Anti-hK2 chimeric antigen receptor (CAR)

Abstract

The present disclosure provides chimeric antigen receptors (CARs) that specifically bind to human kallikrein-2 (hK2) and immunoresponsive cells comprising such CARs for the treatment of cancer.

Description

[Technical Field]

[0001] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on June 30, 2020, is named JBI6124WOPCT1_SL.txt, and is 562,160 bytes in size.

[0002] FIELD OF THE INVENTION The present invention relates to hK2-targeting chimeric antigen receptors (CARs) comprising a single-chain variable fragment that targets hK2, and genetically engineered hK2-targeting immune cells that express the CARs. Also provided are nucleic acids and expression vectors encoding the CARs, recombinant cells containing the vectors, and compositions comprising genetically engineered immune cells that express hK2-targeting CARs. Methods of making the CARs and genetically engineered immune cells, as well as methods of using the genetically engineered immune cells to treat conditions, including cancer, are also provided.

[0003] (background) Prostate cancer is the second most frequently diagnosed cancer and the sixth leading cause of cancer death in men, accounting for 14% (903,500) of all new cancer cases and 6% (258,400) of all cancer deaths in men worldwide. The course of prostate cancer from diagnosis to death is best classified as a series of clinical stages based on the extent of disease, hormonal status, and the presence or absence of detectable metastases: localized disease, rising prostate-specific antigen (PSA) levels without detectable metastases after radiation therapy or surgery, and clinical metastasis in the noncastrated or castrated stages. While surgery, radiation, or a combination of both can be curative for patients with localized disease, a significant proportion of these patients will have recurrent disease as evidenced by rising PSA levels, which may lead to the development of metastases, i.e., progression to terminal disease, particularly in high-risk groups.

[0004] Androgen deprivation therapy (ADT) is the standard of care, with a generally predictable outcome of a decline in PSA, a stable phase in which the tumor does not grow, followed by a rise in PSA and regrowth as castration-resistant disease. For many years, ADT was the standard of care for patients with metastatic prostate cancer.

[0005] Kallikrein-related peptidase 2 (hK2, HK2) is a trypsin-like enzyme specifically expressed in prostate tissue and prostate cancer, driven by the androgen receptor (AR). hK2 is activated by the transmembrane protease serine 2 (TMPRSS2) and secreted into the prostate ducts, where it cleaves semenogelin, an extracellular matrix component in ejaculate, initiating a cascade that enhances sperm motility. hK2 expression is restricted to prostate and prostate cancer tissues, but it has recently been demonstrated that hK2 is detectable in breast cancer lines and primary patient samples after appropriate activation of the AR pathway by steroid hormones (U.S. Patent Application Publication No. 2018 / 0326102). Similar to PSA, retrograde release of catalytically inactive hK2 into the blood occurs when the highly structured organization of the prostate gland is disrupted during hypertrophy or malignant transformation.

[0006] T cell therapy utilizes isolated T cells that have been genetically modified to enhance their specificity for specific tumor-associated antigens. Genetic modification can involve the expression of chimeric antigen receptors (CARs) or exogenous T cell receptors, providing new antigen specificity on the T cells. T cells expressing chimeric antigen receptors (CAR-T cells) can induce tumor immune reactivity. Better cancer therapies utilizing CAR-T cells are needed.

[0007] (overview) Disclosed herein are chimeric antigen receptors (CARs), e.g., CARs that target human kallikrein-2 (hK2), cells comprising the CARs, vectors, e.g., recombinant expression vectors, encoding the CARs, and nucleic acid molecules encoding the CARs, methods of making the CARs, compositions, polypeptides, proteins, nucleic acids, host cells, cell populations, and methods of treating disorders, e.g., cancer, using the disclosed CARs.

[0008] In one aspect, a chimeric antigen receptor (CAR) is provided, comprising: (a) an extracellular domain comprising an scFv that specifically binds to the human kallikrein-2 (hK2) antigen; (b) a transmembrane domain; (c) an intracellular signaling domain that optionally includes at least one costimulatory domain.

[0009] In some embodiments, the CAR is (d) CD8a-hinge region Further comprising: the transmembrane domain comprises a CD8a transmembrane region (CD8A-TM) polypeptide; The intracellular signaling domain includes a costimulatory domain comprising a TNF receptor superfamily member 9 (CD137) component and a primary signaling domain comprising a T cell surface glycoprotein CD3 zeta chain (CD3z) component.

[0010] In some embodiments, the CD8a-hinge region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, 269, 270, 271, or 272. the transmembrane domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 26, and / or The intracellular signaling domain comprises a costimulatory domain having an amino acid sequence at least 90% identical to SEQ ID NO:27 and a primary signaling domain having an amino acid sequence at least 90% identical to SEQ ID NO:28.

[0011] In another aspect, a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain is a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 64, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66; a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 314, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 315, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311; a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 309, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 310, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 73, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 86, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 87, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 88; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 92, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 93, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 94; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 103, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 106, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 107, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 108, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 110, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 111, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 112, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 114, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 115, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 116, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 117; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 118, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 119, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 117; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122; or a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 123, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 124, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122; Including, A CAR is provided, wherein the extracellular antigen-binding domain binds to the hK2 antigen.

[0012] In another aspect, a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain is a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 95, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 96, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 312, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 313, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91; a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 128, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 129, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 130; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 125, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 131, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 132; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 133, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 134, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 135; a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 136, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 137, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 138; or a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 139; Including, A CAR is provided, wherein the extracellular antigen-binding domain binds to the hK2 antigen.

[0013] In some embodiments, the extracellular antigen binding domain comprises: a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; or a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 95, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 96, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 312, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 313, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91; a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 128, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 129, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 130; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 125, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 131, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 132; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 133, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 134, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 135; a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 136, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 137, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 138; or a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 139; Further includes:

[0014] In another aspect, a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain is a light chain variable domain (VL) comprising an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 74, 75, 147, 148, 149, 150, 151, 152, and 318; and / or a heavy chain variable domain (VH) comprising an amino acid sequence selected from SEQ ID NOs: 4, 5, 6, 76, 77, 140, 141, 142, 143, 144, 145, 146, and 317; Including, CARs are provided, wherein the extracellular antigen-binding domain binds to the human kallikrein-2 (hK2) antigen.

[0015] In some embodiments, the extracellular antigen binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 74 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 76; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 77; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 147 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 140; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 141; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 140; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 148 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 142; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 149 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 143; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 150 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 144; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 151 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 145; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 152 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 146; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 318 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 317; Includes.

[0016] In some embodiments, the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv comprises a linker polypeptide between the light chain variable region (VL) and the heavy chain variable region (VH). In some embodiments, the linker polypeptide comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-23, 169-184, and 340-343. In some embodiments, the extracellular antigen-binding domain comprises a signal polypeptide. In some embodiments, the signal polypeptide comprises the amino acid sequence of SEQ ID NO: 24.

[0017] In some embodiments, the intracellular signaling domain comprises a polypeptide component selected from the group consisting of a TNF receptor superfamily member 9 (CD137) component, a T cell surface glycoprotein CD3 zeta chain (CD3z) component, a cluster of differentiation (CD27) component, a cluster of differentiation superfamily member component, and combinations thereof. In some embodiments, the CD137 component comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CD3z component comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the transmembrane domain comprises a CD8a transmembrane region (CD8A-TM) polypeptide. In some embodiments, the CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO: 26.

[0018] In various embodiments, the CAR further comprises a hinge region linking the transmembrane domain to the extracellular antigen-binding domain. In some embodiments, the hinge region is a CD8a hinge region. In some embodiments, the CD8a-hinge region comprises the amino acid sequence of SEQ ID NO: 25, 269, 270, 271, or 272. In some embodiments, the extracellular antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-44, 78-81, 221-236, and 273-308. In some embodiments, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-61, 82-85, and 205-220.

[0019] In another aspect, an isolated lymphocyte is provided that expresses any of the above-mentioned CARs. In some embodiments, the lymphocyte is a T lymphocyte. In some embodiments, the lymphocyte is a natural killer (NK) cell.

[0020] Also provided are isolated nucleic acid molecules encoding any of the above CARs. Vectors containing the nucleic acid molecules are also provided. Additionally, cells expressing the nucleic acid molecules are also provided.

[0021] Additionally, pharmaceutical compositions are provided that include an effective amount of any of the above lymphocytes and a pharmaceutically acceptable excipient.

[0022] In one aspect, the present disclosure provides a CAR according to the present disclosure for use in a method of treatment.

[0023] In one aspect, the present disclosure provides a lymphocyte according to the present disclosure for use in a method of treatment.

[0024] In one aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, according to the present disclosure for use in a method of treatment.

[0025] In one aspect, the present disclosure provides a CAR according to the present disclosure for use in a method of treating cancer.

[0026] In one aspect, the present disclosure provides a lymphocyte according to the present disclosure for use in a method of treating cancer.

[0027] In one aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, according to the present disclosure for use in a method of treating cancer.

[0028] In one embodiment, the cancer is prostate cancer.

[0029] In one embodiment, the cancer is androgen receptor (AR)-expressing breast cancer.

[0030] In another aspect, a method for treating a subject with cancer is provided. The method comprises administering a therapeutically effective amount of any of the lymphocytes described above to a subject in need thereof, thereby causing the lymphocytes to induce the killing of cancer cells in the subject. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is androgen receptor (AR)-expressing breast cancer.

[0031] In another aspect, provided is a method for targeted killing of cancer cells, comprising contacting the cancer cells with any of the lymphocytes described above, thereby causing the lymphocytes to induce the killing of the cancer cells.In some embodiments, the cancer cells are prostate cancer cells.In some embodiments, the cancer is androgen receptor (AR)-expressing breast cancer.

[0032] In another aspect, there is provided a method of detecting the presence of cancer in a subject, comprising: (a) contacting a cell sample obtained from the subject with any of the CARs described above, thereby forming a CAR-cell complex; (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the subject; A method is provided, comprising: [Brief explanation of the drawings]

[0033] The foregoing will be apparent from the following more particular description of exemplary embodiments, as illustrated in the accompanying drawings.

[0034] This patent application file contains at least one drawing executed in color. Copies of this patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1A] Figure 1 shows the construction and expression of hK2 CAR in human T cells. Figure 1A shows a schematic of the construct for hK2 CAR. TM: transmembrane. [Figure 1B] Figure 1B shows the construction and expression of hK2 CAR in human T cells. Figure 1B shows hK2 CAR expression on the T cell surface. Primary human T cells were electroporated with no mRNA (MOCK) or 10 μg of mRNA expressing either the hK2 scFv CAR or an irrelevant control CAR. 24 hours after electroporation, CAR surface expression was measured by flow cytometry after staining with 2 μg / mL biotinylated L protein and streptavidin-conjugated PE (Figure 1B). [Figure 1C] Figure 1C shows the construction and expression of hK2 CAR in human T cells. Figure 1C shows hK2 CAR expression on the T cell surface. Primary human T cells were electroporated with no mRNA (MOCK) or 10 μg of mRNA expressing either the hK2 scFv CAR or an irrelevant control CAR. 24 hours after electroporation, CAR surface expression was measured by flow cytometry after staining with biotinylated hK2 (1 μg / mL) and streptavidin-conjugated PE (Figure 1C). [Figure 2] Figure 1 shows the cytotoxicity of hK2-expressing tumor cells by CAR-T cells in a 20-hour flow-based assay at the indicated effector-to-target cell (E / T) ratios. 24 hours after transient transfection, target cells (Vcap and DU145) were labeled with Cell Trace Violet (CTV) fluorescent dye and then co-cultured with hK2 CAR-T cells. Mock T cells served as a negative effector control. Percent killing (killing) is the ratio of the absolute number of viable (viability dye-negative) target (CTV-positive) cells remaining in the co-culture to the number of viable targets cultured without CAR-T cells. [Figure 3A]Figure 3 shows real-time hK2-specific CAR-T cell-mediated cytotoxicity. Normalized cell index (CI) plots of target cells (Figure 3A) VCap (5E4) incubated with mock, 10 μg mRNA-electroporated (24 h post-transfection) hK2 11B6 CAR LH, or control CAR-T cells at different E / T ratios for approximately 72 hours. When plated alone, target cells adhered to the plate and proliferated, increasing the CI readout. Upon addition of T cells to the target cells, hK2 CAR- and control CAR-T cells caused cytolysis of hK2(+) VCap cells and a subsequent progressive decrease in CI at E / T ratios ranging from 5:1 to 0.156:1. The decrease in CI values ​​after effector cell addition reflects the loss of target cell viability. As shown in (Figure 3B), a parallel experiment using hK2(-)DU145 target cells was also performed; at the E:T ratios tested in this experiment, hK2 and control CAR T cells did not decrease CI after addition and did not exhibit cytolytic activity. The Y axis is the normalized CI generated by the RTCA software and displayed in real time. The X axis is the time of cell culture and treatment time (hours). The mean CI values ​​were plotted ± standard deviation. [Figure 3B]Figure 3B shows real-time hK2-specific CAR-T cell-mediated cytotoxicity. Normalized cell index (CI) plots of target cells (Figure 3B) DU145 (5E3) incubated with mock, 10 μg mRNA-electroporated (24 h post-transfection) hK2 11B6 CAR LH, or control CAR-T cells at different E / T ratios for approximately 72 hours. When plated alone, target cells adhered to the plate and proliferated, increasing the CI readout. Upon addition of T cells to the target cells, hK2 CAR- and control CAR-T cells caused cytolysis of hK2(+)VCap cells and a subsequent progressive decrease in CI at E / T ratios ranging from 5:1 to 0.156:1. The decrease in CI values ​​after effector cell addition reflects the loss of target cell viability. As shown in (Figure 3B), a parallel experiment using hK2(-)DU145 target cells was also performed; at the E:T ratios tested in this experiment, hK2 and control CAR T cells did not decrease CI after addition and did not exhibit cytolytic activity. The Y axis is the normalized CI generated by the RTCA software and displayed in real time. The X axis is the time of cell culture and treatment time (hours). The mean CI values ​​were plotted ± standard deviation. [Figure 4] Figure 3 shows the Th1 cytokine interferon-γ (IFN-γ) production of antigen-stimulated CAR-T cells. Supernatants were collected from xCELLigence-based killing assays (see Figures 3A-3B) after approximately 70 hours of coculture (VCap#5E4, DU145#5E3). hK2 CAR LH and control CAR-modified T cells secrete IFN-γ during coculture with hK2-expressing VCap cells, but not with hK2-negative DU145 cells. The mean IFN-γ concentration ± standard deviation (pg / mL) from duplicate cultures is shown. [Figure 5A]Figure 5A shows that the 11B6 thermostabilized scFv CAR clone was evaluated for antigen-dependent activity in the JNL reporter assay. Figure 5B shows that various hK2CAR constructs were transduced into Jurkat cells (referred to as JNL cells) containing a luciferase gene driven by a signal transduction-responsive NFAT promoter. Expression was determined by biotinylated hK2 followed by streptavidin-conjugated PE. [Figure 5B] Figure 5B shows that the 11B6 heat-stabilized scFv CAR clone was evaluated for antigen-dependent activity in the JNL reporter assay. Figure 5B shows that binding between the CAR construct and its cognate cellular antigen (hK2 on target cells) resulted in luciferase expression in JNL cells. JNL cells containing the indicated CAR clone and untransduced JNL cells (UTD) were co-cultured with target cell lines (VCap or DU145 cells), and luciferase activity was measured as luminescence intensity. A clone was considered active if its luminescence intensity exceeded the level of UTD cells by 1.5-fold in the presence of antigen-expressing cells. [Figure 6] Figure 1 shows hK2 CAR expression on the surface of T cells. Primary human T cells were transduced with 11B6 heat-stable and parental scFv-based CAR lentivirus (multiplicity of infection (MOI): 3), and CAR expression was determined 14 days after transduction by biotinylated hK2 (1 μg / mL) followed by streptavidin-conjugated PE. [Figure 7A] Figure 7 shows that 11B6 heat-stabilized scFv CAR-T cells were evaluated for antigen-dependent cytotoxicity in a real-time IncuCyte killing assay. Heat-stabilized hK2 CAR-T cells and parental 11B6 CAR-T were co-incubated with VCaP cells (Figure 7A) for 88 hours. The effector-to-target (E / T) ratio was calculated based on CAR expression data (see Figure 6). Target cells stably expressed a red nuclear dye, which was measured in real time by an IncuCyte imaging system. Tumor cell growth inhibition (%) = (initial number of viable target cells - current number of viable target cells) / initial number of viable cells × 100 (%). [Figure 7B]Figure 7 shows that 11B6 heat-stabilized scFv CAR-T cells were evaluated for antigen-dependent cytotoxicity in a real-time IncuCyte killing assay. Heat-stabilized hK2 CAR-T cells and parental 11B6 CAR-T were co-incubated with PC3 cells (Figure 7B) for 88 hours. The effector-to-target (E / T) ratio was calculated based on CAR expression data (see Figure 6). Target cells stably expressed a red nuclear dye, which was measured in real time by an IncuCyte imaging system. Tumor cell growth inhibition (%) = (initial number of viable target cells - current number of viable target cells) / initial number of viable cells × 100 (%). [Figure 7C] Figure 1 shows the percent tumor cell growth inhibition of hK2-positive VCaP cells by CAR1-10 or parental 11B6_HL or 11B6_LH-transduced T cells at an effector:target ratio of 1:1 or 0.5:1 in a real-time Incucyte killing assay assessing antigen-dependent cellular cytotoxicity. Tumor cell growth inhibition (%) = (initial number of surviving target cells - current number of surviving target cells) / initial number of surviving cells x 100 (%). [Figure 7D] Figure 1 shows the percent tumor cell growth inhibition of PC3 cells at an effector:target ratio of 1:1 by T cells transduced with CAR1-10 or parental 11B6_HL or 11B6_LH in a real-time Incucyte killing assay assessing antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (initial number of surviving target cells - current number of surviving target cells) / initial number of surviving cells x 100 (%). [Figure 7E] Figure 1 shows the percent tumor cell growth inhibition of hK2-positive VCaP cells by CAR-T cells transduced with CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure) in a real-time Incucyte killing assay assessing antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (initial number of surviving target cells - current number of surviving target cells) / initial number of surviving cells × 100 (%). [Figure 7F]Figure 1 shows the percent tumor cell growth inhibition of hK2-negative DU145 cells by CAR-T cells transduced with CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure) in a real-time Incucyte killing assay assessing antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (initial number of surviving target cells - current number of surviving target cells) / initial number of surviving cells × 100 (%). [Figure 8] Cytokine release by hK2 lenti-CAR-T cells is shown. Supernatants collected from overnight (approximately 20 hours) co-cultures of hK2 CAR-T cells with VCap cells at a 1:1 E / T ratio were analyzed using the 13-plex Milliplex Human High Sensitivity T Cell Kit (HSTCMAG28SPMX13). hK2 CAR-modified T cells secrete cytokines during co-culture with hK2-expressing VCap cells, whereas untransduced T cells (UTD) secrete minimal cytokines. Mean cytokine concentrations ± standard deviation (pg / mL) from duplicate cultures are shown. [Figure 9] IFN-γ release by hK2 lenti-CAR-T cells is shown. Supernatants were collected from overnight (approximately 20 hours) cocultures of hK2 CAR-T cells with VCaP and DU145 (5E4 cells) cells at a 1:1 E / T ratio. hK2 CAR-modified T cells secrete IFN-γ during coculture with hK2-expressing VCaP cells, but not with hK2-negative DU145 cells. Mean IFN-γ concentrations ± standard deviation (pg / mL) from duplicate cultures are shown. CD3 / 28 bead-stimulated T cells and T cells alone were used as positive and negative controls, respectively. [Figure 10A]Results from an hK2 CAR-T cell proliferation assay are shown. (Figures 10A and 10C) hK2 CAR and untransduced (UTD) T cells were labeled with CellTrace Violet (CTV, 5 μM) and cocultured with hK2(+)VCap and hK2(-)DU145 cells. Five days after coculture, cells were harvested and stained with CD3, CD25, NearIR live / dead dye, and hK2CAR. Flow cytometry analysis was performed on a Fortessa flow cytometer equipped with Flowjo software. Lymphocytes were identified by live CD3, and the frequency of CAR+ T cells bearing CTV dye dilution and the activation marker CD25 was determined. (Figures 10B and 10D) CD3 / 28 bead-stimulated T cells and T cells alone were used as positive and negative controls, respectively. The data in Figure 10E show that hK2 CAR-T cells proliferate more robustly than the CD3 / 28 bead-positive control after 5 days of coculture with VCap cells. T cells engineered with different CAR constructs had different proliferative activities and resulted in different CAR+ T cell counts. CAR+ T cell counts were based on the mean absolute cell counts + / - SEM from three technical replicates. [Figure 10B]Results from an hK2 CAR-T cell proliferation assay are shown. (Figures 10A and 10C) hK2 CAR and untransduced (UTD) T cells were labeled with CellTrace Violet (CTV, 5 μM) and cocultured with hK2(+)VCap and hK2(-)DU145 cells. Five days after coculture, cells were harvested and stained with CD3, CD25, NearIR live / dead dye, and hK2CAR. Flow cytometry analysis was performed on a Fortessa flow cytometer equipped with Flowjo software. Lymphocytes were identified by live CD3, and the frequency of CAR+ T cells bearing CTV dye dilution and the activation marker CD25 was determined. (Figures 10B and 10D) CD3 / 28 bead-stimulated T cells and T cells alone were used as positive and negative controls, respectively. The data in Figure 10E show that hK2 CAR-T cells proliferate more robustly than the CD3 / 28 bead-positive control after 5 days of coculture with VCap cells. T cells engineered with different CAR constructs had different proliferative activities and resulted in different CAR+ T cell counts. CAR+ T cell counts were based on the mean absolute cell counts + / - SEM from three technical replicates. [Figure 10C]Results from an hK2 CAR-T cell proliferation assay are shown. (Figures 10A and 10C) hK2 CAR and untransduced (UTD) T cells were labeled with CellTrace Violet (CTV, 5 μM) and cocultured with hK2(+)VCap and hK2(-)DU145 cells. Five days after coculture, cells were harvested and stained with CD3, CD25, NearIR live / dead dye, and hK2CAR. Flow cytometry analysis was performed on a Fortessa flow cytometer equipped with Flowjo software. Lymphocytes were identified by live CD3, and the frequency of CAR+ T cells bearing CTV dye dilution and the activation marker CD25 was determined. (Figures 10B and 10D) CD3 / 28 bead-stimulated T cells and T cells alone were used as positive and negative controls, respectively. The data in Figure 10E show that hK2 CAR-T cells proliferate more robustly than the CD3 / 28 bead-positive control after 5 days of coculture with VCap cells. T cells engineered with different CAR constructs had different proliferative activities and resulted in different CAR+ T cell counts. CAR+ T cell counts were based on the mean absolute cell counts + / - SEM from three technical replicates. [Figure 10D]Results from an hK2 CAR-T cell proliferation assay are shown. (Figures 10A and 10C) hK2 CAR and untransduced (UTD) T cells were labeled with CellTrace Violet (CTV, 5 μM) and cocultured with hK2(+)VCap and hK2(-)DU145 cells. Five days after coculture, cells were harvested and stained with CD3, CD25, NearIR live / dead dye, and hK2CAR. Flow cytometry analysis was performed on a Fortessa flow cytometer equipped with Flowjo software. Lymphocytes were identified by live CD3, and the frequency of CAR+ T cells bearing CTV dye dilution and the activation marker CD25 was determined. (Figures 10B and 10D) CD3 / 28 bead-stimulated T cells and T cells alone were used as positive and negative controls, respectively. The data in Figure 10E show that hK2 CAR-T cells proliferate more robustly than the CD3 / 28 bead-positive control after 5 days of coculture with VCap cells. T cells engineered with different CAR constructs had different proliferative activities and resulted in different CAR+ T cell counts. CAR+ T cell counts were based on the mean absolute cell counts + / - SEM from three technical replicates. [Figure 10E]Results from an hK2 CAR-T cell proliferation assay are shown. (Figures 10A and 10C) hK2 CAR and untransduced (UTD) T cells were labeled with CellTrace Violet (CTV, 5 μM) and cocultured with hK2(+)VCap and hK2(-)DU145 cells. Five days after coculture, cells were harvested and stained with CD3, CD25, NearIR live / dead dye, and hK2CAR. Flow cytometry analysis was performed on a Fortessa flow cytometer equipped with Flowjo software. Lymphocytes were identified by live CD3, and the frequency of CAR+ T cells bearing CTV dye dilution and the activation marker CD25 was determined. (Figures 10B and 10D) CD3 / 28 bead-stimulated T cells and T cells alone were used as positive and negative controls, respectively. The data in Figure 10E show that hK2 CAR-T cells proliferate more robustly than the CD3 / 28 bead-positive control after 5 days of coculture with VCap cells. T cells engineered with different CAR constructs had different proliferative activities and resulted in different CAR+ T cell counts. CAR+ T cell counts were based on the mean absolute cell counts + / - SEM from three technical replicates. [Figure 11A] Figure 11A shows the results of an assay to determine the expression level of hK2 CAR in primary T cells using biotin-labeled hK2 protein. Figure 11A is a schematic diagram of the construct for hK2 CAR. TM: transmembrane. [Figure 11B]Figure 11B shows the results of an assay to determine the expression level of hK2 CAR in primary T cells using biotin-labeled hK2 protein. Primary human T cells were transduced with the novel hK2 scFv-based KL2B413 HL and LH and rehumanized 11B6 KL2B359 HL and LH CARS lentiviruses (MOI 3). 14 days after transduction, CAR expression was assessed using biotinylated hK2 (1 μg / mL) followed by streptavidin-conjugated PE. Figure 11B provides an overview of the percentage of hK2 CAR+ T cells (% positive) detected by the novel KL2B413 HL and LH and rehumanized 11B6 KL2B359 HL and LH CARS clones analyzed. As shown, different clones have different CAR expression levels, ranging from 45.1% to 59.9%. All CAR T+ cells were normalized to equivalent CAR+ T cells for subsequent functional assays. [Figure 12A] Figure 12A shows the results of evaluation of the novel hK2 KL2B413 and rehumanized 11B6 CAR clones in the JNL reporter assay for antigen-dependent activity. In Figure 12A, various hK2CAR constructs were transduced into Jurkat cells (referred to as JNL cells) containing a luciferase gene driven by a signaling-responsive NFAT promoter. Expression was determined by biotinylated hK2 followed by streptavidin-conjugated PE. CAR expression in transduced JNL cells was confirmed for selected clones, with 46-50% of cells expressing the rehumanized 11B6 KL2B359 CAR and 73.7%-96% of cells expressing the KL2B413 CAR. [Figure 12B]Figure 12B shows the results of evaluation of the novel hK2 KL2B413 and rehumanized 11B6 CAR clones in the JNL reporter assay for antigen-dependent activity. The data shown in Figure 12B demonstrate that binding between the hK2 CAR construct and its allogeneic cellular antigen (hK2 on target cells) results in luciferase expression in JNL cells. JNL cells containing the indicated CAR clones and JNL cells (UTD) were cocultured with target cell lines (VCap, LNCap / Hk2, LNCap, C4-2B, 22Rv1, or DU145 cells), and luciferase activity was measured as luminescence intensity. A clone was considered active if its luminescence intensity exceeded the level of UTD cells by 1.5-fold in the presence of antigen-expressing cells. No antigen-dependent activation was observed for the novel KL2B413 and the rehumanized 11B6 HL and LH CARs. [Figure 13A] Figure 13 shows the results of evaluating HK2 CAR-T cells in a real-time IncuCyte killing assay for antigen-dependent cytotoxicity. HK2 CAR-T cells were co-incubated with HK2+VCaP cells (Figure 13A) for 96 hours. The effector-to-target (ET) ratio was calculated based on CAR expression data (Figure 11B). Target cells stably expressed red nuclear dye, which was measured in real time by an IncuCyte imaging system. Tumor cell growth inhibition (%) = (initial number of viable target cells - current number of viable target cells) / initial number of viable cells × 100 (%). [Figure 13B] Figure 13 shows the results of evaluating HK2 CAR-T cells in a real-time IncuCyte killing assay for antigen-dependent cytotoxicity. HK2 CAR-T cells were co-incubated with HK2-DU145 cells (Figure 13B) for 96 hours. The effector-to-target (ET) ratio was calculated based on CAR expression data (Figure 11B). Target cells stably expressed red nuclear dye, which was measured in real time by an IncuCyte imaging system. Tumor cell growth inhibition (%) = (initial number of viable target cells - current number of viable target cells) / initial number of viable cells × 100 (%). [Figure 14]Data are presented showing the production of the Th1 cytokine IFN-gamma by antigen-stimulated CAR-T cells. IFN-γ produced by cytotoxic T cells is crucial for tumor immune surveillance, directly inhibiting proliferation and inducing apoptosis of some malignant tumors in vivo and in vitro. To determine whether hK2 CAR-modified human T cells can be recognized and activated by hK2(+) tumor cells, primary T cells containing the indicated CAR clones and control untransduced T cells (UTD) were cocultured with target cell lines (LNCap / Hk2, LNCap, C4-2B, 22Rv1, or DU145 cells), and supernatants were collected for IFN-γ concentration measurements. As shown, hK2 CAR-modified T cells secrete IFN-gamma during coculture with hK2-expressing LNCap / hK2 cells, even C4-2B and LNCap cells, which express very little HK2, but not hK2-negative DU145 cells. The undisclosed control CAR secreted much more IFN-γ than hK2, due to its much higher antigen expression level. Mean IFN-γ concentrations ± SD (pg / mL) from duplicate cultures are shown. [Figure 15]This figure shows the results of a CD107a degranulation assay to evaluate the anti-tumor activity of hK2 CAR T cells. Target cells (5 × 104) were cocultured with an equal number of effector cells at 0.1 mL / well in a 96-well plate. Control wells contained T cells only. Anti-CD107a (5 μL / well) was added along with 1 μL / sample of monensin (BD Biosciences) and incubated at 37°C for 4 hours. Cells were washed twice with PBS, stained for hK2 CAR, CD3, and CD8 expression, and analyzed using a BD Fortessa flow cytometer. As shown, the presence of hK2 antigen on Vcap cells increased CD107A+ hK2 CAR(+) T cells, but not hK2 CAR(-) and UTD T cells, as measured by flow cytometry, while CAR T cells alone without tumor cell stimulation showed only background staining (<2%). Phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin (eBioscience™ Cell Stimulation Cocktail; 500x) is a consistent inducer of CD107a cell surface expression in this 4-hour cell culture model and was used as a positive control. Results are shown for representative donor T cells and are reproducible across multiple donors. [Figure 16A]Results of an hK2 CAR T cell proliferation assay are shown. hK2 CAR and untransduced (UTD) T cells were labeled with CellTrace Violet (CTV, 5 μM) and co-cultured with HK2(+)VCap and HK2(-)DU145 cells. Five days after co-culture, cells were harvested and stained with CD3, CD25, NearIR live / dead dye, and hK2CAR. Flow cytometry analysis was performed on a Fortessa flow cytometer equipped with Flowjo software. Lymphocytes were identified by live CD3, and the frequency of CAR+ T cells bearing the CTV dye dilution and activation marker CD25 was determined. By gating on CD3+ T cells, hK2(+) VCap cells promoted the proliferation of T cells engineered with all CAR constructs, as shown in Figures 16A-16B, and the upregulation of the activation marker CD25, as shown in Figures 16C-16D, whereas hK2(-) ​​DU145 cells did not. CD3 / 28 bead-stimulated T cells and T cells alone were used as positive and negative controls, respectively. T cells alone without any stimulation did not proliferate, whereas CD3 / 28 bead-stimulated T cells showed equivalent proliferation patterns. As shown, hK2 CAR+ T cells proliferated more robustly than the CD3 / 28 bead-positive control after 5 days of coculture with VCap cells. T cells engineered with different CAR constructs had different proliferative activities and resulted in different numbers of CAR+ T cells. A summary of the percentage of proliferating T cells and CD25-expressing T cells based on mean absolute cell counts + / - SEM from duplicates is shown in Figures 16B and 16D. [Figure 16B]Results of an hK2 CAR T cell proliferation assay are shown. hK2 CAR and untransduced (UTD) T cells were labeled with CellTrace Violet (CTV, 5 μM) and co-cultured with HK2(+)VCap and HK2(-)DU145 cells. Five days after co-culture, cells were harvested and stained with CD3, CD25, NearIR live / dead dye, and hK2CAR. Flow cytometry analysis was performed on a Fortessa flow cytometer equipped with Flowjo software. Lymphocytes were identified by live CD3, and the frequency of CAR+ T cells bearing the CTV dye dilution and activation marker CD25 was determined. By gating on CD3+ T cells, hK2(+) VCap cells promoted the proliferation of T cells engineered with all CAR constructs, as shown in Figures 16A-16B, and the upregulation of the activation marker CD25, as shown in Figures 16C-16D, whereas hK2(-) ​​DU145 cells did not. CD3 / 28 bead-stimulated T cells and T cells alone were used as positive and negative controls, respectively. T cells alone without any stimulation did not proliferate, whereas CD3 / 28 bead-stimulated T cells showed equivalent proliferation patterns. As shown, hK2 CAR+ T cells proliferated more robustly than the CD3 / 28 bead-positive control after 5 days of coculture with VCap cells. T cells engineered with different CAR constructs had different proliferative activities and resulted in different numbers of CAR+ T cells. A summary of the percentage of proliferating T cells and CD25-expressing T cells based on mean absolute cell counts + / - SEM from duplicates is shown in Figures 16B and 16D. [Figure 16C]Results of an hK2 CAR T cell proliferation assay are shown. hK2 CAR and untransduced (UTD) T cells were labeled with CellTrace Violet (CTV, 5 μM) and co-cultured with HK2(+)VCap and HK2(-)DU145 cells. Five days after co-culture, cells were harvested and stained with CD3, CD25, NearIR live / dead dye, and hK2CAR. Flow cytometry analysis was performed on a Fortessa flow cytometer equipped with Flowjo software. Lymphocytes were identified by live CD3, and the frequency of CAR+ T cells bearing the CTV dye dilution and activation marker CD25 was determined. By gating on CD3+ T cells, hK2(+) VCap cells promoted the proliferation of T cells engineered with all CAR constructs, as shown in Figures 16A-16B, and the upregulation of the activation marker CD25, as shown in Figures 16C-16D, whereas hK2(-) ​​DU145 cells did not. CD3 / 28 bead-stimulated T cells and T cells alone were used as positive and negative controls, respectively. T cells alone without any stimulation did not proliferate, whereas CD3 / 28 bead-stimulated T cells showed equivalent proliferation patterns. As shown, hK2 CAR+ T cells proliferated more robustly than the CD3 / 28 bead-positive control after 5 days of coculture with VCap cells. T cells engineered with different CAR constructs had different proliferative activities and resulted in different numbers of CAR+ T cells. A summary of the percentage of proliferating T cells and CD25-expressing T cells based on mean absolute cell counts + / - SEM from duplicates is shown in Figures 16B and 16D. [Figure 16D]Results of an hK2 CAR T cell proliferation assay are shown. hK2 CAR and untransduced (UTD) T cells were labeled with CellTrace Violet (CTV, 5 μM) and co-cultured with HK2(+)VCap and HK2(-)DU145 cells. Five days after co-culture, cells were harvested and stained with CD3, CD25, NearIR live / dead dye, and hK2CAR. Flow cytometry analysis was performed on a Fortessa flow cytometer equipped with Flowjo software. Lymphocytes were identified by live CD3, and the frequency of CAR+ T cells bearing the CTV dye dilution and activation marker CD25 was determined. By gating on CD3+ T cells, hK2(+) VCap cells promoted the proliferation of T cells engineered with all CAR constructs, as shown in Figures 16A-16B, and the upregulation of the activation marker CD25, as shown in Figures 16C-16D, whereas hK2(-) ​​DU145 cells did not. CD3 / 28 bead-stimulated T cells and T cells alone were used as positive and negative controls, respectively. T cells alone without any stimulation did not proliferate, whereas CD3 / 28 bead-stimulated T cells showed equivalent proliferation patterns. As shown, hK2 CAR+ T cells proliferated more robustly than the CD3 / 28 bead-positive control after 5 days of coculture with VCap cells. T cells engineered with different CAR constructs had different proliferative activities and resulted in different numbers of CAR+ T cells. A summary of the percentage of proliferating T cells and CD25-expressing T cells based on mean absolute cell counts + / - SEM from duplicates is shown in Figures 16B and 16D. [Figure 17A] Figure 17A shows a sequence alignment of the VH domains of mu11B6, hu11B6, KL2B357, KL2B358, KL2B359, KL2B360, HCF3, and HCG5. Figure 17A discloses SEQ ID NOs: 317, 5, 140, 141, 77, 140, 6, 4, and 345, respectively, in order of appearance. [Figure 17B]Figure 17B shows the protected segments mapped onto the sequence of hK2 antigen to visualize the binding epitopes of the hK2 antibody identified through HDX-MS. Figure 17B discloses SEQ ID NOs: 351, 351, 351, 351, 351, and 351, respectively, in order of appearance.

[0035] (Detailed description) A description of exemplary embodiments follows.

[0036] The present disclosure provides chimeric antigen receptors (CARs) that target human kallikrein-2 (hK2), cells containing such CARs, and methods of treating cancer (e.g., prostate cancer or AR-expressing breast cancer) using the CARs described herein.

[0037] The CAR of the present invention has antigen specificity for hK2. As used herein, the phrases "having antigen specificity" and "inducing an antigen-specific response" mean that the CAR can specifically bind to and immunologically recognize an antigen, such that the binding of the CAR to the hK2 antigen induces an immune response. Methods for testing CARs for antigen specificity and the ability to recognize target cells are known in the art.

[0038] The disclosure also provides related nucleic acids, recombinant expression vectors, host cells, cell populations, antibodies, or antigen-binding portions thereof, and pharmaceutical compositions related to the CARs of the invention.

[0039] Some aspects of the present invention are described below with reference to examples that are for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are described to provide a thorough understanding of the present invention. However, one of ordinary skill in the art will readily recognize that the present invention can be practiced without one or more of the specific details, or can be practiced with other methods, protocols, reagents, cell lines, and animals. The present invention is not limited by the order of acts or events exemplified, as some acts may occur in a different order and / or simultaneously with other acts or events. Furthermore, not all exemplified acts, steps, or events are required to practice a methodology in accordance with the present invention. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional techniques by those of ordinary skill in the art.

[0040] Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meaning commonly understood by those of ordinary skill in the art to which the present invention pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is generally understood in the art. It will be further understood that terms, such as those defined in commonly used dictionaries, should be construed as having a meaning consistent with their meaning in the context of the relevant art and / or as otherwise defined herein.

[0041] When lists are presented, it is to be understood that each individual element of that list and every combination of that list is a separate embodiment, unless otherwise specified. For example, a list of embodiments presented as "A, B, or C" should be interpreted to include the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0042] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0043] The transitional phrases "comprising," "consisting essentially of," and "consisting" are intended to connote their generally accepted meanings in patent language, i.e., (i) "comprising" is synonymous with "comprising," "containing," or "characterized by" and is inclusive or open-ended and does not exclude other unrecited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not materially affect the "basic and novel characteristic(s)" of the claimed invention. Embodiments described with the phrase "comprising" (or its equivalents) also provide, as embodiments, those described independently with the terms "consisting of" and "consisting essentially of."

[0044] "Activation" or "stimulation" or "activated" or "stimulated" refers to the induction of a change in the biological state of a cell that results in the expression of activation markers, cytokine production, or mediation of target cell proliferation or cytotoxicity. Cells can be activated by a primary stimulatory signal. A costimulatory signal can amplify the magnitude of the primary signal and suppress cell death after initial stimulation, resulting in a more durable activation state and, therefore, greater cytotoxic potential. A "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, causes T cell and / or NK cell proliferation and / or up- or down-regulation of key molecules.

[0045] An "antigen-binding fragment" or "antigen-binding domain" refers to a portion of a protein that binds to an antigen. Antigen-binding fragments may be synthetic, enzymatically obtainable, or genetically engineered polypeptides, and include antigen-binding portions of immunoglobulins, such as VH, VL, VH and VL, Fab, Fab', F(ab')2, Fd, and Fv fragments, domain antibodies (dAbs) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelized VH domains, VHH domains, minimal recognition units consisting of amino acid residues mimicking the CDRs of an antibody, such as the FR3-CDR3-FR4 portion, HCDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3, alternative scaffolds that bind to antigen, and multispecific proteins comprising antigen-binding fragments. Antigen-binding fragments (such as VH and VL) can be linked together via synthetic linkers to form a variety of single-chain antibody designs in which, when the VH and VL domains are expressed as separate single chains, the VH / VL domains can pair intramolecularly or intermolecularly to form monovalent antigen-binding domains, e.g., single-chain Fvs (scFvs) or diabodies. Antigen-binding fragments can also be conjugated to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds, which can be monospecific or multispecific, to engineer bispecific and multispecific proteins.

[0046] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth leads to the formation of malignant tumors that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors.

[0047] A "full-length antibody" is composed of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain is composed of a heavy chain variable domain (VH) and a heavy chain constant domain, which in turn is composed of subdomains CH1, hinge, CH2, and CH3. Each light chain is composed of a light chain variable domain (VL) and a light chain constant domain (CL). VH and VL can be further divided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with framework regions (FRs). Each VH and VL is composed of three CDR and four FR segments, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0048] A "human antibody" refers to an antibody optimized to evoke a minimal immune response when administered to a human subject. The variable regions of a human antibody are derived from human immunoglobulin sequences. If a human antibody contains a constant region or a portion of a constant region, the constant region is also derived from human immunoglobulin sequences. A human antibody contains heavy and light chain variable regions "derived" from sequences of human origin when the variable regions of the human antibody are obtained from a system using human germline immunoglobulins or rearranged immunoglobulin genes. Exemplary such systems include phage-displayed human immunoglobulin gene libraries and transgenic non-human animals, such as mice or rats, carrying human immunoglobulin loci. A "human antibody" typically contains amino acid differences compared to immunoglobulins expressed in humans due to differences in the systems used to obtain human antibodies and human immunoglobulin loci, the introduction of somatic mutations or intentional substitutions into frameworks or CDRs, or both. Typically, a "human antibody" is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. Optionally, a "human antibody" may contain a consensus framework sequence obtained from human framework sequence analysis, e.g., as described in Knappik et al., (2000) J Mol Biol 296:57-86, or a synthetic HCDR3 incorporated into a phage-displayed human immunoglobulin gene library, e.g., as described in Shi et al., (2010) J Mol Biol 397:385-96 and International Publication No. WO2009 / 085462. Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of "human antibody."

[0049] "Humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Humanized antibodies can contain substitutions in the framework, so that the framework may not be an exact copy of an expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0050] "Isolated" refers to a homogenous population of molecules that have been substantially separated and / or purified away from other components of the system in which they are produced, such as in a recombinant cell (e.g., a synthetic polynucleotide or polypeptide), as well as to proteins that have been subjected to at least one purification or isolation step. "Isolated" refers to molecules that are substantially free of other cellular material and / or chemicals and includes molecules isolated to greater degrees of purity, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

[0051] "Modulate" refers to either an enhanced or decreased ability of the test molecule to mediate a greater or lesser response (i.e., a downstream effect) when compared to a control or vehicle-mediated response.

[0052] "Natural killer cells" and "NK cells" are used interchangeably and synonymously herein. NK cells are cells that express CD16 + CD56 + and / or CD57 + TCR - NK cells are differentiated lymphocytes with a phenotype characterized by the ability to bind to and kill cells that fail to express "self" MHC / HLA antigens through the activation of specific cytolytic enzymes, to kill tumor cells or other diseased cells that express ligands for NK-activating receptors, and to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0053] "Specifically binds," "specific binding," "specifically binding," or "binds" refers to a proteinaceous molecule binding to an antigen or an epitope within the antigen with higher affinity than its affinity for other antigens. Typically, a proteinaceous molecule binds to an antigen with an affinity of about 1 x 10 -7 M or less, e.g., about 5 × 10 -8 M or less, approximately 1×10 -8 M or less, approximately 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 M or less, or about 1 x 10 -12 The equilibrium dissociation constant (K D ) binds to an antigen or an epitope within the antigen, typically D is the K for binding to nonspecific antigens (e.g., BSA, casein) D In the context of the prostate neoantigens described herein, "specific binding" refers to the binding of a proteinaceous molecule to a prostate neoantigen without detectably binding to the wild-type protein of which the prostate neoantigen is a variant.

[0054] "Tumor cells" or "cancer cells" refer to cancerous, precancerous, or transformed cells that have spontaneously or induced phenotypic changes, either in vivo, ex vivo, or in tissue culture. These changes do not necessarily involve the incorporation of new genetic material. Transformation can occur through infection with a transforming virus and the incorporation of new genomic nucleic acid, through the incorporation of exogenous nucleic acid, or can occur spontaneously or after exposure to a carcinogen, resulting in mutation of an endogenous gene. Transformation / cancer is exemplified by morphological changes, cellular immortalization, aberrant growth control, formation of foci, proliferation, malignant lesions, modulation of tumor-specific marker levels, invasiveness, and tumor growth in suitable animal hosts, such as nude mice, in vitro, in vivo, and ex vivo.

[0055] The term "chimeric antigen receptor" or "CAR," as used herein, is defined as a cell surface receptor comprising an extracellular target binding domain, a transmembrane domain, and an intracellular signaling domain, all in a combination not naturally found together in a single protein. This particularly includes receptors in which the extracellular domain and intracellular signaling domain are not naturally found together in a single receptor protein. The chimeric antigen receptors of the present invention are intended primarily for use in lymphocytes, such as T cells and natural killer (NK) cells.

[0056] The terms "T cells" and "T lymphocytes" are interchangeable and are used synonymously herein. As used herein, T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells may be T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells may be helper T cells (HTL; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+CD8+ T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells and memory T cells. Also included are "NKT cells," which refer to a special population of T cells that express not only the semi-invariant αβ T cell receptor but also various molecular markers typically associated with NK cells, such as NK1.1. NKT cells include NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+, and CD8 cells. The TCR of NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or harmful effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. Also included are "gamma delta T cells (γδ T cells)." Gamma delta T cells refer to a special population of a small subset of T cells that possess a distinct TCR on their surface, distinct from the majority of T cells, whose TCR is composed of two glycoprotein chains, designated α and β-TCR chains. The TCR in γδ T cells is composed of a γ chain and a δ chain. γδ T cells can play a role in immune surveillance and immune regulation, and have been found to be an important source of IL-17 and to induce vigorous CD8+ cytotoxic T cell responses. Also included are "regulatory T cells" or "Tregs," which refer to T cells that suppress aberrant or excessive immune responses and play a role in immune tolerance. Tregs are typically Foxp3-positive CD4+ T cells, but can also include Foxp3-negative regulatory T cells, which are IL-10-producing CD4+ T cells.

[0057] The terms "natural killer cells" and "NK cells" are interchangeable and are used synonymously herein. As used herein, NK cells refer to differentiated lymphocytes with a CD16+CD56+ and / or CD57+TCR- phenotype. NK cells are characterized by their ability to bind to and kill cells that fail to express "self" MHC / HLA antigens through the activation of specific cytolytic enzymes, to kill tumor cells or other diseased cells that express ligands for NK-activating receptors, and to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0058] As used herein, the term "antigen" refers to any molecule (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portion thereof, or combination thereof) capable of being bound by a T cell receptor. An antigen can also elicit an immune response. Examples of immune responses may involve, but are not limited to, antibody production, or activation of specific immunologically competent cells, or both. Those skilled in the art will understand that an antigen need not necessarily be encoded by a "gene." It is readily apparent that antigens may be synthetically produced or derived from biological samples, or may be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or other biological components, organisms, protein / antigen subunits, killed or inactivated whole cells, or fluids containing lysates.

[0059] The term "antibody" refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFvs), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), intrabodies, minibodies, diabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against antigen-specific TCRs), and epitope-binding fragments of any of the above. The term "antibody" also refers to covalent diabodies, such as those disclosed in U.S. Patent Application Publication No. 2007 / 0004909, and Ig-DARTS, such as those disclosed in U.S. Patent Application Publication No. 2009 / 0060910. Antibodies useful as TCR-binding molecules include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, and IgA2), or subclass.

[0060] The term "host cell" refers to any cell containing heterologous nucleic acid. The heterologous nucleic acid may be a vector (e.g., an expression vector). For example, a host cell may be a cell from any organism that is selected, modified, transformed, grown, used, or manipulated in any way for the cellular production of a substance, e.g., the cellular expression of a gene, DNA or RNA sequence, protein, or enzyme. An appropriate host can be determined. For example, a host cell can be selected based on the vector backbone and the desired result. By way of example, a plasmid or cosmid can be introduced into a prokaryotic host cell to replicate some types of vectors. Bacterial cells, such as, but not limited to, DH5α, JM109, and KCB, SURE® competent cells, and SOLOPACK Gold cells can be used as host cells for vector replication and / or expression. Additionally, bacterial cells, such as E. coli LE392, can be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to, yeast (e.g., YPH499, YPH500, and YPH501), insect, and mammalian cells. Examples of mammalian eukaryotic host cells for replication and / or expression of vectors include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12.

[0061] The host cells of the present disclosure include T cells and natural killer cells that contain a DNA or RNA sequence encoding a CAR and express the CAR on the cell surface. The host cells can be used to enhance T cell activity, natural killer cell activity, treatment of cancer, and treatment of autoimmune diseases.

[0062] "Activation" or "stimulation" refers to the induction of a change in the biological state of a cell (e.g., T cells and NK cells) by which the cell expresses activation markers, produces cytokines, proliferates, and / or becomes cytotoxic to target cells. All of these changes can be caused by a primary stimulatory signal. A costimulatory signal can amplify the magnitude of the primary signal and suppress cell death after the initial stimulation, resulting in a more durable activation state and, therefore, greater cytotoxic potential. A "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, causes proliferation of T cells and / or NK cells and / or up- or down-regulation of key molecules.

[0063] The term "proliferation" refers to an increase in cell division, either symmetric or asymmetric division of cells. The term "expansion" refers to the result of cell division and cell death.

[0064] The term "differentiation" refers to a process that reduces the potency or proliferation of a cell or transitions the cell into a more developmentally restricted state.

[0065] The terms "express" and "expression" mean to allow or cause the information of a gene or DNA sequence to be produced, e.g., to produce a protein by activating cellular functions involved in transcription and translation of the corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product," such as a protein. The expression product itself, e.g., the resulting protein, can also be said to be "expressed" by the cell. Expression products can be characterized as intracellular, extracellular, or transmembrane.

[0066] The term "transfection" refers to the introduction of "foreign" (i.e., exogenous or extracellular) nucleic acid into a cell using recombinant DNA technology. The term "genetic modification" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA, or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. The introduced gene or sequence may also be referred to as a "cloned" or "foreign" gene or sequence and may include regulatory or control sequences operably linked to the polynucleotide encoding the chimeric antigen receptor, such as start, stop, promoter, signal, secretion, or other sequences used by the cell's genetic machinery. The gene or sequence may also include nonfunctional sequences with no known function. A host cell that receives and expresses introduced DNA or RNA is "genetically engineered." The DNA or RNA introduced into a host cell can be from any source, including cells of the same genus or species as the host cell, or from a different genus or species.

[0067] The term "transduction" refers to the introduction of foreign nucleic acid into cells using a viral vector.

[0068] The term "control element" refers to any cis-acting genetic element that controls some aspect of the expression of a nucleic acid sequence. In some embodiments, the term "promoter" essentially includes the minimal sequences necessary to initiate transcription. In some embodiments, the term "promoter" includes sequences for initiating transcription and, in addition, also includes sequences that can upregulate or downregulate transcription, commonly referred to as "enhancer elements" and "repressor elements," respectively.

[0069] As used herein, "operably linked" and similar phrases, when used with reference to nucleic acids or amino acids, refer to the operably linking of nucleic acid or amino acid sequences, respectively, placed in a functional relationship with each other. For example, operably linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the correct production of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements result in the transcription of the open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operably linked peptide refers to functional domains positioned at an appropriate distance from each other to confer their intended function.

[0070] "Enhancement" or "promotion" or "increase" or "expand" or "improvement" generally refers to the ability of a composition contemplated herein to produce, induce, or cause a greater physiological response (i.e., a downstream effect) compared to the response caused by either a vehicle or a control molecule / composition. Measurable physiological responses may include increased T cell expansion, activation, effector function, persistence, and / or increased cancer cell killing capacity, particularly as will be apparent from an understanding of the art and the present disclosure. In certain embodiments, an "increased" or "enhanced" amount may be a "statistically significant" amount and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more (e.g., 500, 1000-fold) (including all integers above 1 and decimal points therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response caused by a vehicle or control composition.

[0071] "Decrease" or "reduction" or "attenuation" or "reduction" or "mitigation" generally refers to the ability of a composition contemplated herein to produce, induce, or cause a smaller physiological response (i.e., a downstream effect) compared to the response caused by either a vehicle or a control molecule / composition. In certain embodiments, a "decreased" or "reduced" amount may be a "statistically significant" amount and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more (e.g., 500, 1000-fold) (including all integers above 1 and decimal points therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response caused by a vehicle or control composition (a reference response) or a response in a particular cell lineage.

[0072] The term "effective" as applied to a dose or amount refers to that amount of a compound or pharmaceutical composition sufficient to produce the desired activity upon administration to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective when administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug used, the mode of administration, etc.

[0073] The phrase "pharmaceutically acceptable," when used in connection with the compositions described herein, refers to molecular entities and other components of such compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to a mammal (e.g., a human). Preferably, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in mammals, more particularly humans.

[0074] The term "protein," as used herein, encompasses all types of modified proteins, including, but not limited to, natural and synthetic proteins, including protein fragments of all lengths, fusion proteins, and glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).

[0075] The terms "nucleic acid," "nucleotide," and "polynucleotide" encompass both DNA and RNA unless otherwise specified. "Nucleic acid sequence" or "nucleotide sequence" refers to a nucleic acid sequence that encodes an amino acid; these terms may also refer to a nucleic acid sequence that includes portions encoding any amino acids added as a cloning artifact, including any amino acids encoded by linkers.

[0076] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water or aqueous solutions, saline, and aqueous dextrose and glycerin solutions are preferably used as carriers, particularly for injectable solutions. Alternatively, the carrier may be a solid dosage form carrier, including, but not limited to, one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavoring agent, and a coloring agent. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0077] The term "about" or "approximately" includes within a statistically meaningful range of values. Such a range can be within one order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term "about" or "approximately" will depend on the specific system under study and can be readily understood by one of ordinary skill in the art.

[0078] "Kallikrein-related peptidase 2," "hK2," or "klk2" refers to a known protein also known as kallikrein-2, granular kallikrein 2, or HK2. hK2 is produced as a preproprotein and is cleaved during proteolysis to generate the active protease. All hK2 isoforms and variants are encompassed by "hK2." The amino acid sequences of various isoforms can be found under GenBank accession numbers NP_005542.1, NP_001002231.1, and NP_001243009. The amino acid sequence of full-length human hK2 is shown in SEQ ID NO: 62. The sequence includes the signal peptide (residues 1-18) and propeptide region (residues 19-24).

[0079] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the indefinite articles "a," "an," and "the" are to be understood as including plural referents unless the context clearly indicates otherwise.

[0080] Chimeric Antigen Receptor The present invention generally relates to the use of T cells genetically engineered to stably express a desired chimeric antigen receptor. Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides containing the antigen-binding domain of an antibody (scFv) linked to a T cell signaling domain. The properties of CARs include their ability to redirect T cell specificity and reactivity toward non-MHC-restricted selected targets, utilizing the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition allows CAR-expressing T cells to recognize antigens independently of antigen processing, thereby bypassing a major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.

[0081] The CARs described herein provide recombinant polypeptide constructs comprising at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain (also referred to herein as a "cytoplasmic signaling domain") comprising a functional signaling domain derived from a stimulatory molecule as defined below. T cells expressing a CAR are referred to herein as CAR T cells, CAR-T cells, or CAR-modified T cells, and these terms are used interchangeably herein. The cells can be genetically engineered to stably express an antibody binding domain on their surface, conferring novel MHC-independent antigen specificity.

[0082] In some cases, T cells are genetically engineered to stably express a CAR in which the antigen recognition domain of a particular antibody is combined with the intracellular domain of the CD3-zeta chain or the FcγRI protein into a single chimeric protein. In one embodiment, the stimulatory molecule is the zeta chain associated with the T cell receptor complex.

[0083] An "intracellular signaling domain" or "cytoplasmic signaling domain," as used herein, refers to the intracellular portion of a molecule. This is a functional portion of a protein that acts by transmitting signals intracellularly to control cellular activity through defined signaling pathways by generating secondary messengers, or by responding to such messengers to function as an effector. The intracellular signaling domain generates signals that promote immune effector functions of the CAR-containing cells (e.g., CAR-T cells). Examples of immune effector functions, for example, in CAR-T cells, include cytolytic activity and helper activity (including cytokine secretion).

[0084] In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Examples of primary intracellular signaling domains include those derived from molecules involved in primary stimulation or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may comprise a costimulatory intracellular domain. Examples of costimulatory intracellular signaling domains include those derived from molecules involved in costimulatory signals or antigen-independent stimulation. For example, in the case of CAR-T, the primary intracellular signaling domain may comprise the cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain may comprise the cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0085] The primary intracellular signaling domain may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3-zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, DAP10, and DAP12.

[0086] The terms "zeta" or alternatively "zeta chain," "CD3-zeta," or "TCR-zeta" are defined as the protein provided as GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species, e.g., murine, rabbit, primate, mouse, rodent, monkey, ape, etc., and a "zeta stimulatory domain" or alternatively "CD3-zeta stimulatory domain" or "TCR-zeta stimulatory domain" is defined as amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit the initial signal required for T cell activation. Sometimes, for example, in aspects and / or embodiments of the present disclosure, a "zeta stimulatory domain" or a "CD3-zeta stimulatory domain" is also referred to as a "primary signaling domain." In one aspect, the cytoplasmic domain of the CD3 zeta stimulatory domain comprises residues 52-164 of GenBank Accession No. BAG36664.1, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., that is a functional ortholog thereof. In a preferred embodiment, the intracellular signaling domain comprises a CD3 zeta stimulatory domain. In one aspect, the "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO:28, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:28.

[0087] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response (such as, but not limited to, proliferation) by the T cell. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137).

[0088] The costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, MyD88, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds to CD83.

[0089] The intracellular signaling domain can comprise the entire intracellular portion of the molecule from which it is derived, or the entire naturally occurring intracellular signaling domain, or a functional fragment thereof.

[0090] The term "4-1BB" or alternatively "CD137" refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank Accession No. AAA62478.2, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and the "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. Sometimes, for example, in aspects and / or embodiments of the present disclosure, the "4-1BB costimulatory domain" is also referred to as the "costimulatory domain."

[0091] In a preferred embodiment, the intracellular signaling domain comprises a costimulatory intracellular signaling domain that is a 4-1BB costimulatory domain or a "CD137 costimulatory domain." In one aspect, the "4-1BB costimulatory domain" or "CD137 costimulatory domain" is the sequence provided as SEQ ID NO:27, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:27. In a preferred embodiment, the 4-1BB costimulatory domain is the sequence provided as SEQ ID NO:27.

[0092] In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used. In another embodiment, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or a different surface membrane protein, minimizing interaction with other members of the receptor complex. In one example embodiment, the transmembrane domain comprises a CD8α hinge domain. In one embodiment, the CAR comprises a CD8α hinge domain and a CD8α transmembrane domain.

[0093] In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined herein. In one embodiment, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, CD3-zeta, and / or CD28. CD28 is a T cell marker important in T cell costimulation. CD27 is a member of the tumor necrosis factor receptor superfamily and functions as a costimulatory immune checkpoint molecule. 4-1BB delivers potent costimulatory signals to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes. CD3-zeta associates with TCRs to generate signals and contains immunoreceptor tyrosine-based activation motifs (ITAMs). In another embodiment, the costimulatory molecule is MyD88 or CD40.

[0094] In one embodiment, the CAR comprises an intracellular hinge domain comprising CD8 and an intracellular T cell receptor signaling domain comprising CD28, 4-1BB, and CD3-zeta. In another embodiment, the CAR comprises an intracellular hinge domain comprising all or a portion of the extracellular region of CD8, CD4, or CD28, all or a portion of an antibody constant region, an FcRIIIA receptor, an IgG hinge, an IgM hinge, an IgA hinge, an IgD hinge, an IgE hinge, or an Ig hinge. The IgG hinge may be derived from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or chimeras thereof.

[0095] The CARs described herein provide recombinant polypeptide constructs that include at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain (also referred to herein as a "cytoplasmic signaling domain") that includes a functional signaling domain derived from, for example, a stimulatory molecule as defined below.

[0096] In one embodiment, a CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one embodiment, a CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one embodiment, a CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule.

[0097] The CARs of the present invention may be designed to contain only the CD28 and / or 4-1BB signaling domains, or to be combined with any other desired cytoplasmic domain useful in the context of the CARs of the present invention. In one embodiment, the cytoplasmic domain of the CAR may further contain the signaling domain of CD3-zeta. For example, the cytoplasmic domain of the CAR may include, but is not limited to, CD3-zeta, 4-1BB, and CD28 signaling modules, and combinations thereof.

[0098] In one embodiment, the CAR comprises a hinge domain comprising a CD8α hinge domain, a CD8α transmembrane domain, and an intracellular signaling domain comprising 4-1BB and CD3-zeta. In one embodiment, the CAR comprises a hinge domain comprising a CD8α hinge domain, a CD8α transmembrane domain, and an intracellular signaling domain comprising CD28, 4-1BB, and CD3-zeta.

[0099] In another embodiment, the CAR comprises a CD8α hinge domain, a CD8α transmembrane domain, and an intracellular signaling domain comprising the signaling domain of CD3-zeta and a 4-1BB costimulatory domain.

[0100] In another embodiment, the CAR comprises a CD8a-hinge region comprising the amino acid sequence of SEQ ID NO: 25, a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 26, an intracellular signaling domain comprising a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 27, and a primary signaling domain comprising the amino acid sequence of SEQ ID NO: 28.

[0101] Thus, the present invention provides CAR T cells and methods of using them for adoptive therapy.

[0102] The present disclosure further provides variants (e.g., functional variants) of the CARs, nucleic acids, polypeptides, and proteins described herein. A "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications, such as, for example, a substitution, insertion, or deletion. As used herein, the term "functional variant" refers to a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity with a parent CAR, polypeptide, or protein, and these functional variants retain the biological activity of the CAR, polypeptide, or protein from which they are variant. Functional variants include, for example, variants of a CAR, polypeptide, or protein described herein (parent CAR, polypeptide, or protein) that retain the ability to recognize target cells to a similar extent, the same extent, or a greater extent than the parent CAR, polypeptide, or protein. With respect to a parent CAR, polypeptide, or protein, a functional variant can be, for example, at least about 30%, about 40%, about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the parent CAR, polypeptide, or protein.

[0103] f) As used herein, the structure of a polypeptide is appropriately defined based on the % sequence identity with a listed reference sequence (with a given SEQ ID NO). In this context, the % sequence identity between two amino acid sequences can be determined by comparing the two sequences aligned in an optimal manner, and the compared amino acid sequence may contain additions or deletions relative to the reference sequence for optimal alignment between the two sequences. The percentage of identity is calculated by determining the number of identical positions where the amino acid residues between the two sequences are identical, dividing this number of identical positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of identity between the two sequences. Typically, the comparison window corresponds to the entire length of the sequences being compared. For example, the BLAST program "BLAST 2 Sequences" (Tatusova et al., "Blast 2 Sequences - A New Tool for Comparing Protein and Nucleotide Sequences," FEMS Microbiol Lett. 174:247-250) can be used, available at the website http: / / www.ncbi.nlm.nih.gov / gorf / bl2.html, using the default parameters (specifically, the parameters "Open Gap Penalty": 5 and "Extension Gap Penalty": 2; the selected matrix is, for example, the matrix "BLOSUM 62" proposed by the program), and the percentage of identity between the two sequences being compared is calculated directly by the program. Determining the sequence identity of a query sequence to a reference sequence is within the capabilities of those skilled in the art and can be performed using commercially available analysis software such as BLAST (trademark).

[0104] A functional variant can, for example, comprise the amino acid sequence of a parent CAR, polypeptide, or protein with at least one conservative amino acid substitution. In another embodiment, a functional variant can comprise the amino acid sequence of a parent CAR, polypeptide, or protein with at least one non-conservative amino acid substitution. In this case, the non-conservative amino acid substitution cannot suppress or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can improve the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent CAR, polypeptide, or protein.

[0105] The amino acid substitutions in the CAR of the present invention may be conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions that replace one amino acid with a specific physical and / or chemical property with another amino acid with the same or similar chemical or physical property. For example, conservative amino acid substitutions may be an acidic amino acid (e.g., Asp or Glu) substituted with another acidic amino acid, an amino acid having a non-polar side chain substituted with another amino acid having a non-polar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid (Lys, Arg, etc.) substituted with another basic amino acid, an amino acid having a polar side chain substituted with another amino acid having a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

[0106] A CAR, polypeptide, or protein can consist essentially of a particular amino acid sequence or sequences described herein, such that other components, e.g., other amino acids, do not substantially alter the biological activity of the functional variant.

[0107] The CARs, polypeptides, and proteins (including functional portions and functional variants) of the presently disclosed embodiments can be of any length, i.e., can contain any number of amino acids, provided that the CAR, polypeptide, or protein (or functional portion or functional variant thereof) retains their biological activity, such as the ability to specifically bind to an antigen, detect diseased cells (e.g., cancer cells) within a host, or treat or prevent disease within a host. For example, a polypeptide can be about 50 to about 5,000 amino acids in length, e.g., about 50, about 70, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1,000 or more amino acids in length. Polypeptides of the present invention also include oligopeptides.

[0108] The CARs, polypeptides, and proteins of the present embodiments (including functional portions and functional variants of the present invention) can include synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β- Hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, and α-tert-butylglycine.

[0109] The CARs, polypeptides, and proteins (including functional portions and functional variants) of the present embodiments can be post-translationally modified. They can be glycosylated, esterified, N-acylated, amidated, carboxylated, phosphorylated, esterified, cyclized, for example, via disulfide bridges, or converted into acid addition salts. In some embodiments, they are dimerized or polymerized, or conjugated.

[0110] The CARs, polypeptides, and / or proteins (including functional portions and functional variants thereof) of the present embodiments can be obtained by methods known in the art. Suitable methods for de novo synthesis of polypeptides and proteins are described in references such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; and Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001. Polypeptides and proteins can also be produced recombinantly using the nucleic acids described herein using standard recombinant methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, portions of the CARs, polypeptides, and proteins (including functional portions and functional variants thereof) of the present invention can be isolated and / or purified from sources such as plants, bacteria, insects, mammals, etc. Methods of isolation and purification are known in the art. Alternatively, the CARs, polypeptides, and / or proteins (including functional portions and functional variants thereof) described herein can be commercially synthesized. In this regard, the CARs, polypeptides, and proteins can be synthesized, recombinant, isolated, and / or purified.

[0111] Examples of modified nucleotides that can be used to generate recombinant nucleic acids utilized to produce the polypeptides described herein include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, N-(2-aminomethyl-4-methyl-2-oxo-2 ... 6 -substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5"-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -Isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, beta-D-galactosylqueosine, inosine, N 6 -isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

[0112] The nucleic acid can comprise any isolated or purified nucleotide sequence that encodes a CAR, a polypeptide or protein, or a functional portion or functional variant thereof. Alternatively, the nucleotide sequence can comprise a nucleotide sequence that is degenerate to any of the sequences, or a combination of degenerate sequences.

[0113] Some embodiments of the present invention also provide isolated or purified nucleic acids, which comprise a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein, or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0114] Nucleotide sequences that hybridize under stringent conditions may also hybridize under high stringency conditions. "High stringency conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of a nucleic acid described herein) in a detectably stronger manner than nonspecific hybridization. High stringency conditions include conditions that distinguish polynucleotides with exactly complementary sequences or polynucleotides containing only scattered mismatches from random sequences that end up with several small regions (e.g., 3-12 bases) of matched nucleotide sequences. Such small regions of complementarity are more easily melted than full-length complements of 14-17 or more bases, making them readily distinguishable by high stringency hybridization. Relatively high stringency conditions include, for example, low salt and / or high temperature conditions (such as those provided by about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70°C). Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the CARs described herein, It is generally understood that conditions can be made more stringent by the addition of increasing amounts of formamide.

[0115] In one embodiment, the nucleic acids of the present invention can be incorporated into a recombinant expression vector. The present disclosure provides recombinant expression vectors comprising any of the nucleic acids of the present invention. As used herein, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that enables expression of an mRNA, protein, polypeptide, or peptide by a host cell, where the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the cell. The vectors described herein are not naturally occurring in their entirety. However, portions of the vectors may be naturally occurring. The described recombinant expression vectors may contain any type of nucleotide, including, but not limited to, DNA and RNA (which may be single-stranded or double-stranded, synthetic, or derived in part from natural sources, and may contain natural, non-natural, or modified nucleotides). The recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Non-naturally occurring or altered nucleotides or internucleotide linkages do not inhibit the transcription or replication of the vector.

[0116] In one embodiment, the recombinant expression vector of the present invention can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include vectors designed for propagation and propagation, or for expression, or both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λEMBL4, λNM1149, and λZapII (Stratagene) can also be used. Examples of plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, e.g., a retroviral vector, e.g., a gamma retroviral vector.

[0117] In one embodiment, the recombinant expression vectors of the invention are prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al. (supra) and Ausubel et al. (supra). Expression vector constructs, whether circular or linear, can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColE1, SV40, 2μ plasmid, λ, bovine papilloma virus, etc.

[0118] Recombinant expression vectors may, where appropriate, include regulatory sequences, such as transcription and translation initiation and termination codons, specific to the type of host (e.g., bacteria, plants, fungi, or animals) into which the vector will be introduced, taking into account whether the vector is DNA- or RNA-based.

[0119] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.), complementation to provide prototrophy in auxotrophic hosts, etc. Suitable marker genes for the described expression vectors include, for example, neomycin / G418 resistance gene, histidinol x resistance gene, histidinol resistance gene, tetracycline resistance gene, and ampicillin resistance gene.

[0120] The recombinant expression vector can include a native or canonical promoter operably linked to a nucleotide sequence encoding a CAR, polypeptide, or protein (including functional portions and functional variants thereof), or to a nucleotide sequence that is complementary to or hybridizes to the nucleotide sequence encoding the CAR, polypeptide, or protein. The selection of a promoter (e.g., strong, weak, tissue-specific, inducible, and developmentally specific) is within the skill of one in the art. Similarly, combining a nucleotide sequence with a promoter is also within the skill of one in the art. The promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, a RSV promoter, an SV40 promoter, or a promoter found in the long terminal repeat of murine stem cell virus.

[0121] Recombinant expression vectors can be designed for either transient expression, stable expression, or both, and can be made for constitutive or inducible expression.

[0122] Additionally, recombinant expression vectors can be engineered to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes cells expressing the suicide gene to die. A suicide gene can be a gene that confers sensitivity to an agent (e.g., a drug) on ​​a cell expressing the gene, causing the cell to die when contacted with or exposed to the agent. Suicide genes are known in the art and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0123] Conjugates, e.g., bioconjugates, comprising a CAR, polypeptide, or protein (including any functional portion or variant thereof), host cell, nucleic acid, recombinant expression vector, host cell population, or antibody, or any antigen-binding portion thereof, are included within the scope of the present invention. Conjugates, as well as methods for synthesizing conjugates, are generally known in the art (see, e.g., Hudecz, F., Methods Mol. Biol. 298:209-223 (2005) and Kirin et al., Inorg Chem. 44(15):5405-5415 (2005)).

[0124] One embodiment of the present invention further provides an antibody, or antigen-binding portion thereof, that binds, e.g., specifically binds, to an epitope of a CAR of the invention.

[0125] The antibody may be any type of immunoglobulin known in the art. Immunoglobulins may be assigned to five major classes: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subdivided into isotypes: IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Antibody light chains of vertebrate species may be assigned to one of two types, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain. Antibodies may be of any class or isotype.

[0126] Antibodies include immunoglobulin molecules, including monoclonal antibodies, including murine, human, humanized, and chimeric monoclonal antibodies, polyclonal, antigen-binding fragments, bispecific or multispecific antibodies, monomeric, dimeric, tetrameric, or multimeric antibodies, single-chain antibodies, domain antibodies, and any other modified configuration of immunoglobulin molecule that contains an antigen-binding site of the required specificity. The antibody may be a naturally occurring antibody, e.g., an antibody isolated and / or purified from a mammal (e.g., murine, primate, mouse, rabbit, goat, horse, chicken, hamster, human, etc.). Alternatively, the antibody may be a genetically engineered (e.g., genetically engineered) antibody.

[0127] A humanized antibody has an antigen-binding site derived from a non-human species and the variable region framework is derived from a human immunoglobulin sequence. A human antibody has heavy and light chain variable regions in which both the framework and the antigen-binding site are derived from sequences of human origin.

[0128] Additionally, antibodies can have any level of affinity or avidity for the functional portion of a CAR. In some embodiments, antibodies have a range of affinities (K D In one embodiment according to the invention, and in some embodiments of each and all of the numbered embodiments listed below, the antibody can bind to the hK2 antigen with high affinity, e.g., about 10, as determined by surface plasmon resonance or Kinexa methods performed by one skilled in the art. -7 KD less than or equal to M, for example, but not limited to, 1 to 9.9 (or any value therein, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9) x 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15 M or any range or value of K therein D An example of an affinity is 1 x 10-8 Another example of affinity is 1 × 10 -9 It is below M.

[0129] Methods for testing antibodies for the ability to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as a radioimmunoassay (RIA), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and competitive inhibition assays.

[0130] Suitable methods for producing antibodies are known in the art, e.g., standard hybridoma techniques are described in, e.g., Koehler and Milstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001). Alternatively, other methods are known in the art, such as the EBV-hybridoma method (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), and Roder et al., Methods Enzymol., 121, 140-67 (1986)) and bacteriophage vector expression systems (see, e.g., Huse et al., Science, 246, 1275-81 (1989)). Additionally, methods for producing antibodies in non-human animals are described, for example, in U.S. Patent Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. Patent Application Publication No. 2002 / 0197266(A1).

[0131] Antibodies can also be generated using phage display. In this regard, phage libraries encoding antibody antigen-binding variable (V) domains can be generated using standard molecular biology and recombinant DNA techniques (see, e.g., Sambrook et al., supra, and Ausubel et al., supra). Phage encoding variable regions with the desired specificity are selected for specific binding to the desired antigen (i.e., hK2), and complete or partial antibodies comprising the selected variable domains are reconstituted. Nucleic acid sequences encoding the reconstituted antibodies are introduced into a suitable cell line, such as myeloma cells used for hybridoma production, so that the cells secrete antibodies with the properties of monoclonal antibodies (see, e.g., Janeway et al., supra, Huse et al., supra, and U.S. Pat. No. 6,265,150).

[0132] Antibodies can be produced by transgenic mice that are transgenic for particular heavy and light chain immunoglobulin genes. Such methods are known in the art and are described, for example, in U.S. Patent Nos. 5,545,806 and 5,569,825, and Janeway et al., supra.

[0133] Methods for producing humanized antibodies are known in the art and are described, for example, in Janeway et al., supra, U.S. Patent Nos. 5,225,539, 5,585,089, and 5,693,761, European Patent No. 0239400(B1), and British Patent No. 2188638. Humanized antibodies can also be produced using antibody resurfacing techniques described in U.S. Patent No. 5,639,641 and Pedersen et al., J. Mol. Biol., 235, 959-973 (1994).

[0134] Antibodies, as used herein, may be multi- or single-chain or intact immunoglobulins and may be derived from natural or recombinant sources. An antibody may be a tetramer of immunoglobulin molecules.

[0135] The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof that retains the antigen-binding properties of the parent full-length antibody. It refers, for example, to an antigen-binding domain (e.g., the antigen-determining variable region of an intact antibody) that is sufficient to confer recognition and binding (e.g., specific binding of the antibody fragment to a target such as an antigen). An "antigen-binding fragment" refers to a portion of an immunoglobulin molecule. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), and Fv fragments, single-chain antibodies (scFv), linear antibodies, single-domain antibodies (either VL or VH) such as sdAbs, camelid VHH domains, and multispecific antibodies formed from antibody fragments.

[0136] The term "scFv" refers to a protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region. In some embodiments, the light chain variable region and the heavy chain variable region are contiguously linked via a short, flexible polypeptide linker and can be expressed as a single-chain polypeptide, with the scFv retaining the specificity of the intact antibody from which it was derived. Unless specified, as used herein, an scFv may have the VL and VH variable regions in either order; for example, with respect to the N- and C-terminal ends of the polypeptide, an scFv may comprise VL-linker-VH or VH-linker-VL.

[0137] One embodiment of the present invention also provides an antigen-binding portion of any of the antibodies described herein, which can be any moiety having at least one antigen-binding site, such as a Fab, F(ab')2, dsFv, sFv, diabodies, and triabodies.

[0138] In some embodiments, the antigen-binding fragment is a domain antibody (dAb) comprising (e.g., consisting of) one, two, and / or three heavy chain complementarity determining regions (HCDRs), one, two, and / or three light chain complementarity determining regions (LCDRs), a heavy chain variable region (VH), or a light chain variable region (VL), Fab, F(ab'), Fd, and Fv fragments, and either one VH domain or one VL domain. The VH and VL domains may be linked together via a linker, e.g., a synthetic linker.

[0139] "Complementarity determining regions (CDRs)" are antigen-binding sites in antibodies. CDRs can be defined using various terms: (i) The complementarity determining regions (CDRs), of which there are three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3), are based on sequence diversity (Wu and Kabat, J Exp Med 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) "Hypervariable regions," "HVRs," or "HVs," three in VH (H1, H2, H3) and three in VL (L1, L2, L3), refer to regions of antibody variable domains that are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk Mol Biol 196:901-17, 1987). The International ImMunoGeneTics (IMGT) database (http: / / www_imgt_org) provides standard numbering and definitions for antigen-binding sites. The correspondence between CDRs, HVs, and IMGT profiles is provided in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003. As used herein, the terms "CDR," "HCDR1," "HCDR2," "HCDR3," "LCDR1," "LCDR2," and "LCDR3," unless otherwise specified herein, include CDRs defined by any of the methods described by Kabat, Chothia, or IMGT, supra.

[0140] Antibodies or antigen-binding portions thereof can also be modified to contain detectable labels, such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0141] Nucleic acids comprising a nucleotide sequence encoding any of the CARs, polypeptides, or proteins (including functional portions and functional variants thereof) described herein are provided by the present disclosure.

[0142] The portion of the CAR comprising an antibody or antibody fragment thereof may exist in various forms, where the antigen-binding domain is expressed as part of a continuous polypeptide chain, such as, for example, a single-domain antibody fragment (sdAb), an scFv, and a human chimeric or humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen-binding domain of the CAR composition of the invention comprises an antibody fragment. In one embodiment, the CAR comprises an antibody fragment comprising an scFv.

[0143] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed in a bacteriophage or yeast expression system. The term should be taken to mean an antibody that has been produced by the synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein, or an amino acid sequence that specifies the antibody, where the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence technology that is available and known in the art.

[0144] The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA will encode an "antigen" as that term is used herein by including a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences can be arranged in various combinations to encode polypeptides that elicit a desired immune response.

[0145] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain binds to an hK2 antigen.

[0146] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises: light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 1, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 4; light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:2, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:4; a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 6; a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 4; light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:2, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:6; light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 1, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 6; light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:3, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:5; light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 1, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 5; light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:2, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:5; a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 74, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 76; a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 75, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 77; a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 147, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 140; a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 75, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 141; a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 75, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 140; a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 148, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 142; a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 149, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 143; a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 150, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 144; a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 151, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 145; a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 152, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 146; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 318, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 317 Including, The extracellular antigen-binding domain binds to the hK2 antigen.

[0147] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 1, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0148] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO:2, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO:4.

[0149] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO:3, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO:6.

[0150] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO:3, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO:4.

[0151] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO:2, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO:6.

[0152] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 1, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 6.

[0153] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO:3, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO:5.

[0154] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 1, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 5.

[0155] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO:2, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO:5.

[0156] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 74, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 76.

[0157] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 75, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 77.

[0158] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 147, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 140.

[0159] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 75, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 141.

[0160] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 75, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 140.

[0161] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 148, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 142.

[0162] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 149, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 143.

[0163] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 150, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 144.

[0164] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 151, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 145.

[0165] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 152, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 146.

[0166] In one embodiment, the extracellular antigen-binding domain comprises a light chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 318, and a heavy chain CDR1, CDR2, and CDR3 from a variable region comprising the amino acid sequence of SEQ ID NO: 317.

[0167] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises: The extracellular antigen-binding domain comprises a heavy chain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:63, SEQ ID NO:72, SEQ ID NO:86, SEQ ID NO:92, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:110, SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:123, SEQ ID NO:309, and SEQ ID NO:314, and conservative modifications thereof, and the extracellular antigen-binding domain binds to the hK2 antigen.

[0168] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises: The extracellular antigen-binding domain comprises a heavy chain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:87, SEQ ID NO:93, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:124, SEQ ID NO:310, and SEQ ID NO:315, and conservative modifications thereof, and the extracellular antigen-binding domain binds to the hK2 antigen.

[0169] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises: The extracellular antigen-binding domain comprises a heavy chain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:66, SEQ ID NO:88, SEQ ID NO:94, SEQ ID NO:104, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:117, SEQ ID NO:122, and SEQ ID NO:311, and conservative modifications thereof, and the extracellular antigen-binding domain binds to the hK2 antigen.

[0170] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises: a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 64, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66; a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 314, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 315, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311; a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 309, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 310, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 73, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66; or a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 86, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 87, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 88; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 92, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 93, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 94; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 103, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 106, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 107, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 108, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 110, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 111, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 112, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 114, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 115, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 116, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 117; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 118, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 119, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 117; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122; or a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 123, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 124, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122; Including, The extracellular antigen-binding domain binds to the hK2 antigen.

[0171] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 64, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66.

[0172] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 314, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 315, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311.

[0173] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 309, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 310, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311.

[0174] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66.

[0175] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 73, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66.

[0176] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 86, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 87, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 88.

[0177] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 92, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 93, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 94.

[0178] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 103, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104.

[0179] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 106, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104.

[0180] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 107, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 108, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109.

[0181] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 110, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 111, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109.

[0182] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 112, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113.

[0183] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 114, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113.

[0184] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 115, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 116, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 117.

[0185] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 118, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 119, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 117.

[0186] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122.

[0187] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 123, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 124, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122.

[0188] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises: The extracellular antigen-binding domain comprises a light chain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:89, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:125, SEQ ID NO:128, SEQ ID NO:133, SEQ ID NO:136, and SEQ ID NO:312, and conservative modifications thereof, and the extracellular antigen-binding domain binds to the hK2 antigen.

[0189] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises: The extracellular antigen-binding domain comprises a light chain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:90, SEQ ID NO:96, SEQ ID NO:126, SEQ ID NO:137, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:134, SEQ ID NO:137, and SEQ ID NO:313, and conservative modifications thereof, and the extracellular antigen-binding domain binds to the hK2 antigen.

[0190] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises: The extracellular antigen-binding domain comprises a light chain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:91, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, and SEQ ID NO:139, and conservative modifications thereof, and the extracellular antigen-binding domain binds to the hK2 antigen.

[0191] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises: a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 95, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 96, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 312, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 313, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91; a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 128, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 129, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 130; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 125, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 131, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 132; a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 133, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 134, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 135; a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 136, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 137, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 138; or a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 139; Including, The extracellular antigen-binding domain binds to the hK2 antigen.

[0192] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71.

[0193] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71.

[0194] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91.

[0195] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 95, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 96, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71.

[0196] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 312, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 313, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91.

[0197] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91.

[0198] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71.

[0199] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 128, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 129, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 130.

[0200] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 125, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 131, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 132.

[0201] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 133, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 134, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 135.

[0202] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 136, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 137, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 138.

[0203] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 139.

[0204] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises: a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 64, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 314, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 315, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 312, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 313, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 103, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 128, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 129, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 130; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 107, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 108, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 125, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 131, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 132; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 112, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 133, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 134, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 135; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 115, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 116, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 117, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 136, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 137, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 138; or a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 138; Including, The extracellular antigen-binding domain binds to the hK2 antigen.

[0205] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises: a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 64, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 314, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 315, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 312, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 313, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 103, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 128, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 129, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 130; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 107, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 108, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 125, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 131, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 132; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 112, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 133, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 134, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 135; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 115, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 116, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 117, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 136, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 137, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 138; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 138; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 73, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 73, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 73, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 73, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 73, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 309, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 310, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 312, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 313, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 106, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 128, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 129, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 130; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 110, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 111, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 125, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 131, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 132; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 114, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 133, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 134, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 135; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 118, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 119, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 117, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 136, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 137, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 138; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 123, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 124, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 139; a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 139; or a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 86, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 87, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 88, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91; Including, The extracellular antigen-binding domain binds to the hK2 antigen.

[0206] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 64, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71.

[0207] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71.

[0208] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71.

[0209] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71.

[0210] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 65, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71.

[0211] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 314, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 315, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 312, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 313, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91.

[0212] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 103, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 128, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 129, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 130.

[0213] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 107, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 108, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 125, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 131, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 132.

[0214] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 112, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 133, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 134, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 135.

[0215] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 115, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 116, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 117, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 136, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 137, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 138.

[0216] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 138.

[0217] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 73, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71.

[0218] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 73, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71.

[0219] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 73, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71.

[0220] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 73, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71.

[0221] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 73, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71.

[0222] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 309, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 310, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 312, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 313, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91.

[0223] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 106, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 128, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 129, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 130.

[0224] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 110, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 111, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 125, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 131, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 132.

[0225] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 114, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 133, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 134, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 135.

[0226] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 118, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 119, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 117, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 136, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 137, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 138.

[0227] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 123, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 124, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 139.

[0228] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 139.

[0229] In one embodiment, the extracellular antigen binding domain comprises: It comprises a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 86, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 87, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 88, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91.

[0230] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises: The extracellular antigen-binding domain comprises a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3, or a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 6, or a combination of an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 and an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 6, and the extracellular antigen-binding domain binds to the hK2 antigen.

[0231] In one embodiment, the extracellular antigen binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 74 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 76; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 77; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 147 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 140; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 141; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 140; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 148 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 142; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 149 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 143; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 150 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 144; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 151 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 145; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 152 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 146; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 318 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 317; Includes.

[0232] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4.

[0233] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4.

[0234] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6.

[0235] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4.

[0236] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6.

[0237] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6.

[0238] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5.

[0239] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5.

[0240] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5.

[0241] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:74 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:76.

[0242] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 77.

[0243] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 147 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 140.

[0244] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 141.

[0245] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 140.

[0246] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 148 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 142.

[0247] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 149 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 143.

[0248] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 150 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 144.

[0249] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 151 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 145.

[0250] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 152 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 146.

[0251] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 318 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 317.

[0252] In one embodiment, the extracellular antigen binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:1, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:4; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:2, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:4; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:3, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:6; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:3, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:4; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:2, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:6; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:3, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:4; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:1, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:6; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:3, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:5; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:1, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:5; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:2, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:5; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:74, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:76; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:75, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:77; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 147, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 140; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:75, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:141; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:75, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:140; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 148, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 142; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 149, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 143; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 150, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 144; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 151, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 145; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 152, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 146; a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:318, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:317. Includes.

[0253] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:1, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:4.

[0254] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:2, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:4.

[0255] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:3, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:6.

[0256] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:3, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:4.

[0257] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:2, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:6.

[0258] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:3, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:4.

[0259] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:1, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:6.

[0260] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:3, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:5.

[0261] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:1, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:5.

[0262] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:2, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:5.

[0263] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:74, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:76.

[0264] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:75, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:77.

[0265] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 147, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 140.

[0266] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 75, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 141.

[0267] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 75, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 140.

[0268] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 148, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 142.

[0269] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 149, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 143.

[0270] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 150, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 144.

[0271] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 151, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 145.

[0272] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 152, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 146.

[0273] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:318, and a heavy chain variable region comprising an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:317.

[0274] In one embodiment, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the scFv comprises a linker polypeptide between the light chain variable region and the heavy chain variable region. In a specific embodiment, the extracellular antigen-binding domain is an scFv that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-23, 169-184, and 340-343 and specifically binds to an hK2 polypeptide (e.g., a human hK2 polypeptide having the amino acid sequence of SEQ ID NO: 62 or a fragment thereof).

[0275] In recombinant expression systems, the linker is a peptide linker and can contain any naturally occurring amino acid. Exemplary amino acids that can be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker must be of an appropriate length to link VH and VL in a manner that allows them to form the correct conformation relative to each other so as to maintain the desired activity, such as binding to hK2.

[0276] The linker can be about 5 to 50 amino acids in length. In some embodiments, the linker is about 10 to 40 amino acids in length. In some embodiments, the linker is about 10 to 35 amino acids in length. In some embodiments, the linker is about 10 to 30 amino acids in length. In some embodiments, the linker is about 10 to 25 amino acids in length. In some embodiments, the linker is about 10 to 20 amino acids in length. In some embodiments, the linker is about 15 to 20 amino acids in length. In some embodiments, the linker is 6 amino acids in length. In some embodiments, the linker is 7 amino acids in length. In some embodiments, the linker is 8 amino acids in length. In some embodiments, the linker is 9 amino acids in length. In some embodiments, the linker is 10 amino acids in length. In some embodiments, the linker is 11 amino acids in length. In some embodiments, the linker is 12 amino acids in length. In some embodiments, the linker is 13 amino acids in length. In some embodiments, the linker is 14 amino acids in length. In some embodiments, the linker is 15 amino acids in length. In some embodiments, the linker is 16 amino acids in length. In some embodiments, the linker is 17 amino acids in length. In some embodiments, the linker is 18 amino acids in length. In some embodiments, the linker is 19 amino acids in length. In some embodiments, the linker is 20 amino acids in length. In some embodiments, the linker is 21 amino acids in length. In some embodiments, the linker is 22 amino acids in length. In some embodiments, the linker is 23 amino acids in length. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker is 25 amino acids in length. In some embodiments, the linker is 26 amino acids in length. In some embodiments, the linker is 27 amino acids in length. In some embodiments, the linker is 28 amino acids in length. In some embodiments, the linker is 29 amino acids in length. In some embodiments, the linker is 30 amino acids in length.In some embodiments, the linker is 31 amino acids in length. In some embodiments, the linker is 32 amino acids in length. In some embodiments, the linker is 33 amino acids in length. In some embodiments, the linker is 34 amino acids in length. In some embodiments, the linker is 35 amino acids in length. In some embodiments, the linker is 36 amino acids in length. In some embodiments, the linker is 37 amino acids in length. In some embodiments, the linker is 38 amino acids in length. In some embodiments, the linker is 39 amino acids in length. In some embodiments, the linker is 40 amino acids in length. Exemplary linkers that can be used are GIy-rich linkers, GIy- and Ser-containing linkers, GIy- and Ala-containing linkers, Ala- and Ser-containing linkers, and other flexible linkers.

[0277] In one embodiment, the linker polypeptide comprises the amino acid sequence of SEQ ID NO: 7. In one embodiment, the linker polypeptide comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 7. In one embodiment, the linker polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 237-268. In one embodiment, the linker polypeptide comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to any one of SEQ ID NOs: 237-268.

[0278] In one embodiment, the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-23, 169-184, and 340-343. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 8, or the sequence of SEQ ID NO: 8. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 9, or the sequence of SEQ ID NO: 9. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 10, or the sequence of SEQ ID NO: 10. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 11, or the sequence of SEQ ID NO: 11.In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 12, or the sequence of SEQ ID NO: 12. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 13, or the sequence of SEQ ID NO: 13. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 14, or the sequence of SEQ ID NO: 14. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 15, or the sequence of SEQ ID NO: 15.In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 16, or the sequence of SEQ ID NO: 16. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 17, or the sequence of SEQ ID NO: 17. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 18, or the sequence of SEQ ID NO: 18. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 19, or the sequence of SEQ ID NO: 19.In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 20, or the sequence of SEQ ID NO: 20. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 21, or the sequence of SEQ ID NO: 21. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 22, or the sequence of SEQ ID NO: 22. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 23, or the sequence of SEQ ID NO: 23.In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 169, or the sequence of SEQ ID NO: 169. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 170, or the sequence of SEQ ID NO: 170. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 171, or the sequence of SEQ ID NO: 171. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 172, or the sequence of SEQ ID NO: 172.In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 173, or the sequence of SEQ ID NO: 173. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 174, or the sequence of SEQ ID NO: 174. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 175, or the sequence of SEQ ID NO: 175. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 176, or the sequence of SEQ ID NO: 176.In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 177, or the sequence of SEQ ID NO: 177. In one embodiment, the scFv comprises SEQ ID NO: 1. 78, or the sequence of SEQ ID NO: 178. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 179, or the sequence of SEQ ID NO: 179. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 180, or the sequence of SEQ ID NO: 180. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 181, or the sequence of SEQ ID NO: 181. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 182, or the sequence of SEQ ID NO: 182.In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 183, or the sequence of SEQ ID NO: 183. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 184, or the sequence of SEQ ID NO: 184. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 340, or the sequence of SEQ ID NO: 340. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 341, or the sequence of SEQ ID NO: 341.In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 342, or the sequence of SEQ ID NO: 342. In one embodiment, the scFv comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 343, or the sequence of SEQ ID NO: 343.

[0279] In one embodiment, the extracellular antigen-binding domain comprises a signal polypeptide. The signal polypeptide may be located at the N-terminus of the extracellular antigen-binding domain that binds to hK2. The signal polypeptide may optionally be cleaved from the extracellular antigen-binding domain during cellular processing and localization of the CAR to the cell membrane. Any of a variety of signal polypeptides known to those skilled in the art may be used as the signal polypeptide. Non-limiting examples of peptides from which the signal polypeptide may be derived include FcεR, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8α, or any of a variety of other proteins secreted by T cells. In various embodiments, the signal polypeptide is compatible with the secretory pathway of T cells. In some embodiments, the signal polypeptide comprises the amino acid sequence of SEQ ID NO: 24. In one embodiment, the signal polypeptide comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:24.

[0280] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-44, 78-81, 221-236, and 273-308. Another feature of a CAR having an extracellular antigen-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-44, 78-81, 221-236, and 273-308 is that the extracellular antigen-binding domain binds to the hK2 antigen.

[0281] In one embodiment, the intracellular signaling domain comprises a polypeptide component selected from the group consisting of a TNF receptor superfamily member 9 (CD137) component, a T cell surface glycoprotein CD3 zeta chain (CD3z) component, a cluster of differentiation (CD27) component, a cluster of differentiation superfamily member (such as, for example, CD28 or inducible T cell costimulator (ICOS)) component, and combinations thereof.

[0282] In one embodiment, the CD137 component comprises the amino acid sequence of SEQ ID NO: 27. In one embodiment, the CD137 component comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:27.

[0283] In one embodiment, the CD3z component comprises the amino acid sequence of SEQ ID NO: 28. In one embodiment, the CD3z component comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:28.

[0284] In one embodiment, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 45. In one embodiment, the intracellular signaling domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:45.

[0285] In one embodiment, the transmembrane domain comprises a CD8a transmembrane region (CD8A-TM) polypeptide. In some embodiments, the CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the CD8a-TM polypeptide comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 26.

[0286] In one embodiment, the transmembrane domain comprises at least the transmembrane region of the α, β, or ζ chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154. In another embodiment, the transmembrane domain comprises at least the transmembrane domain of ζ, η, or FcεR1γ and -β, MB1 (Igα), B29, or CD3-γ, ζ, or η. In another embodiment, the transmembrane domain is synthetic and comprises primarily hydrophobic residues, such as, for example, a triplet of leucine and valine, phenylalanine, or tryptophan.

[0287] In one embodiment, the CAR further comprises a hinge region linking the transmembrane domain to the extracellular antigen-binding domain. In some embodiments, the hinge region is a CD8a hinge region. In some embodiments, the CD8a-hinge region comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CD8a-hinge region comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 25. In some embodiments, the hinge region comprises the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 269) or an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to EPKSCDKTHTCPPCP (SEQ ID NO: 269). In some embodiments, the hinge region comprises the sequence ERKCCVECPPCP (SEQ ID NO: 270) or an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to ERKCCVECPPCP (SEQ ID NO: 270).In some embodiments, the hinge region comprises the sequence ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO: 271) or an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO: 271). In some embodiments, the hinge region comprises the sequence ESKYGPPCPSCP (SEQ ID NO:272) or an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to ESKYGPPCPSCP (SEQ ID NO:272).

[0288] g) In one embodiment, the CAR comprises an extracellular antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain. In one such embodiment, the hinge region is a CD8α hinge region, the transmembrane domain is a CD8α-TM domain, and the intracellular signaling domain comprises a CD3-zeta domain and a 4-1BB / CD137 domain.

[0289] h) In one such embodiment, the hinge region is a CD8α hinge region comprising the amino acid sequence of SEQ ID NO: 25, the transmembrane domain is a CD8α-TM domain comprising the amino acid sequence of SEQ ID NO: 26, and the intracellular signaling domain comprises a CD3-zeta domain and a 4-1BB / CD137 domain comprising the amino acid sequence of SEQ ID NO: 45.

[0290] i) In another embodiment, the hinge region is a CD8α hinge region comprising the amino acid sequence of SEQ ID NO: 25, the transmembrane domain is a CD8α-TM domain comprising the amino acid sequence of SEQ ID NO: 26, and the intracellular signaling domain comprises a 4-1BB / CD137 domain comprising the amino acid sequence of SEQ ID NO: 27 and a CD3-zeta domain comprising the amino acid sequence of SEQ ID NO: 28.

[0291] In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-44, 78-81, 221-236, and 273-308. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 29, or the sequence of SEQ ID NO: 29. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 30, or the sequence of SEQ ID NO: 30. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 31, or the sequence of SEQ ID NO: 31. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 32, or the sequence of SEQ ID NO: 32.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 33, or the sequence of SEQ ID NO: 33. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 34, or the sequence of SEQ ID NO: 34. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 35, or the sequence of SEQ ID NO: 35. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 36, or the sequence of SEQ ID NO: 36.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 37, or the sequence of SEQ ID NO: 37. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 38, or the sequence of SEQ ID NO: 38. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 39, or the sequence of SEQ ID NO: 39. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 40, or the sequence of SEQ ID NO: 40.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 41, or the sequence of SEQ ID NO: 41. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 42, or the sequence of SEQ ID NO: 42. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 43, or the sequence of SEQ ID NO: 43. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 44, or the sequence of SEQ ID NO: 44.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 78, or the sequence of SEQ ID NO: 78. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 79, or the sequence of SEQ ID NO: 79. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 80, or the sequence of SEQ ID NO: 80. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 81, or the sequence of SEQ ID NO:81.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 221, or the sequence of SEQ ID NO: 221. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 222, or the sequence of SEQ ID NO: 222. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 223, or the sequence of SEQ ID NO: 223. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 224, or the sequence of SEQ ID NO: 224. In one embodiment, the extracellular antigen-binding domain comprises at least 50, at least 55, to SEQ ID NO: 225. , at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 226, or the sequence of SEQ ID NO: 225. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 226, or the sequence of SEQ ID NO: 226. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 227, or the sequence of SEQ ID NO: 227. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 228, or the sequence of SEQ ID NO: 228.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 229, or the sequence of SEQ ID NO: 229. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 230, or the sequence of SEQ ID NO: 230. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 231, or the sequence of SEQ ID NO: 231. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 232, or the sequence of SEQ ID NO: 232.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 233, or the sequence of SEQ ID NO: 233. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 234, or the sequence of SEQ ID NO: 234. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 235, or the sequence of SEQ ID NO: 235. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 236, or the sequence of SEQ ID NO: 236.

[0292] CAR constructs and CAR-expressing immunoresponsive cells The term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, in a cell or other biological system, a gene, cDNA, or RNA encodes a protein when transcription and translation of mRNA corresponding to that gene produces the protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0293] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes nucleotide sequences that are degenerate versions of each other and all nucleotide sequences that encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" can also include introns to the extent that in some cases the nucleotide sequence encoding the protein may contain introns.

[0294] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0295] In one embodiment, the disclosure provides a cell that expresses a nucleic acid molecule encoding a CAR. In one embodiment, a CAR of the disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-61, 82-85, and 205-220. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 46, or the sequence of SEQ ID NO: 46. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 47, or the sequence of SEQ ID NO: 47. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 48, or the sequence of SEQ ID NO: 48. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO:49, or the sequence of SEQ ID NO:49.In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 50, or the sequence of SEQ ID NO: 50. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 51, or the sequence of SEQ ID NO: 51. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 52, or the sequence of SEQ ID NO: 52. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 53, or the sequence of SEQ ID NO: 53.In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 54, or the sequence of SEQ ID NO: 54. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 55, or the sequence of SEQ ID NO: 55. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 56, or the sequence of SEQ ID NO: 56. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 57, or the sequence of SEQ ID NO: 57.In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 58, or the sequence of SEQ ID NO: 58. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 59, or the sequence of SEQ ID NO: 59. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 60, or the sequence of SEQ ID NO: 60. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 61, or the sequence of SEQ ID NO: 61.In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 82, or the sequence of SEQ ID NO: 82. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 83, or the sequence of SEQ ID NO: 83. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 84, or the sequence of SEQ ID NO: 84. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 85, or the sequence of SEQ ID NO: 85.In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 205, or the sequence of SEQ ID NO: 205. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 206, or the sequence of SEQ ID NO: 206. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 207, or the sequence of SEQ ID NO: 207. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 208, or the sequence of SEQ ID NO: 208. In some embodiments, the present disclosure The illustrated CAR comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 209, or the sequence of SEQ ID NO: 209. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 210, or the sequence of SEQ ID NO: 210. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 211, or the sequence of SEQ ID NO: 211. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 212, or the sequence of SEQ ID NO: 212.In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 213, or the sequence of SEQ ID NO: 213. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 214, or the sequence of SEQ ID NO: 214. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 215, or the sequence of SEQ ID NO: 215. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 216, or the sequence of SEQ ID NO: 216.In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 217, or the sequence of SEQ ID NO: 217. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 218, or the sequence of SEQ ID NO: 218. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 219, or the sequence of SEQ ID NO: 219. In some embodiments, a CAR of the disclosure comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 220, or the sequence of SEQ ID NO: 220.

[0296] In certain embodiments, the CAR may comprise a signal polypeptide. In certain embodiments, the signal peptide is at the N-terminus of the CAR sequence. In certain embodiments, the signal peptide is at the C-terminus of the CAR sequence. In certain embodiments, the signal peptide comprises or consists of the amino acid sequence of SEQ ID NO: 24.

[0297] Herein, the structure of a nucleic acid molecule is appropriately defined based on its % sequence identity with a listed reference sequence (with a given SEQ ID NO). In this context, % sequence identity with respect to a nucleic acid molecule refers to the similarity between at least two different nucleic acid sequences. If both positions of two compared sequences are occupied by the same base, for example, if each position of two DNA molecules is occupied by adenine, the molecules are identical at that position, and the percentage of identity between two sequences is a function of the number of matches or homologies at positions shared by the two sequences, divided by the number of compared positions, multiplied by 100. For example, if 6 out of 10 positions of two sequences are matched or homologous, the two sequences are 60% identical (or homologous). As an example, the DNA sequences ATTGCC and TATGGC share 50% identity (or homology). Generally, comparisons are performed when the two sequences are aligned to maximize homology. The percent identity of each can be determined by standard alignment algorithms, such as the Basic Local Alignment Search Tool (BLAST) described by Altshul et al. ((1990) J. MoI. Biol. 215:403-10); the algorithm of Needleman et al. ((1970) J. MoI. Biol. 48:444-53); or the algorithm of Meyers et al. ((1988) Comput. Appl. Biosci. 4:11-17). The parameter set can be a Blosum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller ((1989) CABIOS 4:11-17) incorporated into the ALIGN program (version 2.0), with a gap length penalty of 12 and a gap penalty of 4 using a PAM120 weighted residue table. The percent identity is usually calculated by comparing sequences of similar length.

[0298] In various embodiments, the nucleic acid molecule encoding the CAR comprises the nucleic acid sequence of any one of SEQ ID NOs: 153-160. In various embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence comprising a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to any one of SEQ ID NOs: 153-160.

[0299] In various embodiments, the nucleic acid molecule encoding the CAR comprises the nucleic acid sequence of any one of SEQ ID NOs: 161-168. In various embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence comprising a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to any one of SEQ ID NOs: 161-168.

[0300] In various embodiments, the nucleic acid molecule encoding the CAR comprises the nucleic acid sequence of any one of SEQ ID NOs: 195-204. In various embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence comprising a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to any one of SEQ ID NOs: 195-204.

[0301] In various embodiments, the nucleic acid molecule encoding the CAR comprises the nucleic acid sequence of any one of SEQ ID NOs: 320-325. In various embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence comprising a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to any one of SEQ ID NOs: 320-325.

[0302] In various embodiments, the nucleic acid molecule encoding the CAR comprises the nucleic acid sequence of any one of SEQ ID NOs: 326-331. In various embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence comprising a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to any one of SEQ ID NOs: 326-331.

[0303] In various embodiments, the nucleic acid molecule encoding the CAR comprises the nucleic acid sequence of any one of SEQ ID NOs: 336-339. In various embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence comprising a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to any one of SEQ ID NOs: 336-339.

[0304] In one embodiment, a nucleic acid molecule encoding a CAR comprises a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to, or a nucleic acid sequence comprising, the sequence of SEQ ID NO: 153. In one embodiment, a nucleic acid molecule encoding a CAR comprises a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to, or a nucleic acid sequence comprising, the sequence of SEQ ID NO: 154. In one embodiment, a nucleic acid molecule encoding a CAR comprises a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to, or a nucleic acid sequence comprising, the sequence of SEQ ID NO: 155. In one embodiment, a nucleic acid molecule encoding a CAR comprises a sequence having at least 50, at least 55, at least 60,...

Claims

1. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, 13, 14, 18, 19, 21, and 23, and binds to a human kallikrein-2 (hK2) antigen; The CAR.

2. 2. The CAR of claim 1, wherein the intracellular signaling domain comprises a polypeptide component selected from the group consisting of a TNF receptor superfamily member 9 (CD137) component, a T cell surface glycoprotein CD3 zeta chain (CD3z) component, a cluster of differentiation (CD27) component, a cluster of differentiation superfamily member component, and combinations thereof.

3. The CAR of claim 2, wherein the CD137 component comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that is at least 90% identical to said amino acid sequence.

4. The CAR of claim 2 or 3, wherein the CD3z component comprises the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 90% identical to said amino acid sequence.

5. The CAR of any one of claims 2 to 4, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 45, or optionally, the intracellular signaling domain comprises a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a primary signaling domain comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 90% identical to said amino acid sequence.

6. The CAR according to any one of claims 1 to 5, wherein the transmembrane domain comprises a CD8a transmembrane region (CD8a-TM) polypeptide.

7. The CAR of claim 6, wherein the CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that is at least 90% identical to said amino acid sequence.

8. The CAR of any one of claims 1 to 7, further comprising a hinge region linking the transmembrane domain to the extracellular antigen-binding domain.

9. The CAR according to claim 8, wherein the hinge region is a CD8a-hinge region.

10. The CAR of claim 9, wherein the CD8a-hinge region comprises the amino acid sequence of SEQ ID NO: 25, 269, 270, 271, or 272, or an amino acid sequence which is at least 90% identical to said amino acid sequence.

11. The CAR of any one of claims 1 to 10, wherein the extracellular antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 274, 278, 279, 283, 284, 286, and 288.

12. The CAR according to any one of claims 1 to 11, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 51, 52, 56, 57, 59, and 61.

13. An isolated lymphocyte expressing the CAR according to any one of claims 1 to 12.

14. 14. The isolated lymphocyte of claim 13, which is a T lymphocyte.

15. 14. The isolated lymphocyte of claim 13, which is a natural killer (NK) cell.

16. An isolated nucleic acid molecule encoding the CAR of any one of claims 1 to 12.

17. A vector comprising the nucleic acid molecule of claim 16.

18. A cell expressing the nucleic acid molecule of claim 16.

19. A pharmaceutical composition comprising an effective amount of lymphocytes according to any one of claims 13 to 15.

20. A pharmaceutical composition comprising an effective amount of lymphocytes according to any one of claims 13 to 15 and a pharmaceutically acceptable excipient.

21. 21. A pharmaceutical composition according to claim 19 or 20 for use in therapy.

22. 21. A pharmaceutical composition according to claim 19 or 20 for use in treating a subject with cancer.

23. 21. The pharmaceutical composition of claim 19 or 20, for use in treating a subject with cancer, wherein the lymphocytes induce the killing of cancer cells in the subject.

24. 24. The pharmaceutical composition of claim 22 or 23, wherein the cancer is prostate cancer or androgen receptor-expressing breast cancer.

25. 21. The pharmaceutical composition of claim 19 or 20, for use in targeted killing of cancer cells, wherein the lymphocytes induce killing of the cancer cells.

26. 26. The pharmaceutical composition of claim 25, wherein the cancer cells are prostate cancer cells or androgen receptor-expressing breast cancer cells.

27. A pharmaceutical composition for use in detecting the presence of cancer in a subject, the pharmaceutical composition comprising the CAR according to any one of claims 1 to 12.

28. The CAR according to any one of claims 1 to 12 for use in therapy.

29. The CAR according to any one of claims 1 to 12, for use in treating a subject with cancer.

30. The CAR of claim 28 or 29, wherein the cancer is prostate cancer or androgen receptor-expressing breast cancer.

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