated herein by reference in its entirety) and T cell receptor fusion construct (TRuC) (for example, as disclosed in Baeuerle et al., " Synthetic TRuC receptors engage 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 the CD3ζ polypeptide associated with the antigen-binding chain upon binding to an antigen. In certain embodiments, activation of the CD3ζ polypeptide can activate immune response cells. In certain embodiments, the TCR-like fusion molecule can associate 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 (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 comprises the V 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 dimerize 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 can 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 presently disclosed subject matter provides cells comprising a CD371-targeting antigen-recognizing receptor of the present disclosure (e.g., those disclosed in Section 5.3). In certain embodiments, the cells are selected from the group consisting of lymphoid lineage cells and myeloid lineage cells. In certain embodiments, the cells are immune response cells. In certain embodiments, the immune response cells are lymphoid lineage cells.

[0242] In certain embodiments, the cell is a lymphoid lineage cell. Cells of the lymphoid lineage 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 cells are T cells. T cells may 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 presently disclosed subject matter may 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 cells or tumor cells. A patient's own T cells may be genetically modified to target specific antigens by introducing an antigen-recognition receptor, e.g., a CAR or TCR. 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 cells are NK cells. Natural killer (NK) cells are lymphocytes that are part of cell-mediated immunity and act during innate immune responses. NK cells do not require prior activation to perform 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 peripheral donor lymphocytes genetically modified to express T cell receptor complexes that recognize 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 selective in vitro expanded antigen-specific peripheral blood leukocytes using artificial antigen presenting cells (AAPCs) or pulsed dendritic cells).

[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 a myeloid lineage cell.Non-limiting examples of myeloid lineage cells include monocytes, macrophages, neutrophils, dendritic cells, basophils, neutrophils, eosinophils, megakaryocytes, mast cells, erythrocytes, platelets, and stem cells from which myeloid cells can differentiate.In certain embodiments, the stem cell is a pluripotent stem cell (for example, an embryonic stem cell or an 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" is characterized by the infiltration regulatory CD4 + The tumor microenvironment contains various immunosuppressive factors, including T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), immunosuppressive cytokines including TGF-β, and expression of ligands targeting immunoinhibitory 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 antitumor immune responses. Collectively, 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-targeting 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 with 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 diminished immune response. Polypeptides known to suppress or diminish 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, immunostimulatory 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 increased immune response. Immunostimulatory activity can include pro-inflammatory activity. Polypeptides known to stimulate or increase immune responses 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 responses 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-recognizing 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. 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 positioned to drive IL-18 gene expression.

[0257] Cells containing an antigen-recognizing receptor (e.g., a CAR) and engineered to express IL-18, e.g., cells containing an exogenous IL-18 polypeptide or an altered promoter / enhancer in the IL-18 locus, are disclosed in International Patent Publication No. WO 2018 / 027155, which is incorporated herein 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 comprising an antigen recognition receptor (e.g., a CAR) and engineered to express IL-33, e.g., cells comprising an exogenous IL-33 polypeptide or an engineered promoter / enhancer at the IL-33 locus, are disclosed in International Patent Publication No. 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 modified promoter / enhancer at the IL-36 locus, which can increase IL-36 gene expression, for example, a constitutive promoter or an inducible promoter is configured to drive IL-36 gene expression. Cells comprising an antigen-recognition receptor (e.g., CAR) and engineered to express IL-36, for example, cells comprising an exogenous IL-36 polypeptide or a modified promoter / enhancer at the IL-36 locus, are disclosed in International Patent Publication No. 2019 / 099483, the entire contents of which are incorporated herein by reference. 5.5. Compositions and Vectors

[0262] The presently disclosed subject matter provides compositions comprising the CD371-targeting antigen-recognizing receptors 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-recognizing receptor of the present disclosure is operably linked to a promoter.

[0264] Furthermore, the presently disclosed subject matter provides nucleic acid compositions comprising polynucleotides encoding the CD371-targeting antigen-recognizing receptors 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 a CD371-targeting antigen-recognizing 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 group consisting of elongation factor (EF)-1 promoter, cytomegalovirus immediate early promoter (CMV) promoter, simian virus 40 early promoter (SV40) promoter, phosphoglycerate kinase (PGK) promoter and metallothionein promoter.In certain embodiments, the promoter is an inducible promoter.In certain embodiments, the inducible promoter is selected from the group consisting of NFAT transcription response element (TRE) promoter, CD69 promoter, CD25 promoter and IL-2 promoter.

[0267] The composition and nucleic acid composition 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 also be used.

[0268] For the initial genetic modification of cells to contain the CD371-targeting antigen-recognizing receptors (e.g., CARs or TCRs) of the present disclosure, retroviral vectors can be used for transduction, however, any other suitable viral vector or non-viral delivery system can be used. The antigen-recognizing receptors can be constructed in a single multicistronic expression cassette, in multiple expression cassettes on 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, wherein the capsid protein is functional to infect human cells. A variety of amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller et al., (1985) Mol Cell Biol (1985); 5:431-437); PA317 (Miller, et al., Mol Cell Biol (1986); 6:2895-2902); and CRIP (Danos et al., Proc Natl Acad Sci USA (1988); 85:6460-6464). Non-amphotropic particles, such as those pseudotyped with VSVG, RD114, or GALV envelopes, and any others known in the art, are also suitable.

[0269] Potential methods of transduction also include direct co-culturing of cells with producer cells (Bregni et al., Blood (1992); 80:1418-1422), or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations (Xu et al., Exp Hemat (1994); 22:223-230; and Hughes et al. J Clin Invest (1992); 89:1817).

[0270] Other transduction virus vectors can be used to modify cells.In certain embodiments, the vector selected shows high-efficiency infection, and stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8:423-430,1997; Kido et al., Current Eye Research 15:833-844,1996; Bloomer et al., Journal of Virology 71:6641-6649,1997; Naldini et al., Science 272:263-267,1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319,1997). Other viral vectors that can be used include, for example, adenoviral, lentiviral and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, or herpes viruses, such as Epstein-Barr virus (see, e.g., Miller, Human Gene Thera (1990); 15-14; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques (1988); 6:608-614; Tolstoshev et al., Cur Opin Biotechnol (1990); 1:55-61; Sharp, The Lancet (1991); 337:1277-78; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-22, 1987; Anderson, Science (1984);226:401-409; Moen, Blood Cells 17:407-16, 1991; Miller et al., Biotechnol (1989);7:980-90; LeGal La Salle et al., Science (1993);259:988-90; and Johnson, Chest (1995)107:77S-83S (see also the vectors).Retroviral vectors are 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 methods can also be used for genetic modification of cells.For example, nucleic acid molecules can be introduced into cells by administering nucleic acid in the presence of lipofection (Feigner et al., Proc Natl Acad Sci USA (1987);84:7413;Ono et al., Neurosci Lett (1990);17:259;Brigham et al., Am J Med Sci (1989);298:278;Staubinger et al., Methods in Enzymol (1983);101:512;Wu et al., J Biol Chem (1988);263:14621;Wu et al., J Biol Chem (1989);264:16985) or by microinjection under surgical conditions (Wolff et al., Science (1990);247:1465). Other non-viral methods for gene transfer include in vitro transfection using calcium phosphate, DEAE-dextran, electroporation, and protoplast fusion. Liposomes can also potentially be useful for delivering DNA to cells. Transplantation of normal genes into affected tissues of a subject can also be achieved by transferring normal nucleic acids into ex vivo culturable cell types (e.g., autologous or heterologous primary cells or their progeny), and then injecting the cells (or their progeny) into targeted tissues or systemically injecting them. 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 also be obtained by RNA electroporation.

[0272] Any target genome editing method can also be used to deliver the antigen-recognizing receptor of the present disclosure to cell or subject.In certain embodiments, 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, 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 discovered in prokaryotic cells. When utilized for genome editing, this system includes Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (crRNA, which contains the RNA used by Cas9 to guide it to the correct section of host DNA along 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, which binds to crRNA and forms an active complex with Cas9), and an optional portion of a DNA repair template (DNA that guides the cellular repair process, allowing for the insertion of a specific DNA sequence). CRISPR / Cas9 often uses a plasmid to transfect target cells. The crRNA is the sequence that Cas9 uses to identify and directly bind to target DNA in cells, so it 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 ligated with the Cas9 gene into a plasmid for transfection into cells.

[0274] Zinc finger nucleases (ZFNs) are artificial restriction enzymes 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, allowing the zinc finger nuclease to target desired sequences within 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 nonspecific cleavage domain derived 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) consist 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 polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV) promoter, the simian virus 40 (SV40) promoter, or the metallothionein promoter) and can be regulated by any appropriate mammalian regulatory element or intron (e.g., an 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. 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 agents / systems can be varied 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, impale infection, hydrostatic pressure, continuous infusion, sonication, magnetofection, adeno-associated virus, pseudotyping of viral vector envelope proteins, 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 presently disclosed subject matter provides methods for optimizing amino acid or nucleic acid sequences by generating alterations in the sequence. Such alterations may include certain mutations, deletions, insertions, or post-translational modifications. The presently disclosed subject matter further includes analogs of any naturally occurring polypeptide disclosed herein (including, but not limited to, CD371, CD8, CD28, 4-1BB, and CD3ζ). Analogs may differ from the naturally occurring polypeptides disclosed herein by differences in amino acid sequence, by post-translational modifications, or both. Analogs may exhibit at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology or identity to all or a portion of the naturally occurring amino acid sequence of the presently disclosed subject matter. 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 approach to determining the degree of identity, the BLAST program uses a BLAST index to identify closely related sequences. -3 ~e -100The probability score can be used. Modifications include in vivo and in vitro chemical derivatization of polypeptides, such as acetylation, carboxylation, phosphorylation, or glycosylation; such modifications can occur during polypeptide synthesis or processing, or after treatment with isolated modifying enzymes. Analogs can also differ from naturally occurring polypeptides by changes in the primary sequence. These include both natural and induced genetic variants (resulting from random mutagenesis, e.g., 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 containing 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 presently disclosed subject matter also provides fragments of any of the polypeptides disclosed herein. As used herein, the term "fragment" refers to 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 due to alternative mRNA splicing or alternative protein processing events). 5.7. Formulation and Administration

[0279] The presently disclosed subject matter also provides compositions comprising the cells of the present disclosure. Compositions comprising the cells of the present disclosure can be conveniently provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are generally 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 longer contact periods with specific tissues. Liquid or viscous compositions can contain a carrier, which can be a solvent or dispersion medium, for example, water, saline, phosphate-buffered saline, polyols (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 the required amount of an appropriate solvent with various amounts of other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, dextrose, or the like. 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 buffering agents, gelling or thickening additives, preservatives, flavoring agents, coloring agents, and the like. Suitable preparations can be prepared without undue experimentation by reference to standard texts such as "REMINGTON'S PHARMACEUTICAL SCIENCE," 17th edition, 1985, 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 absorption-delaying agents, 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 composition can be isotonic, i.e., it can have the same osmotic pressure as blood and tears.The desired isotonicity of the composition can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes.Sodium chloride can be used, particularly for buffers containing sodium ions.

[0283] The viscosity of the composition can be maintained at a selected level by using a pharmaceutically acceptable thickening agent, if desired. For example, methylcellulose is readily available economically and is 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 properties 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] Compositions comprising the cells of the present disclosure can be provided systemically or directly to a subject to induce and / or enhance an immune response to an antigen and / or treat and / or prevent neoplasia. In certain embodiments, the cells of the present disclosure or a composition comprising the same are directly injected into an organ of interest (e.g., an organ affected by neoplasia). Alternatively, the cells of the present disclosure or a composition comprising the same are indirectly provided to an organ of interest, for example, by administration into the circulatory system (e.g., tumor vasculature). Expansion and differentiation agents can be provided before, during, or after administration of the cells or composition to increase the production 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 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, at least about 1 x 10 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 In certain embodiments, about 1 x 10 cells of the present disclosure are administered to a subject. 6 The cells of the present disclosure are administered to the subject.The exact dose that will be considered as an effective dose can be determined based on the individual factors of each subject, including the size, age, sex, weight and condition of the specific subject.The dosage can be easily determined by those skilled in the art based on the present disclosure and knowledge in the art.

[0287] The cells of the present disclosure can include purified populations of cells. Those skilled 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 skilled in the art (e.g., a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, or the like.

[0288] Those skilled in the art can readily determine the amounts of cells and optional additives, vehicles, and / or carriers to be administered in the compositions and methods. Typically, any additives (in addition to the active cells and / or drugs) are present in an amount of 0.001 to 50% (by weight) of the solution in phosphate-buffered saline, with the active ingredient being present in microgram to milligram amounts, 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., a rodent such as a mouse; the dosage of the composition, the concentration of components therein, and the timing of administration of the composition that will elicit 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 pharmaceutically 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 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., a 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 present 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 presently disclosed subject matter provides methods 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 presently disclosed subject matter provides various methods 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 a composition comprising the same (e.g., a pharmaceutical composition) 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 the desired effect. An effective amount can be provided in one or a series of administrations. An effective amount can be provided by bolus or continuous perfusion.

[0294] The subject matter of the present disclosure provides various methods for using cells (e.g., T cells) or compositions comprising the same.For example, the subject matter of the present disclosure provides a method for reducing tumor burden in a subject.In certain embodiments, the method for 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 tumors 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, stomach carcinoma, and bile duct carcinoma.

[0296] The presently disclosed subject matter 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 the tumor burden in the subject. In addition, the presently disclosed subject matter 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 presently disclosed subject matter 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), cancer of the uterus, rectum, anal region, bladder, brain, stomach, testicle, fallopian tube, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, leukemia (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 include Waldenstrom's hypergammaglobulinemia, heavy chain disease, and sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, 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, cholangiocarcinoma (nile carcinoma) solid tumors such as duct carcinoma, choriocarcinoma, seminoma, 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, 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 wide range of immune cells (tumor-infiltrating lymphocytes, NK cells, NKT cells, dendritic cells, and macrophages) that participate in physiological antitumor or antiviral responses.

[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 enable T cell elimination by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). The EGFRt can be covalently attached upstream of an antigen-recognition receptor (e.g., CAR). The suicide gene can be included within a vector containing 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, which can activate iCasp-9) designed to activate the suicide gene during malignant T cell transformation (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 confers an additional level of safety by its ability to eliminate the majority of receptor-expressing cells within a very short period of time. Cells (e.g., T cells) of the present disclosure incorporating the suicide gene can be proactively 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 kits for inducing and / or enhancing an immune response in a subject, treating and / or preventing a tumor or neoplasm in a subject, reducing tumor burden in a subject, and / or increasing or extending the survival time of a subject with a tumor or neoplasm in a subject. In certain embodiments, the kit includes a cell of the present disclosure or a composition comprising the same. In certain embodiments, the kit includes a sterile container, which may be a box, an ampoule, a bottle, a vial, a tube, a bag, a sachet, a blister pack, or other suitable container form known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceuticals. In certain non-limiting embodiments, the kit includes a nucleic acid molecule encoding a CD371-targeting antigen-recognition receptor (e.g., a 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 using the composition for the treatment and / or prevention of tumors or neoplasms. In certain embodiments, the instructions include at least one of the following: a 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), as a label affixed to the container, or as a separate sheet, pamphlet, card, or folder provided in or with the container. [Example]

[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 those in the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual," second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" and "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); and "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 that are particularly useful for particular 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 structures of the CARs are illustrated in Figure 1.

[0307] As shown in Figure 2, CARs were 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 CARs in B10-based constructs. Controls included non-transduced human T cells expressing neither EGFRt nor Myc tags, as well as the 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 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 and 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 co-cultures of non-functional CD371-targeted CAR T cells (based on B10L4H but without the 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 and 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. Because U937 cells also express CD33, "Et.M195MTh28Z" was used as a positive control. CD371-targeted CAR T cells derived from healthy human donors (i.e., donor C and donor D) were co-cultured with CD33 / CD371 U937 cells (U937gL) expressing GFP and firefly luciferase at different effector:tumor ratios. After 24 hours, bioluminescence was measured 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 lacking the CD28 or CD3 zeta signaling domain) 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 cells / 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 the signaling domain). "Et.C1HVh28Z" represents a CD371-targeted CAR derived from the murine anti-human CD371 antibody 1075.7, and "EtM195MTh28Z" or "M19528" represent CD33-targeted CAR T cells derived from the murine 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 cocultured 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 coculture and plotted as the percentage of signal detected in cocultures of nonfunctional CD371-targeted CAR T cells (based on B10L4H but lacking the CD28 or CD3 zeta signaling domain) 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 is evaluated.Human CD371 (hCD371) CRISPR knockout cell line is generated.Human CD371 is knocked out from HL60gL and U937gL using CRISPR, and knockout is 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 cocultured with antigen-negative U937 cells or HL60 (U937RFPcyp.371KO or HLgL.371KO) expressing RFP and Cypridina luciferase at different effector:tumor ratios. After 24 hours, bioluminescence was measured and plotted as a percentage of the signal detected in cocultures of nonfunctional CD371-targeted CAR T cells (based on B10L4H but lacking the 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 did not exhibit significant cytotoxicity against CD371-negative U937 cells and showed 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 the 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+ cells / 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 anti-tumor activity of B10-based CD371-targeted CAR T cells was evaluated. Figure 17 shows a xenograft model of AML. NCG mice were treated with 5.0 x 10 4AML FAB-M5 cell line of U937gL tumor cells was inoculated via the tail vein, and after 3 days, approximately 1.25 × 10 6 10HL-based CAR T cells. 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 demonstrated superior survival in AML xenografted mice compared with B10LH-based CAR T cells.

[0317] Next, we assessed whether the in vivo activity was dose-dependent. NCG mice were administered 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] Both orientations (V H -V L or V L -V H The binding of B10 (also referred to as "1B10") scFv of 1B10 to cells expressing CD371 was evaluated. The results are shown in Figure 21. As shown in Figure 21, binding to cells was assessed by V L -V H However, the V H -V L No binding of the oriented B10 scFv was observed by flow cytometry, suggesting low affinity and therefore the need 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 containing scFvs with lower binding affinities outperformed CARs containing scFvs with higher binding affinities in vitro and in vivo, 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 L It contained a B10-based scFv (designated as B10H1L) with an orientation and a 5 amino acid long G4S linker (GGGGS [SEQ ID NO: 93]) and a CD28Z-based signaling domain. ·Et.B10H2L_MT_h28Z is V 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 comprised a B10-based scFv (designated as B10H6L) with a CD28Z-based signaling 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 Figure 22, V H -V LCAR 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 B10-based CAR T cells were injected into NCG mice. 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, 5x10 5 At a dose of 2.5 x 10 CAR T cells, human CD371-targeting CAR T cells based on B10H3L, B10H5L, and IL33-secreting B10H4L outperformed B10H4L-based CAR T cells. As shown in Figure 24, 5 At a dose of 1.0 x 10 CAR T cells, B10H4L-based human CD371-targeted CAR T cells secreting IL18 and IL33 outperformed B10H4L-based CAR T cells. As shown in Figure 25, 5 At low doses of CAR T cells, IL18-secreting B10H4L human CD371-targeting CAR T cells outperformed other CAR T cells. Embodiments of the subject matter of the present disclosure

[0324] From the foregoing description, it will be apparent that variations and modifications may be made to the subject matter of the present disclosure to adapt it for various uses and conditions. Such embodiments also fall 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 or 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) 2. The antigen-recognizing receptor according to item 1, wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv). (Item 3) 3. The antigen-recognizing receptor according to item 2, wherein the extracellular antigen-binding domain is a human scFv. (Item 4) 2. The antigen-recognizing receptor according to item 1, wherein the extracellular antigen-binding domain is an optionally cross-linked Fab. (Item 5) The extracellular antigen-binding domain is F(ab) 2 2. The antigen-recognition receptor according to Item 1, (Item 6) scFv, Fab and F(ab) 2 6. The antigen-recognizing receptor according to any one of items 2 to 5, wherein one or more of the following are included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. (Item 7) the extracellular antigen-binding domain (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 Aspects 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 (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 the amino acid sequence set forth in 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. 9. 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 Aspects 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 (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 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 Aspects 1 to 11, 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; a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 30; 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 13) 13. The antigen-recognizing receptor according to any one of Aspects 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 set forth in SEQ ID NO:1. (Item 16) 16. The antigen-recognizing receptor according to any one of Aspects 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 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 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 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 selected 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) 19. The antigen-recognizing receptor according to any one of Items 1 to 18, comprising 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 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 (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 of SEQ ID NO: 1; and a light chain variable region comprising the amino acid sequence of 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 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. (Item 25) 24. The antigen recognition receptor according to item 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 V 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 located 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 . (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-recognizing receptor of 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-recognizing receptor according to any one of items 1 to 35, wherein the antigen-recognizing 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-recognizing receptor. (Item 43) 43. The cell of item 41 or 42, wherein the antigen-recognizing receptor is constitutively expressed on the surface of the cell. (Item 44) 44. The cell of any one of items 41 to 43, wherein the cell is engineered to express a cytokine or a fragment thereof. (Item 45) 45. The cell of item 44, wherein the cell comprises an exogenous polypeptide of the cytokine or a fragment thereof. (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 of 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 cells are embryonic-like stem cells or induced pluripotent stem cells. (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 of 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 of 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 of item 59, wherein the vector is a gamma-retroviral vector. (Item 61) 59. A host cell expressing the nucleic acid molecule of 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 cells 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 pharmaceutically acceptable carrier. (Item 65) 65. A method for reducing tumor burden in a subject, comprising administering to the subject an effective amount of the cell of any one of items 41 to 55 or the composition of 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) 64. A method for increasing or prolonging the survival time of a subject having a tumor or neoplasm, the method comprising administering an effective amount of the cell according to any one of items 41 to 55 or the composition according to item 63 or 64. (Item 68) 64. A method for treating and / or preventing a tumor or neoplasm in a subject, the method comprising administering an effective amount of the cell according to any one of items 41 to 55 or the 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 containing 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 prolonging survival 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 item 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

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, and comprising: (a) a heavy chain variable region (VH) comprising CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 28, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 29, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 30; and (b) a light chain variable region (VL) comprising CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 31, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 32, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:

33.

2. The antigen-recognizing receptor of claim 1 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).

3. The antigen-recognizing receptor according to claim 2 , wherein the extracellular antigen-binding domain is a human scFv.

4. The antigen-recognizing receptor according to claim 1 , wherein the extracellular antigen-binding domain is a Fab.

5. The antigen-recognizing receptor according to claim 4, wherein the extracellular antigen-binding domain is a cross-linked Fab.

6. The extracellular antigen-binding domain is F(ab) ２ The antigen-recognition receptor according to claim 1,

7. The scFv, Fab and F(ab) ２ The antigen-recognizing receptor according to any one of claims 2 to 6, wherein one or more of:

8. The antigen-recognizing receptor according to any one of claims 1 to 7, 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.

9. The antigen-recognizing receptor according to any one of claims 1 to 8, 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.

10. The antigen-recognizing receptor according to claim 9, wherein the linker has an 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.

11. The antigen-recognizing receptor according to claim 9 , wherein the linker has the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO:

14.

12. The antigen-recognizing receptor according to any one of claims 1 to 11, wherein the extracellular antigen-binding domain comprises a signal peptide covalently joined to the 5' end of the extracellular antigen-binding domain.

13. The extracellular antigen-binding domain is V from the N-terminus to the C-terminus Ｈ -V Ｌ The antigen-recognizing receptor according to any one of claims 1 to 12, comprising a heavy chain variable region and a light chain variable region located at

14. The extracellular antigen-binding domain is 1×10 －８ M or higher dissociation constant (K ｄ The antigen recognition receptor according to any one of claims 1 to 12, which binds to CD371 at the .

15. The antigen-recognizing receptor of any one of claims 1 to 14, 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.

16. The antigen-recognition receptor of claim 15, wherein the transmembrane domain comprises a CD28 polypeptide.

17. The antigen-recognition receptor according to any one of claims 1 to 16, wherein the intracellular signaling domain comprises a CD3ζ polypeptide.

18. The antigen-recognizing receptor according to any one of claims 1 to 17, wherein the intracellular signaling domain further comprises at least one costimulatory signaling region.

19. The antigen-recognizing receptor of claim 18, 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.

20. The antigen-recognizing receptor according to claim 18 or 19, wherein the at least one costimulatory signaling region comprises a CD28 polypeptide or a 4-1BB polypeptide.

21. The antigen-recognizing receptor according to any one of claims 1 to 20, wherein the antigen-recognizing receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a T cell-like fusion protein.

22. The antigen recognition receptor according to any one of claims 1 to 21, wherein the antigen recognition receptor is a CAR.

23. The antigen-recognizing receptor according to any one of claims 1 to 22, wherein the antigen-recognizing receptor is recombinantly expressed.

24. The antigen-recognizing receptor according to any one of claims 1 to 23, wherein the antigen-recognizing receptor is expressed from a vector.

25. The antigen-recognizing receptor according to claim 24, wherein the vector is a gamma-retroviral vector.

26. A cell comprising the antigen-recognizing receptor according to any one of claims 1 to 25.

27. The cell of claim 26 , wherein the cell is transduced with the antigen-recognizing receptor.

28. The cell of claim 26 or 27, wherein the antigen-recognizing receptor is constitutively expressed on the surface of the cell.

29. The cell of any one of claims 26 to 28, wherein the cell is engineered to express a cytokine or a fragment thereof.

30. 30. The cell of claim 29, wherein the cell comprises an exogenous polypeptide of the cytokine or a fragment thereof.

31. 31. The cell of claim 29 or 30, wherein the cell comprises a nucleic acid molecule encoding the cytokine or a fragment thereof.

32. The cell of any one of claims 29 to 31, wherein the cytokine is selected from the group consisting of IL-18, IL-33, IL-36, and combinations thereof.

33. The cell according to any one of claims 29 to 32, wherein the cytokine is IL-18.

34. The cell of any one of claims 26 to 33, wherein the cell is an immune response cell.

35. The cell according to any one of claims 26 to 34, wherein the cell is a lymphoid or myeloid lineage cell.

36. The cell of any one of claims 26 to 35, wherein the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, and a stem cell from which a lymphoid cell can be differentiated.

37. The cell of any one of claims 26 to 36, wherein the cell is a T cell.

38. 38. The cell of claim 36 or 37, wherein the T cell is a cytotoxic T lymphocyte (CTL) or a regulatory T cell.

39. 37. The cell of claim 36, wherein the stem cell is a pluripotent stem cell.

40. 40. The cell of claim 39, wherein the pluripotent stem cell is an embryonic-like stem cell or an induced pluripotent stem cell.

41. A nucleic acid molecule encoding the antigen-recognizing receptor according to any one of claims 1 to 25.

42. 42. The nucleic acid molecule of claim 41, wherein the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:

22.

43. A vector comprising the nucleic acid molecule of any one of claims 41 to 42.

44. The vector of claim 43, wherein the vector is a gamma-retroviral vector.

45. A host cell expressing the nucleic acid molecule of any one of claims 41 to 42.

46. 46. ​​The host cell of claim 45, wherein the host cell is a T cell.

47. A composition comprising the cells of any one of claims 26 to 40.

48. 48. The composition of claim 47, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

49. A composition comprising a cell according to any one of claims 26 to 40 or a composition according to claim 47 or 48 for reducing tumor burden in a subject.

50. 50. The composition of claim 49, wherein the composition reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject.

51. A composition comprising a cell according to any one of claims 26 to 40 or a composition according to claim 47 or 48 for increasing or prolonging the survival of a subject having a tumor or neoplasm.

52. A composition comprising a cell according to any one of claims 26 to 40 or a composition according to claim 47 or 48 for treating and / or preventing a tumor or neoplasm in a subject.

53. 53. The composition of claim 51 or 52, 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 myelogenous leukemia (CML).

54. 54. The composition of any one of claims 51 to 53, wherein the tumor or neoplasm is acute myeloid leukemia (AML).

55. An in vitro or ex vivo method for generating a cell comprising the antigen-recognizing receptor according to any one of claims 1 to 25, comprising introducing into the cell a nucleic acid molecule encoding the antigen-recognizing receptor.

56. 41. 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 prolonging the survival of a subject having a tumor or neoplasm, comprising the cells of any one of claims 26 to 40.

57. 57. The kit of claim 56, wherein the kit further comprises 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.

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