Anti-hk2 chimeric antigen receptor (CAR)

Abstract

To provide chimeric antigen receptors (CARs) that specifically bind to a human Kallikrein-2 (hK2), and immunoresponsive cells comprising such CARs, for the treatment of cancer.SOLUTION: Provided is a chimeric antigen receptor (CAR) comprising: (a) an extracellular domain that specifically binds to a human Kallikrein-2 (hK2) antigen, (b) a transmembrane domain, and (c) an intracellular signaling domain optionally comprising at least one costimulatory domain.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] (Sequence Listing) This application has been filed electronically in ASCII format and is incorporated by reference in its entirety. The above ASCII copy was last updated on June 30, 2020. The file is created in , named JBI6124WOPCT1_SL.txt, and is 562 bytes in size. ,160 bytes.

[0002] FIELD OF THE INVENTION The present invention provides a chimeric antigen receptor targeting hK2, which comprises a single-chain variable fragment targeting hK2. Chimeric antigen receptors (CARs), and genetically engineered cells expressing CARs The present invention relates to immune cells that target hK2. The present invention also relates to nucleic acids and expression vectors encoding CAR. The vectors are used to express CARs targeting hK2. Compositions comprising the CAR and the engineered immune cells are also provided. Methods for producing immune cells and genetically engineered immune cells to treat conditions including cancer A method for using the method is also provided.

[0003] (background) Prostate cancer is the second most commonly diagnosed cancer and the sixth leading cause of cancer death in men. 14% (903,500) of all new cancer cases and 6% of all cancer deaths in men worldwide The progression from diagnosis to death of prostate cancer depends on the severity of the disease, hormone levels, and Mon status and presence or absence of detectable metastases: localized disease, detectable after radiation therapy or surgery Elevated prostate-specific antigen (PSA) levels without metastasis and non-castration stage or is best classified as a series of clinical stages based on clinical progression in the castration stage. Surgery, radiation, or a combination of both may be curative for patients with localized disease. Although it is possible that a significant proportion of these patients will not undergo treatment with PSA levels as evidence. The risk of recurrent disease is particularly high in high-risk groups, which may lead to the development of metastases, i.e., the end of the disease. This may lead to progression to end-stage disease.

[0004] Androgen deprivation therapy (ADT) is the standard of care, with generally predictable outcomes. A decrease in PSA, a stable phase in which the tumor does not grow, followed by an increase in PSA and castration-resistant disease. For many years, ADT has been the standard treatment for patients with metastatic prostate cancer. there were.

[0005] Kallikrein-related peptidase 2 (hK2, HK2) is a phosphodiesterase that mediates the androgen receptor (AR). hK is a trypsin-like enzyme that is specifically expressed in prostate tissue and prostate cancer. 2 is activated by the transmembrane protease, serine 2 (TMPRSS2), and It is secreted into the ducts where it cleaves semenogelin, an extracellular matrix in the ejaculate, and hK2 expression is expressed in prostate and prostate cancer tissues, initiating a cascade that enhances the motility of Limited but recent evidence suggests that breast cancer may be a consequence of steroid hormone-mediated activation of the AR pathway. hK2 was demonstrated to be detectable in strains and primary patient samples (U.S. Patent Application Publication No. 2005 / 0100234). Similar to PSA, the prostate gland is involved in hyperplasia or malignant transformation. Retrograde migration of catalytically inactive hK2 to the blood when highly structured organization is impaired Sexual emission occurs.

[0006] T cell therapy involves genetically modifying cells to enhance their specificity for specific tumor-associated antigens. Genetic modification can be achieved by using chimeric antigen receptor (CAR) or exogenous T cells. This involves the expression of a chimeric antigen receptor, which can provide new antigen specificity on T cells. T cells expressing CAR-T cells can induce tumor immune reactivity. There is a need for better T cell-based cancer therapies.

[0007] (overview) Chimeric antigen receptors (CARs), such as those targeting human kallikrein-2 (hK2), CAR, cells containing the CAR, vectors encoding the CAR, e.g., recombinant expression vectors The present invention also provides vectors and nucleic acid molecules encoding the CAR, methods for producing the CAR, and compositions thereof. Compositions, polypeptides, proteins, nucleic acids, host cells, cell populations, and the disclosed CARs Disclosed herein are methods for treating disorders, such as cancer, using the compounds of the present invention.

[0008] In one aspect, a chimeric antigen receptor (CAR) is provided, comprising: (a) Extracellular scFv containing specific binding to human kallikrein-2 (hK2) antigen The domain and (b) a transmembrane domain; (c) an intracellular signaling domain, optionally including 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 is TNF receptor superfamily member 9 (C D137) component and the costimulatory domain of the T cell surface glycoprotein CD3 zeta chain (CD3 zeta chain) 3z) components of the primary signaling domain.

[0010] In some embodiments, the CD8a-hinge region is selected from the group consisting of SEQ ID NOs: 25, 269, 270, 271 or 272, comprising an amino acid sequence at least 90% identical to 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 an amino acid sequence at least 90% identical to SEQ ID NO:27. a costimulatory domain having the sequence, and an amino acid sequence at least 90% identical to SEQ ID NO:28 The primary signaling domain has the sequence

[0011] In another aspect, the extracellular antigen binding domain, the transmembrane domain, and the intracellular signaling domain are A chimeric antigen receptor (CAR) comprising a domain, wherein the extracellular antigen-binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 4, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 66. CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 314, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 315, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 309, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 310, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 7 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:3, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66 CDR3; heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 86, SEQ ID NO: 8 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 88 CDR3; heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 92, SEQ ID NO: 9 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:3, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:94 CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 103, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 106, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 107, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 108, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 110, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 111, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 112, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 114, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 115, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 116, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 118, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 119, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, and a heavy chain CDR3 comprising: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 123, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 124, and heavy chain CDR3, Including, A CAR is provided, wherein the extracellular antigen-binding domain binds to the hK2 antigen.

[0012] In another aspect, the extracellular antigen binding domain, the transmembrane domain, and the intracellular signaling domain are A chimeric antigen receptor (CAR) comprising a domain, wherein the extracellular antigen-binding domain comprises: Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, SEQ ID NO: 6 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 9, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 7 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 71. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, SEQ ID NO: 9 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 91. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 95, SEQ ID NO: 9 6, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 71. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 312, SEQ ID NO: a light chain CDR2 having an amino acid sequence of SEQ ID NO: 313, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 91; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, SEQ ID NO: 9 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 91. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, SEQ ID NO: 1 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 26, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 128, SEQ ID NO: 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; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 125, SEQ ID NO: 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; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 133, SEQ ID NO: 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; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 136, SEQ ID NO: 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 light chain CDR3 comprising: Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, SEQ ID NO: 1 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 139; and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 140. light chain CDR3, 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: Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, SEQ ID NO: 6 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 9, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; CDR3; or Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 7 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 71. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, SEQ ID NO: 9 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 91. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 95, SEQ ID NO: 9 6, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 71. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 312, SEQ ID NO: a light chain CDR2 having an amino acid sequence of SEQ ID NO: 313, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 91; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, SEQ ID NO: 9 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 91. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, SEQ ID NO: 1 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 26, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 128, SEQ ID NO: 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; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 125, SEQ ID NO: 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; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 133, SEQ ID NO: 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; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 136, SEQ ID NO: 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 light chain CDR3 comprising: Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, SEQ ID NO: 1 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 139; and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 140. light chain CDR3, Further includes:

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

[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 chain variable region; 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 chain variable region; 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 chain variable region; 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 chain variable region; 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 chain variable region; 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 chain variable region; 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 chain variable region; 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 chain variable region; 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 chain variable region; 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 chain variable region; 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 chain variable region; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 74 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 76; heavy chain variable region; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 77; heavy chain variable region; A light chain variable region comprising the amino acid sequence of SEQ ID NO: 147 and the amino acid sequence of SEQ ID NO: 140 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and the amino acid sequence of SEQ ID NO: 141 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and the amino acid sequence of SEQ ID NO: 140 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 148 and the amino acid sequence of SEQ ID NO: 142 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 149 and the amino acid sequence of SEQ ID NO: 143 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 150 and the amino acid sequence of SEQ ID NO: 144 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 151 and the amino acid sequence of SEQ ID NO: 145 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 152 and the amino acid sequence of SEQ ID NO: 146 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 318 and the amino acid sequence of SEQ ID NO: 317 a heavy chain variable region comprising Includes:

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

[0017] In some embodiments, the intracellular signaling domain is a TNF receptor superfamily. CD3 zeta chain (CD3), a component of the T cell surface glycoprotein CD3 (CD137) z) component, cluster of differentiation (CD27) component, 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 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 this embodiment, the transmembrane domain is a CD8a transmembrane region (CD8A-TM) polypeptide. In some embodiments, the CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO:26. It contains the amino acid sequence.

[0018] In various embodiments, the CAR links a transmembrane domain to an extracellular antigen-binding domain. In some embodiments, the hinge region is a CD8a hinge region. In some embodiments, the CD8a-hinge region is selected from the group consisting of SEQ ID NOs: 25, 269, 27 In some embodiments, the extracellular antigen binding fragment comprises an amino acid sequence of 0, 271, or 272. The binding domains are SEQ ID NOs: 29 to 44, 78 to 81, 221 to 236, and 273 to 308. In some embodiments, the CAR comprises an amino acid sequence selected from the group consisting of: Amino acids selected from the group consisting of sequence numbers 46 to 61, 82 to 85, and 205 to 220 Contains arrays.

[0019] In another embodiment, an isolated lymphocyte expressing any of the above CARs is provided. In some embodiments, the lymphocytes are T lymphocytes. , natural killer (NK) cells.

[0020] Also provided is an isolated nucleic acid molecule encoding any of the above CARs. Also provided are vectors comprising the nucleic acid molecules. Also provided are cells expressing the nucleic acid molecules.

[0021] Further, a pharmaceutical composition comprising an effective amount of any of the above lymphocytes and a pharmaceutically acceptable excipient. Compositions are also provided.

[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. A composition is provided.

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

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

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

[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 of treating a subject having cancer is provided. The method comprises administering to a subject a therapeutically effective amount of Any of the lymphocytes described above can be administered to a subject in need thereof, thereby In some embodiments, the spheres induce the killing of cancer cells in the subject. The cancer is prostate cancer. In some embodiments, the cancer is androgen receptor (AR)-expressing She currently has breast cancer.

[0031] In another aspect, there is provided a method for targeted killing of cancer cells, comprising administering the cancer cells to any of the lymphocytes described above. and contacting the lymphocytes with the cancer cells, thereby inducing the lymphocytes to kill 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 above-described CARs; This allows the formation of a CAR-cell complex, and (b) detecting the complex, wherein detecting the complex determines whether the complex is present in the subject. indicates the presence of cancer, 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. It will be.

[0034] This patent application file contains at least one drawing executed in color. Copies of this patent application will be provided by the Patent Office upon request and payment of the necessary fee. Provided. [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 a chimeric antigen receptor (CAR) targeting human kallikrein-2 (hK2). , cells containing such CARs, and the CARs described herein can be used to treat cancer (e.g., The present invention provides methods for treating cancers of the breast (prostate cancer or AR-expressing breast cancer).

[0037] The CAR of the present invention has antigen specificity for hK2. The phrase "elicits an antigen-specific response" as used herein means that the CAR is specific to the antigen. The CAR binds to the hK2 antigen and can be immunologically recognized, resulting in binding of the CAR to the hK2 antigen. This means that an immune response is induced by the antigen specificity and ability to recognize target cells. Methods for testing CAR for are known in the art.

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

[0039] Some aspects of the present invention are described below with reference to examples, which are for illustrative purposes only. Numerous specific details, relationships and methods are set forth in order to provide a thorough understanding of the present invention. However, it will be understood by those skilled in the art that the present invention may be practiced without the aid of specific details. can be performed without one or more of the following, or other methods, protocols, or reagents It will be readily apparent that the present invention can be practiced in various cell lines, cell lines, and animals. Because acts may occur in different orders and / or concurrently with other acts or events, the present invention The present invention is not limited by the order of acts or events illustrated. Not all illustrated acts, steps, or events are required to implement the invention. Many of the techniques and procedures described or referenced herein may be readily understood by those skilled in the art. are well understood and commonly used using conventional techniques.

[0040] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein or technical terms have the meanings commonly understood by those skilled in the art to which the present invention pertains. In some cases, terms with their commonly understood meanings have been omitted for the sake of clarity and and / or are defined herein for ready reference and include such definitions herein. The inclusion of a Terms such as those defined in commonly used dictionaries are not to be construed as meaningless or unrelated words. having a meaning consistent with their meaning in the context of the relevant art, and / or It is further understood that the terms "above" and "below" should be construed as if otherwise defined in the specification. It will be.

[0041] Where a list is presented, unless otherwise specified, each individual element of the list and its It should be understood that all combinations of the above are separate embodiments. A list of embodiments presented as "A," "B," "C," or "D" is not intended to be limiting. shall be interpreted to include "A or B," "A or C," "B or C," or "A, B, or C." should be.

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

[0043] The transitional phrases "comprising" and "consisting essentially of" "ly of" and "consisting of" are generally accepted terms in patent language. is intended to imply the meanings set forth therein, namely, (i) "comprises" or "contains" "featuring" is synonymous with "including," "containing," or "featuring" and is inclusive or non-exclusive. It is intended to be limiting and not to exclude other unrecited elements or method steps. and (ii) "consisting of" is not specified in the claims. and (iii) "consisting essentially of" The patent defines the specified materials or steps, as well as the "basic and novel characteristics ( The phrase "comprises" limits the scope of the claim to "not substantially affecting the An embodiment described in terms of "comprising / comprising" (or its equivalents) also includes an embodiment. As a form, "consisting of" and "consisting essentially of" are independently described. Provide.

[0044] "Activation" or "stimulation" or "activated" or "stimulated" means the activation marker expression of ATP, cytokine production, or mediation of target cell proliferation or cytotoxicity refers to the induction of a change in the biological state of a cell. A cell is activated by a primary stimulatory signal. Costimulatory signals can amplify the magnitude of the primary signal and suppress cell death after the initial stimulus. This results in a more durable activation state and therefore a higher cytotoxic potential. "Co-stimulatory signals" are signals that are produced by primary signals such as TCR / CD3 ligation. In combination with T cell and / or NK cell proliferation and / or upregulation of key molecules It refers to a signal that causes pre-regulation or down-regulation.

[0045] An "antigen-binding fragment" or "antigen-binding domain" refers to a portion of a protein that binds to an antigen. An antigen-binding fragment may be a synthetic polypeptide, an enzymatically obtainable polypeptide, or a genetically The polypeptide may be an engineered polypeptide, including an immunoglobulin polypeptide that binds to an antigen. Parts such as VH, VL, VH and VL, Fab, Fab', F(ab')2, Fd and and Fv fragments, domain antibodies (dAbs) consisting of one VH domain or one VL domain. ), shark variable IgNAR domain, camelized VH domain, VHH domain, FR3-C A minimal recognition unit consisting of amino acid residues that mimic the CDR of an antibody, such as the DR3-FR4 portion. HCDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3, alternative scaffolds that bind antigens, and antigen binding The antigen-binding fragments (such as VH and VL) include: The VH and VL domains are expressed as separate single chains linked together via a synthetic linker. When the VH / VL domains are combined, they form a monovalent antigen-binding domain by intramolecular or intermolecular pairing. For example, single chain Fv (scFv) or diabodies can be formed. A variety of single chain antibody designs can be created, including those that can be used in combination with other antigen-binding fragments. Monospecific or multispecific proteins can be engineered to engineer polyspecific and multispecific proteins. other antibodies, proteins, antigen-binding fragments, or alternative scaffolds that may It may be conjugated.

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

[0047] A "full-length antibody" is a molecule that consists of two heavy chains (HC) interconnected by disulfide bonds. Each heavy chain is composed of two light chains (LC) and two light chains (LC), and multimers of these (e.g., IgM). The chain is composed of a heavy chain variable domain (VH) and a heavy chain constant domain, Each light chain is composed of the subdomains CH1, hinge, CH2, and CH3. VH and VL are composed of a frame constant domain (VL) and a light chain constant domain (CL). Complementarity determining regions (CDRs) are interspersed in the framework region (FR). Each V can be further divided into regions of hypervariability called CDRs (compatibility determining regions). H and VL are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1 , FR2, CDR2, FR3, CDR3, and FR4, It consists of two FR segments.

[0048] A "human antibody" is an antibody that is optimized to evoke a minimal immune response when administered to a human subject. Human antibodies refer to antibodies that have been engineered to be human. The variable regions of a human antibody are derived from human immunoglobulin sequences. When the human antibody contains a constant region or a part of a constant region, the constant region is also a human immunoglobulin sequence. Human antibodies are antibodies in which the variable regions of the human antibody are derived from human germline immunoglobulins or rearrangements. If the sequence is derived from a system that uses immunoglobulin genes engineered to be human, the sequence will be labeled "derived from" An exemplary such system comprises a heavy chain variable region and a light chain variable region that are "transfected" into a phage. Splayed human immunoglobulin gene libraries and human immunoglobulin gene loci "Human antibodies" include transgenic non-human animals, such as mice or rats, carrying the " refers to the system used to obtain human antibodies and human immunoglobulin loci. differences, introduction of somatic mutations or intentional introduction of substitutions into the framework or CDRs or both, resulting in a lower amino acid sequence when compared to human-expressed immunoglobulins. Typically, a "human antibody" is a human antibody derived from human germline immunoglobulins or rearrangements. The amino acid sequence encoded by the immunoglobulin gene is Sequences 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 some cases, a "human antibody" is a human antibody, e.g., K et al., (2000) J Mol Biol 296:57-86 Consensus framework sequences obtained from analysis of human framework sequences, e.g. Shi et al., (2010) J Mol Biol 397:385-96 and and human phage-displayed antibodies described in International Publication No. WO2009 / 085462. The immunoglobulin gene library may contain a synthetic HCDR3. Antibodies in which one CDR is derived from a non-human species are not included in the definition of "human antibody."

[0049] A "humanized antibody" is an antibody in which at least one CDR is derived from a non-human species and at least one FDR is derived from a non-human species. A humanized antibody refers to an antibody whose framework is derived from human immunoglobulin sequences. The framework can contain substitutions so that the framework is compatible with expressed human immunoglobulins. may not be an exact copy of the human immunoglobulin germline gene sequence .

[0050] "Isolated" refers to a molecule produced in a recombinant cell or other component of the system in which it is produced. A homogenous population of molecules that have been substantially isolated and / or purified away from In addition to the peptide or polypeptide, the peptide has been subjected to at least one purification or isolation step. "Isolated" refers to a protein that is substantially free from other cellular material and / or chemicals. refers to molecules that are free of 80%, 81%, 82%, 83%, 84% ,85%,86%,87%,88%,89%,90%,91%,92%,93%,94% Molecules isolated to 95%, 96%, 97%, 98%, 99%, or 100% purity Contains.

[0051] "Modulate" means to induce a greater increase in a response when compared to a control or vehicle-mediated response. Enhanced or decreased activity of the test molecule mediating a weaker or weaker response (i.e., downstream effect) This refers to one of the abilities.

[0052] "Natural killer cells" and "NK cells" are used interchangeably and synonymously herein. NK cells express CD16 + CD56 + and / or CD57 + TCR - have a phenotype NK cells are differentiated lymphocytes that can destroy "self" M cells by activating specific cytolytic enzymes. Ability to bind to and kill cells that cannot express HC / HLA antigens, tumor cells or NK activation The ability to kill other diseased cells that express the receptor's ligand, and to stimulate or inhibit the immune response They are characterized by their ability to release protein molecules called cytokines that act against the immune system.

[0053] "Specifically binds," "specific binding," "specifically bound," or "binds" " refers to a protein molecule that binds to an antigen or an epitope within the antigen and has affinity to other antigens. Typically, proteinaceous molecules bind with an affinity greater than about 1× 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 More than M The equilibrium dissociation constant (K D ) binds to an antigen or an epitope within the antigen, typically The K D is the K for binding to nonspecific antigens (e.g., BSA, casein) D Less than In the context of the prostate neoantigens described herein, "specific binding" refers to binding of a specific antigen to a prostate gland. "Synthesis" means that the proteinaceous molecule is a wild-type protein of which the prostate neoantigen is a variant. This refers to binding to prostate neoantigens without detectably binding to proteins.

[0054] "Tumor cells" or "cancer cells" refer to cells that grow in the body, either in vivo, ex vivo, or in tissue culture. cancerous, precancerous, or transformed cells with spontaneous or induced phenotypic changes These changes do not necessarily involve the incorporation of new genetic material. Transformation involves infection with a transforming virus and the integration of new genomic nucleic acid, as well as the uptake of exogenous nucleic acid. It can also be caused by carcinogens, occur spontaneously, or after exposure to carcinogens. This may result in mutations in endogenous genes. Transformation / cancer can be caused by a variety of factors, including in vitro and in vivo. Morphological changes, cell immortalization, abnormal growth control, lesion formation in vivo and ex vivo Growth, proliferation, malignant lesions, regulation of tumor-specific marker levels, invasiveness, and preference for nude mice This is exemplified by the growth of tumors in a suitable animal host.

[0055] The term "chimeric antigen receptor" or "CAR" as used herein refers to a chimeric antigen receptor (CAR) that is not all naturally occurring. Extracellular target-binding domains in combinations not found together in a single protein It is defined as a cell surface receptor containing a membrane domain, a transmembrane domain, and an intracellular signaling domain. This includes, in particular, those in which the extracellular domain and the intracellular signaling domain are not naturally occurring. Receptors that are not found together in a single receptor protein are included. Chimeric antigen receptors are primarily expressed in lymphocytes such as T cells and natural killer (NK) cells. The use of is intended.

[0056] The terms "T cell" and "T lymphocyte" are interchangeable and are used synonymously herein. As used herein, T cells include thymocytes, naive T lymphocytes, immature T T lymphocytes include mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) 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 (TI L; 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: These include naive T cells and memory T cells. Also included are "NKT cells," which Not only do they express semi-invariant αβ T cell receptors, but also express the NK1.1 and other NK cell-associated proteins. NKT cells are a special group of T cells that express various molecular markers related to immune responses. NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+, and CD8 cells The TCR of NKT cells is presented by the MHC I-like molecule CD Id. NKT cells are unique in that they recognize glycolipid antigens. These cells can have either protective or harmful effects depending on their ability to produce cytokines that promote Also included are "gamma delta T cells (γδ T cells)." Gamma delta T cells refers to a specialized population of small subsets of T cells that have distinct TCRs on their surface. 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. T cells may play a role in immune surveillance and immune regulation, and IL-17 is an important It has been found to be a source of IgG and to induce a vigorous CD8+ cytotoxic T cell response. It also refers to T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Tregs also include "regulatory T cells" or "Tregs," which typically express the transcription factor F oxp3-positive CD4+ T cells and IL-10-producing CD4+ T cells. It may also include offspring Foxp3-negative regulatory T cells.

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

[0058] As used herein, the term "antigen" refers to any molecule capable of being bound by a T cell receptor. Any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, An antigen is a molecule that can elicit an immune response. Examples of immune responses include, but are not limited to, antibody production, or the production of specific immunological components. Those skilled in the art will appreciate that antigens are never "genes." It will be understood that the antigen need not be encoded by a specific polypeptide. The antigen may be synthetically produced. or may be derived from a biological sample, or may be a macromolecule other than a polypeptide It is readily apparent that such biological samples may include tissue samples. Samples, tumor samples, cells, or other biological components, organisms, protein / antigen subpopulations The invention also includes fluids containing killed or inactivated whole cells or lysates. It can be, but is not limited to these.

[0059] The term "antibody" includes monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, Monomeric antibodies, single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide fragments Sulfide-linked Fv (sdFv), intrabodies, minibodies, diabodies, and anti- diotypic (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 the antigen-binding fragments of ... covalent diabodies such as those disclosed in JP 2007 / 0004909; and Ig- such as those disclosed in U.S. Patent Application Publication No. 2009 / 0060910. Also referred to as DARTS. Antibodies useful as TCR binding molecules include immunoglobulin molecules and Immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain the antigen-binding site, are Immunoglobulin molecules include any type (e.g., IgG, IgE, IgM, IgE ... IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG 4, IgM1, IgM2, IgA1, and IgA2), or subclasses.

[0060] The term "host cell" refers to any cell that contains heterologous nucleic acid. The heterologous nucleic acid may be a vector. For example, the host cell may be a vector (e.g., an expression vector) that is capable of directing the production of a substance by the cell. , e.g., the expression by a cell of a gene, a DNA or RNA sequence, a protein, or an enzyme Any organism that is selected, modified, transformed, grown, used, or manipulated in any way for The host cell may be a cell derived from a vector. The vector backbone and desired outcome can be selected based on the vector backbone and the desired outcome. For example, a plasmid or cosmid can be introduced into a prokaryotic host cell for replication of some types of vectors. Bacterial cells such as, but not limited to, H5α, JM109, and KCB, SUR E® competent cells, as well as SOLOPACK Gold cells, were used to It can be used as a host cell for replication and / or expression. Bacterial cells such as 92 can be used as host cells for phage viruses. Eukaryotic cells that can be used as cells include yeast (e.g., YPH499, YP H500, and YPH501), insects, and mammals. Examples of mammalian eukaryotic host cells for replication and / or expression of vectors include HeLa , NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12. Examples include, but are not limited to:

[0061] Host cells of the present disclosure include those containing a DNA or RNA sequence encoding a CAR, Host cells include T cells and natural killer cells that express CAR on their cell surface. Enhances T cell activity, natural killer cell activity, treatment of cancer, and treatment of autoimmune diseases It can be used to

[0062] "Activation" or "stimulation" refers to the process by which cells (e.g., T cells and NK cells) become active. expressing activation markers, producing cytokines, proliferating, and / or targeting cells This refers to the induction of changes in the biological state of cells that result in cytotoxicity. Co-stimulatory signals increase the magnitude of the primary signal. This can suppress cell death after initial stimulation, resulting in a more durable activated state. The "costimulatory signal" activates the TCR / CD3 receptor, resulting in a higher cytotoxicity. In combination with primary signals such as irrigation, proliferation of T cells and / or NK cells and / or up-regulation or down-regulation of key molecules It refers to the signal that causes it.

[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 the process of reducing the potency or proliferation of a cell or making the cell more developmentally restricted. This refers to a method of transitioning to a specified state.

[0065] The terms "express" and "expression" refer to the ability of a gene or DNA sequence information to be produced. To enable or produce, for example, the transcription and translation of the corresponding gene or DNA sequence D means producing proteins by activating cellular functions involved in The NA 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, is said to be "expressed" by the cell. Expression products can be characterized as intracellular, extracellular, or transmembrane. do.

[0066] The term "transfection" refers to the introduction of "exogenous" ( That is, the introduction of exogenous or extracellular (exogenous) nucleic acid. A gene, DNA, or RNA that is "foreign" (i.e., exogenous or extracellular) to a host cell A sequence is introduced so that the host cell expresses the introduced gene or sequence to produce the desired product. a protein, typically a protein or enzyme, encoded by the introduced gene or sequence The term "cloned" refers to the production of a gene or sequence. " or "foreign" gene or sequence, and Chimeric antigen receptors, such as null, secreted, or other sequences used by the cell's genetic machinery. a regulatory or control sequence operably linked to the encoding polynucleotide The gene or sequence may include non-functional sequences that have 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 is transmitted to other cells, including cells of the same genus or species as the host cell. The species may be of any origin, including the same species, or from different genera or species.

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

[0068] The term "control element" refers to any cis-acting molecule that controls some aspect of the expression of a nucleic acid sequence. In some embodiments, the term "promoter" refers to a genetic element that, in essence, In some embodiments, the term "promoter" refers to a sequence that contains the minimal sequence necessary to initiate transcription. "-" contains sequences for initiating transcription, and in addition, each is generally called an "enhancer." upregulating or suppressing transcription, called "repressor elements" and "repressor elements" Also included are sequences that can be downregulated.

[0069] As used herein, "operably linked" and similar phrases refer to nucleic acids or amino acids. When used with respect to nucleic acids, each refers to nucleic acid sequences arranged in a functional relationship to each other or Refers to an operably linked amino acid sequence. For example, an operably linked promoter, enhancer element, open reading frame, 5' and 3' UTR, and The transinator sequence provides for the accurate production of nucleic acid molecules (e.g., RNA). In embodiments, the operably linked nucleic acid elements comprise an open reading frame transcription of the open reading frame and ultimately the production of a polypeptide (i.e., In another example, operably linked peptides may be peptides in which the functional domains are linked to each other. This refers to the fact that each domain is arranged at an appropriate distance to provide the intended function of the domain.

[0070] "Enhancement" or "promotion" or "increase" or "enhancement" or "improvement" generally refers to the enhancement of a vehicle. or a control molecule / composition. produce, induce, or cause a physiological response (i.e., downstream effects), A measurable physiological response refers to the ability of a composition contemplated herein to As will be apparent from the understanding of the present disclosure, the expansion, activation, and effector function of T cells In certain embodiments, the therapeutic effect of the present invention may include increased efficacy, increased persistence, and / or increased cancer cell killing capacity. An "increased" or "enhanced" amount may be a "statistically significant" amount, and may be greater than vehicle or control 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, of the response caused by the lighting composition 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (above 1 Includes all integers and decimal points between them, e.g., 1.5, 1.6, 1.7, 1.8, etc. This may include an increase in

[0071] "Reduction" or "decrease" or "attenuation" or "reduction" or "mitigation" generally refers to the reduction of a or a control molecule / composition. produce, induce, or cause a physiological response (i.e., downstream effects), In certain embodiments, "reduced" or "reduced" refers to the ability of the composition to The amount of "resulting" may be a "statistically significant" amount, and is not related to the amount of "resulting" produced by the vehicle or control composition. Responses in specific cell lines (reference responses) or responses in specific cell lines (1.1, 1.2, 1.5, 2, 3) , 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500 , 1000 times) (all integers greater than 1 and decimal points between them, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

[0072] The term "effective" as applied to a dose or amount means that the desired amount is effective when administered to a subject in need thereof. It refers to the amount of a compound or pharmaceutical composition sufficient to produce the desired activity. When administered in combination, an effective amount of the combination is equal to the amount of each component that would be effective when administered individually. Please note that the exact amount may or may not be included. The required dose will depend on the species, age, and general condition of the subject, the severity of the condition being treated, and the specific Varies from subject to subject depending on drug, mode of administration, etc.

[0073] The phrase "pharmaceutically acceptable" when used in connection with the compositions described herein means: Physiologically tolerated and typically do not cause adverse reactions when administered to mammals (e.g., humans) Preferably, the term "pharmaceutical" refers to the molecular entities and other components of such compositions that do not cause "Pharmaceutically acceptable" means a substance that meets the requirements of the Federal Register for use in mammals, and more specifically, humans. or approved by a state government regulatory agency, or in compliance with the United States Pharmacopoeia or other general regulations. This means that it is listed in a globally recognized pharmacopoeia.

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

[0075] The terms "nucleic acid," "nucleotide," and "polynucleotide" are used herein unless otherwise specified. "Nucleic acid sequence" or "nucleotide sequence" refers to an RNA or DNA fragment. These terms also refer to nucleic acid sequences encoding amino acids that are linked together by linkers. Any amino acids added as artifacts of cloning, including any amino acids encoded in the It may also refer to a nucleic acid sequence that includes a portion encoding any amino acid.

[0076] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers may be derived from petroleum, animal, or plant oils, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The liquid may be a sterile liquid such as water or oil, including those of natural or synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions are particularly useful carriers for injectable solutions. Alternatively, the carrier may be a binder (for compressed pills), a glidant, a sealant, or the like. solid dosage carriers, including, but not limited to, one or more of: additives, flavoring agents, and coloring agents Suitable pharmaceutical carriers are described in "Remington 's Pharmaceutical Sciences.

[0077] The terms "about" or "approximately" include within a statistically meaningful range of a value. Such ranges are within an order of magnitude, preferably within 50%, and more preferably within 20% of a given value or range. It may be within 0%, even more preferably within 10%, and even more preferably within 5%. The acceptable variation encompassed by the words "about" or "approximately" is intended to be within the scope of the specific system under study. It will depend on the system and will be readily understood by one skilled in the art.

[0078] "Kallikrein-related peptidase 2," "hK2," or "klk2" refers to a kallikrein-related peptidase This refers to a known protein also known as granular kallikrein-2, HK2, or HK2. K2 is produced as a preproprotein and is cleaved during proteolysis to produce the active protein. All hK2 isoforms and variants are encompassed by "hK2." The amino acid sequences of the various isoforms are available 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. contains a signal peptide (residues 1-18) and a propeptide region (residues 19-24) .

[0079] The terminology used herein is for the purpose of describing particular embodiments only. As used herein, unless the context clearly indicates otherwise, Unless otherwise specified, the indefinite articles "a," "an," and "the" are understood to include plural referents. It should be understood.

[0080] Chimeric Antigen Receptor The present invention generally relates to a method for producing a chimeric antigen receptor (CAGR) comprising: The present invention relates to the use of T cells containing chimeric antigen receptors (CARs) that are capable of expressing T cell signaling domains. An artificially constructed hybrid containing the antigen-binding domain of an antibody (scFv) linked to CAR is a lid protein or polypeptide. The characteristics of CAR include non-MHC-restricted selection. Redirecting T cell specificity and reactivity toward selected targets and monoclonal antibodies Non-MHC-restricted antigen recognition allows for the T cells expressing AR can recognize antigens independently of antigen processing. Therefore, it bypasses a major mechanism of tumor escape. Furthermore, when expressed in T cells, C The AR interacts with the endogenous T cell receptor (TCR) alpha and beta chains. does not dimerize.

[0081] The CARs described herein comprise at least an extracellular antigen-binding domain, a transmembrane domain, and , an intracellular signaling molecule comprising a functional signaling domain derived from a stimulatory molecule as defined below. and a cytoplasmic signaling domain (also referred to herein as a "cytoplasmic signaling domain"). The present invention provides a recombinant polypeptide construct comprising a CAR. T cells expressing a CAR are described herein. In the literature, these are referred to as CAR T cells, CAR-T cells, or CAR-modified T cells, and their use The terms are used interchangeably herein. Cells have antibody binding domains on their surface. Can be genetically engineered for stable expression and novel MHC-independent antigen specificity is granted.

[0082] In some cases, T cells recognize that the antigen recognition domain of a particular antibody binds to the CD3-zeta chain or F C, which combines with the intracellular domain of the cγRI protein to form a single chimeric protein In one embodiment, the stimulatory molecule is a T The zeta chain is associated with the cell receptor complex.

[0083] "Intracellular signaling domain" or "cytoplasmic signaling domain" as used herein When used, it refers to the intracellular portion of the molecule, which is responsible for producing secondary messengers. Transmitting information within cells to control cellular activities through specific signaling pathways These act as effectors by acting through or responding to such signaling substances. It is the functional part of a protein that acts by functioning as a signaling pathway. The domain promotes immune effector function of CAR-containing cells (e.g., CAR-T cells) For example, the immune effector function of CAR-T cells is These include cytolytic activity and helper activity (including secretion of cytokines).

[0084] In one embodiment, the intracellular signaling domain comprises a primary intracellular signaling domain. Examples of primary intracellular signaling domains include primary stimulation or antigen-dependent signaling domains. In one embodiment, the signaling pathway is derived from a molecule involved in intracellular signaling. The core transduction domain may comprise a costimulatory intracellular domain. Examples of genes 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 is a T cell The co-stimulatory intracellular signaling domain may comprise the cytoplasmic sequence of a co-receptor or co-stimulatory domain. It may contain cytoplasmic sequences from the stimulatory molecule.

[0085] The primary intracellular signaling domain is an immunoreceptor tyrosine-based activation motif or It may contain signaling motifs known as ITAMs. Primary cytoplasmic signaling sequences Examples of ITAMs containing α-aminomethyl-2-methyl-1 ... 3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79 b, and CD66d derived from DAP10 and DAP12, Not limited to:

[0086] "Zeta" or alternatively "Zeta chain", "CD3-zeta" or "TCR-zeta" The term is provided as GenBank accession number BAG36664.1 Proteins, or non-human species, e.g., murine, rabbit, primate, mouse, rodent, monkey , and is defined as the equivalent residues from apes, etc., and is referred to as the "zeta stimulatory domain" or alternatively as " The "CD3-zeta stimulatory domain" or "TCR-zeta stimulatory domain" is involved in T cell activation. The access from the cytoplasmic domain of the zeta chain is sufficient to functionally transmit the necessary initial signal. Sometimes, for example, in aspects and / or embodiments of the present disclosure, The "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is a "primary signal transduction domain" In one embodiment, the cytoplasmic domain of the CD3 zeta stimulatory domain is , residues 52 to 164 of GenBank accession number BAG36664.1, or functional orthologs of non-human species, e.g., mice, rodents, monkeys, apes, etc. In a preferred embodiment, the intracellular signaling domain comprises equivalent residues derived from C In one embodiment, the "zeta stimulatory domain" or "CD3-zeta stimulatory domain" The "data stimulatory domain" may be the sequence provided as SEQ ID NO: 28, or at least SEQ ID NO: 28. at least 50, at least 55, at least 60, at least 65, at least 70, at least At least 75, at least 80, at least 85, at least 90, at least 91, at least At least 92, at least 93, at least 94, at least 95, at least 96, at least and sequences that share 97, at least 98, or at least 99% sequence identity with each other. .

[0087] The term "costimulatory molecule" refers to a molecule that specifically binds to a costimulatory ligand, thereby inducing T cell proliferation. Allogeneic binding on T cells mediating costimulatory responses (e.g., but not limited to, proliferation) by Costimulatory molecules are antigen receptors or their ligaments required for an efficient immune response. Co-stimulatory molecules include 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) These include, but are not limited to:

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

[0089] An intracellular signaling domain may refer to the entire intracellular portion of the molecule from which it is derived, or to the portion of the molecule from which it is derived. It may comprise the entire intracellular signaling domain, or a functional fragment thereof.

[0090] The term "4-1BB" or alternatively "CD137" refers to the GenBank accession The amino acid sequence provided under the number AAA62478.2, or a sequence from a non-human species, e.g., mouse The TNFR superfamily has equivalent residues from mice, rodents, monkeys, and apes. and "4-1BB costimulatory domain" refers to members of GenBank accession no. Amino acid residues 214 to 255 of No. AAA62478.2, or a non-human species, e.g., mouse , rodents, monkeys, and apes. In aspects and / or embodiments of the present disclosure, a "4-1BB costimulatory domain" refers to a "co-stimulatory domain" Also called "Main".

[0091] In a preferred embodiment, the intracellular signaling domain is a 4-1BB costimulatory domain or contains a costimulatory intracellular signaling domain, the "CD137 costimulatory domain." In such a case, the "4-1BB costimulatory domain" or "CD137 costimulatory domain" is represented by SEQ ID NO: 27, or sequences from non-human species, e.g., mice, rodents, monkeys, apes, etc. or 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 In a preferred embodiment, the 4-1BB codon is a sequence having at least 99% sequence identity. The stimulatory 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 is selected or modified by amino acid substitution to avoiding binding of such domains to transmembrane domains of the same or different surface membrane proteins, Interactions with other members of the receptor complex can be minimized. In one embodiment, the transmembrane domain comprises a CD8α hinge domain. , the CD8α hinge domain and the CD8α transmembrane domain.

[0093] In some embodiments, the cytoplasmic signaling domain is at least one of the nucleotides defined herein. The antibody further comprises one or more functional signaling domains derived from at least one costimulatory molecule. In one embodiment, the costimulatory molecule is 4-1BB (i.e., CD137), CD27, CD 3-zeta, and / or CD28. CD28 is important in T cell costimulation. CD27 is a member of the tumor necrosis factor receptor superfamily. 4-1BB is a co-stimulatory immune checkpoint molecule that acts as a co-stimulatory immune checkpoint molecule. Delivering potent costimulatory signals, promoting differentiation and enhancing long-term survival of T lymphocytes. 3-Zeta associates with TCR to generate signals and initiate immunoreceptor tyrosine-based activation Contains immunoreceptor tyrosine-based activation motifs (ITAM) 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 hinge domain comprising CD28, 4-1 BB, and an intracellular T cell receptor signaling domain including CD3-zeta. In another embodiment, the CAR comprises an intracellular hinge domain and a CAR comprising ... and an intracellular T cell receptor signaling domain containing D3-zeta, The antibody may be a fragment of all or part of the extracellular region of CD8, CD4, or CD28, or a fragment of the entire antibody constant region. or part of FcRIIIA receptor, IgG hinge, IgM hinge, IgA hinge, Ig The hinge may comprise all or part of a D hinge, an IgE hinge, or an Ig hinge. G1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, Ig It may be derived from IgD, IgE, or chimeras thereof.

[0095] The CARs described herein comprise at least an extracellular antigen-binding domain, a transmembrane domain, and , e.g., an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule as defined below. a signaling domain (also referred to herein as a "cytoplasmic signaling domain"); and a recombinant polypeptide construct comprising:

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

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

[0098] In one embodiment, the CAR comprises a hinge domain comprising a CD8α hinge domain and a CD8 α transmembrane domain and intracellular signaling domains including 4-1BB and CD3-zeta In one embodiment, the CAR comprises a hinge domain comprising a CD8α hinge domain. and cells containing the CD8α transmembrane domain, CD28, 4-1BB, and CD3-zeta. and an inner signaling domain.

[0099] In another embodiment, the CAR comprises a CD8α hinge domain, a CD8α transmembrane domain, and , an intracellular synthase comprising the signal transduction domain of CD3-zeta and the 4-1BB costimulatory domain. and a signal transduction 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 and a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 27 a costimulatory domain comprising the amino acid sequence of SEQ ID NO:28 and a primary signaling domain comprising the amino acid sequence of SEQ ID NO:29 and an intracellular signaling domain containing a nucleotide.

[0101] Thus, the present invention provides CAR T cells and methods for their use for adoptive therapy. Provide.

[0102] The present disclosure provides variants of the CARs, nucleic acids, polypeptides, and proteins described herein. The present invention further provides variants (e.g., functional variants). "Variant" refers to, for example, substitutions, insertions, or a reference polypeptide or a reference polynucleotide by one or more modifications, such as deletions. As used herein, "functional" refers to a polypeptide or polynucleotide that is distinct from The term "variant" refers to a polypeptide that is substantially or significantly different from the parent CAR, polypeptide, or protein. refers to a CAR, polypeptide, or protein having little sequence identity or similarity, A functional variant of a CAR, polypeptide, or protein is a variant of the CAR. Functional variants include, for example, CARs, polypeptides described herein, and the like. or a variant of the parent CAR, polypeptide, or protein, Targeted to a similar extent, to the same extent, or to a greater extent than the parent CAR, polypeptide, or protein. With respect to the parent CAR, polypeptide, or protein, Functional variants can be, for example, amino acids or amino acids that are different from the parent CAR, polypeptide, or protein. At least about 30%, about 40%, about 50%, about 60%, about 75%, about 80% of the amino acid sequence is Approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, It may be about 97%, about 98%, about 99% or more identical.

[0103] f) As used herein, the structure of a polypeptide is represented by a recited reference sequence (having a given SEQ ID NO). In this context, the percentage of sequence identity between two amino acids The % sequence identity between sequences is calculated by comparing these two sequences aligned in an optimal way. The amino acid sequences to be compared can be determined by comparing these two sequences The sequence may contain additions or deletions relative to the reference sequence for optimal alignment between the two sequences. The percentage of identity determines the number of identical positions where the amino acid residues between the two sequences are identical. Divide the number of identical positions by the total number of positions in the comparison window and multiply the result by 100. to obtain the percentage identity between the two sequences. Typically, the comparison window corresponds to the entire length of the sequences being compared. For example, in BLAS T program, "BLAST 2 Sequences" (Tatusova et al., "Blast 2Seq - A new tool for comparing protein and nucleotide sequences," FEMS Microbiol Lett.174:247-250) can be used. and the website http: / / www.ncbi.nlm.nih.gov / gorf / bl 2.html and the parameters used are the defaults (specifically, Parameter "Open Gap Penalty": 5, and "Extension Gap Penalty": 2. The selected matrix is, for example, the matrix proposed by the program. are parameters specified in the BLOSUM 62 standard and are used to compare the two The percentage of identity between the sequences is calculated directly by the program. Determining sequence identity to a reference sequence is within the capabilities of those skilled in the art and can be accomplished using BLAST™ or similar techniques. This can be done using commercially available analysis software such as

[0104] A functional variant can be, for example, a parent CAR with at least one conservative amino acid substitution. In another embodiment, the amino acid sequence of a functional A variant is a polypeptide of the parent CAR having at least one non-conservative amino acid substitution. or the amino acid sequence of a protein, in which case non-conservative amino acid substitutions may be made to maintain function. Non-conservative amino acid substitutions cannot suppress or inhibit the biological activity of the target variant. This can improve the biological activity of the functional variant, resulting in The biological activity of the CAR is increased compared to the parent CAR, polypeptide, or protein.

[0105] The amino acid substitutions in the CAR of the present invention can be conservative amino acid substitutions. Substitutes are known in the art and are compounds that have particular physical and / or chemical properties. Replacing one amino acid with another amino acid that has the same or similar chemical or physical properties For example, a conservative amino acid substitution would be one in which an amino acid is replaced with another acidic amino acid. a polar amino acid (e.g., Asp or Glu), substituted with another amino acid having a nonpolar side chain Amino acids with nonpolar side chains (e.g., Ala, Gly, Val, Ile, Leu, M et, Phe, Pro, Trp, Val, etc.), basic amino acids replaced with other basic amino acids Amino acids (e.g., Lys, Arg), polar side chains replaced with another amino acid with a polar side chain and amino acids having the formula (Asn, Cys, Gln, Ser, Thr, Tyr, etc.). do.

[0106] A CAR, polypeptide, or protein may be a specific amino acid sequence or a polypeptide as described herein. The sequence may consist essentially of a sequence such that other components, e.g., other amino acids, are not required to provide a functional balance. does not substantially alter the biological activity of the ant.

[0107] CARs, polypeptides, and proteins (including functional portions and functional blocks) of the disclosed embodiments the amino acid sequence (including variants) can be of any length, i.e., can contain any number of amino acids, However, CAR, polypeptide, or protein (or functional part or functional variant thereof) Antibodies (antigens) are those that express their biological activity, such as the ability to specifically bind to antigens and to target disease cells in the host. The ability to detect cells (e.g., cancer cells) or to treat or prevent disease in a host. For example, the polypeptide may be about 50 to about 5,000 amino acids in length, e.g., Approximately 50, approximately 70, approximately 75, approximately 100, approximately 125, approximately 150, approximately 175, approximately 200, approximately 22 5, approx. 250, approx. 275, approx. 300, approx. 325, approx. 350, approx. 375, approx. 400, approx. 42 5, approx. 450, approx. 475, approx. 500, approx. 525, approx. 550, approx. 575, approx. 600, approx. 62 5, approx. 650, approx. 675, approx. 700, approx. 725, approx. 750, approx. 775, approx. 800, approx. 82 5, about 850, about 875, about 900, about 925, about 950, about 975, about 1000 or more The polypeptides of the present invention also include oligopeptides.

[0108] CARs, polypeptides, and proteins of the present invention (including functional moieties and (including functional variants) are those that substitute 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, Examples include aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, and phosphatase. Moserin, 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-dia Aminopropionic acid, homophenylalanine, 4-chlorophenylalanine, 4-carboxy β-phenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine Lysine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, N '-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxy Cylidridine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexa carboxylic acid, α-aminocycloheptanecarboxylic acid, indoline-2-carboxylic acid, 1 ,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid Examples include glycerol monosodium benzoate, glycerol monosodium benzoate, and alpha-tert-butylglycine.

[0109] CARs, polypeptides, and proteins (functional portions and functional blocks) of the present embodiments Proteins (including ribonucleotides) can undergo post-translational modifications. These include glycosylation, esterification, and cleavage. Termination, N-acylation, amidation, carboxylation, phosphorylation, esterification, e.g., di- It can be cyclized via a sulfide bridge or converted into an acid addition salt. In some embodiments, they are dimerized or polymerized, or conjugated. It is being done.

[0110] CARs, polypeptides, and / or proteins (including functional portions thereof) of the present invention Such proteins (including functional variants thereof) can be obtained by methods known in the art. Suitable methods for the de novo synthesis of polypeptides and proteins can be found in the literature, e.g. han et al.,Fmoc Solid Phase Peptide Synt hesis,Oxford University Press,Oxford,Uni ted Kingdom,2000;Peptide and Protein Dru g Analysis, ed. Reid, R., Marcel Dekker, Inc. ,2000;and Epitope Mapping,ed.Westwood et. al.,Oxford University Press,Oxford,Unit ed. Kingdom, 2001. Also, polypeptides and proteins can be recombinantly synthesized using the nucleic acids described herein using standard recombinant methods. See, e.g., Sambrook et al., Molecular Clo ning:A Laboratory Manual,3rd ed.,Cold Sp ring 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 . In addition, the CARs, polypeptides, and proteins (functional moieties) of the present invention Some of the genes (including functional variants thereof) are derived from sources such as plants, bacteria, insects, and mammals. The method for isolation and purification can be described in the art. Alternatively, the CARs, polypeptides, and / or proteins described herein The substance (including functional portions and functional variants thereof) can be commercially synthesized. In this regard, CARs, polypeptides, and proteins may be synthesized, recombinant, isolated, and and / or purified.

[0111] To generate the recombinant nucleic acids utilized to produce the polypeptides described herein, Examples of modified nucleotides that can be used for this purpose include 5-fluorouracil, 5- Bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, carboxy Methylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, Dihydrouracil, N 6 -substituted adenine, 7-methylguanine, 5-methylaminomethyl Uracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosyl -osine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methyl Lucio-N 6 -Isopentenyladenine, uracil-5-oxyacetic acid (v), Wibtki Sosin (wybutoxosine), pseudouracil, queusin, beta-D-galactosyl Queuosine, Inosine, N6 -Isopentenyladenine, 1-methylguanine, 1-methyl Chilinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiocytosine uracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-ol hydroxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil Examples of benzodiazepines include, but are not limited to, benzodiazepines ... and 2,6-diaminopurine.

[0112] The nucleic acid may be a CAR, a polypeptide or protein, or a functional portion or function thereof. Any isolated or purified nucleotide sequence encoding any of the genetic variants of Alternatively, the nucleotide sequence may be a nucleotide sequence degenerate to any of the sequences: or may comprise a combination of degenerate sequences.

[0113] Some embodiments of the present invention also provide isolated or purified nucleic acids, the nucleic acids comprising the A nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein. or any of the nucleotide sequences of the nucleic acids described herein under stringent conditions. The term "nucleotide sequence" includes a nucleotide sequence that hybridizes under certain conditions.

[0114] Nucleotide sequences that hybridize under stringent conditions are called highly stringent. "High stringency conditions" refer to conditions under which the nucleotide sequence can hybridize. The sequence hybridizes to the target sequence (herein) in a detectable amount that is stronger than non-specific hybridization. specifically hybridize to any of the nucleotide sequences of the nucleic acids set forth in High stringency conditions are defined as conditions under which polynucleotides with exactly complementary sequences are required. A polynucleotide containing only a few mismatches, or scattered mismatches, is called a nucleotide The resulting markers had several small regions (e.g., 3 to 12 bases) that matched the marker sequence. The complementary small region is 14 to 17 or more. It melts more easily than the full-length complement of more bases, and is a high-stringency hybrid. The high stringency conditions make them easily distinguishable. The conditions include, for example, low salt and / or high temperature conditions (about 0.02 to 0.02°C at a temperature of about 50 to 70°C). Such high-strength solutions include those provided by 0.1 M NaCl or equivalent. The congruency condition is the mismatch between the nucleotide sequence and the template strand or the target strand. little, if any, tolerance to detect expression of any of the CARs described herein The conditions are made more stringent by the addition of increasing amounts of formamide. It is generally understood that this can be an agent.

[0115] In one embodiment, the nucleic acids of the invention can be incorporated into a recombinant expression vector. The disclosure provides recombinant expression vectors comprising any of the nucleic acids of the invention. As used herein, the term "recombinant expression vector" refers to the expression of mRNA, tandem repeats, or a combination thereof by a host cell. A genetically engineered oligonucleotide that allows for the expression of a protein, polypeptide, or peptide. means a nucleotide or polynucleotide construct, where the construct is Nucleotide sequences encoding mRNA, proteins, polypeptides, or peptides The vector expresses mRNA, a protein, a polypeptide, or a peptide in a cell. The vectors described herein are contacted with the cells under conditions sufficient to allow the vectors to transfect the cells. However, parts of the vector may be naturally occurring. The recombinant expression vectors used can be DNA and RNA (which can be single-stranded or double-stranded, and can be synthetic). or may be derived in part from natural sources, and may contain natural, non-natural or modified nucleotides. It may contain any type of nucleotide, including but not limited to, Recombinant expression vectors do not contain naturally occurring internucleotide linkages or naturally occurring It may contain non-naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. The modified nucleotide or internucleotide linkage does not inhibit the transcription or replication of the vector. stomach.

[0116] In one embodiment, the recombinant expression vector of the present invention is any suitable recombinant expression vector. and can be used to transform or transfect any suitable host. Suitable vectors include plasmids and viruses, which are suitable for propagation and multiplication. Vectors include vectors designed for transcription or expression, or both. UC Series (Fermentas Life Sciences, Glen Burn i.e., Md.), pBluescript series (Stratagene, LaJol la, Calif.), pET series (Novagen, Madison, Wis.) , pGEX series (Pharmacia Biotech, Uppsala, Sweden) en), and pEX series (Clontech, Palo Alto, Calif.) λGT10, λGT11, λEMBL4, and λNM11 49, using bacteriophage vectors such as λZapII (Stratagene). Examples of plant expression vectors include pBI01, pBI01.2, pB Examples include I121, pBI101.3, and pBIN19 (Clontech). Examples of expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clo Recombinant expression vectors include viral vectors, e.g., retroviral vectors. The vector may be a retroviral vector, for example a gammaretroviral vector.

[0117] In one embodiment, the recombinant expression vector of the invention is a vector of the type described, for example, in Sambrook, supra. et al. (supra) and Ausubel et al. (supra). It is prepared using recombinant DNA technology. The constructs of expression vectors can be circular or linear. The construct can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems include, for example, ColE1, SV40, 2μ plasmid, λ, bovine papillomavirus It may be derived from Rus or something similar.

[0118] The recombinant expression vector may be DNA-based or RNA-based, as appropriate. The type of host into which the vector will be introduced (e.g., bacterial, plant, fungal, or animal) will be considered. The gene may include regulatory sequences, such as transcription and translation initiation and termination codons, specific for the target gene (e.g., a target gene).

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

[0120] The recombinant expression vector can express a CAR, polypeptide, or protein (including functional portions and or to a nucleotide sequence encoding a CAR, polypeptide, or ... complementary to or equivalent to a nucleotide sequence encoding a peptide or protein A natural or canonical promoter operably linked to a nucleotide sequence that hybridizes to it. The promoter may include a promoter of choice (e.g., strong, weak, tissue-specific, inducible). The use of promoters (e.g., promoter-specific and development-specific) is within the skill of those in the art. It is within the skill of one in the art to combine a promoter with a non-viral promoter. promoters or viral promoters, e.g., cytomegalovirus rus, CMV) promoter, RSV promoter, SV40 promoter, or murine It may be the promoter found in the stem cell virus long terminal repeat.

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

[0122] Additionally, recombinant expression vectors can be engineered to contain a suicide gene. When used, the term "suicide gene" refers to a gene that causes the death of a cell that expresses the suicide gene. A suicide gene is a gene that makes cells that express the gene resistant to an agent (e.g., a drug). These drugs confer susceptibility to certain diseases, leading to cell death when the cells come into contact with or are exposed to the drug. Suicide genes are known in the art and can be, for example, Herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase enzymes, purine nucleoside phosphorylases, and nitroreductases.

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

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

[0125] The antibody can be any type of immunoglobulin known in the art. Globulins are divided into five major classes: IgA, IgD, IgE, IgG, and Ig IgA and IgG can be assigned to the isotypes IgA1, IgA2, IgG IgG1, IgG2, IgG3, and IgG4 are further subdivided. Based on the amino acid sequences of their constant domains, two types of ATP are known: kappa (κ) and lambda (λ). Antibodies can be of any class or isotype. obtain.

[0126] The antibodies may be monoclonal antibodies, including murine, human, humanized, and chimeric monoclonal antibodies. antibodies, polyclonal, antigen-binding fragments, bispecific or multispecific antibodies, monomers, dimers, Tetrameric or multimeric antibodies, single chain antibodies, domain antibodies, and antigen binding antibodies of the required specificity Immunoglobulins, including immunoglobulin molecules of any other modified configuration containing binding sites. Antibodies include naturally occurring antibodies, e.g., antibodies from mammals (e.g., murine, primate, etc.). isolated and / or from animals (such as mice, rabbits, goats, horses, chickens, hamsters, and humans) The antibody may be a purified antibody. Alternatively, the antibody may be a genetically engineered (e.g., genetically engineered) antibody. The antibody may be an engineered (antibody).

[0127] Humanized antibodies have an antigen-binding site derived from a non-human species, and the variable region framework is Human antibodies are derived from human immunoglobulin sequences. Human antibodies contain identical sequences in both the framework and antigen-binding sites. Each of these antibodies has heavy and light chain variable regions derived from sequences of human origin.

[0128] The antibody may also have any level of affinity or avidity for a functional portion of a CAR. In some embodiments, antibodies are obtained with a range of affinities (K D ) binds to the hK2 antigen In one embodiment according to the present invention, and in each of the numbered embodiments listed below: In some embodiments and all embodiments, the present invention may be implemented by those skilled in the art. The antibodies have high affinity, e.g., as determined by surface plasmon resonance or Kinexa. , about 10 -7 KD of 1 to 9.9 (or 1, 2, 3, 4, 5, 6, 7, 8, or 9, or any other value therein) 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 Binds to hK2 antigen An example of affinity is 1 x 10 -8 Another example of affinity is 1 × 10 -9 More than M Below.

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

[0130] Suitable methods for producing antibodies are known in the art. For example, standard human immunodeficiency virus (HIV) antibodies can be used. The hybridoma method is described, for example, in Koehler and Milstein, Eur. J. Immunol.,5,511-519(1976),Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CS H Press (1988), and CA Janeway et al. (eds.) ,Immunobiology,5th Ed.,Garland Publishing g, New York, NY (2001). Alternatively, other methods For example, the EBV-hybridoma method (Haskard and Archer, J.I. mmunol.Methods,74(2),361-67(1984),and Ro der et al.,Methods Enzymol.,121,140-67(1 986)), and bacteriophage vector expression systems (e.g., Huse et al. , Science, 246, 1275-81 (1989)) is the subject of the present invention. Additionally, methods for producing antibodies in non-human animals are known in the art. Patent Nos. 5,545,806, 5,569,825, and 5,714,352 No. 6,299,262, as well as U.S. Patent Application Publication No. 2002 / 0197266A1.

[0131] Phage display can also be used to generate antibodies. Phage libraries encoding antigen-binding variable (V) domains of can be produced using chemical and recombinant DNA techniques (e.g., Sambrook et al. (supra), and Ausubel et al. (supra). Phage encoding variable regions with desired specificity are then directed to the desired antigen (i.e., hK2), and a complete or partial antibody containing the selected variable domain The nucleic acid sequence encoding the rearranged antibody is then purified from the hybridoma. The antibody is introduced into a suitable cell line, such as a myeloma cell line, which is then used to generate a monoclonal antibody. Antibodies with specific properties are secreted by cells (e.g., Janeway et al. (supra), Huse et al. (supra), and U.S. Pat. No. 6,265,150. See the reference.

[0132] Antibodies are transgenic antibodies that are transgenic for specific heavy and light chain immunoglobulin genes. Such methods can be used in transgenic mice. See, for example, U.S. Patent Nos. 5,545,806 and 5,569,825, and and Janeway et al., supra.

[0133] Methods for producing humanized antibodies are known in the art, see, e.g., Janew ay et al., supra, U.S. Pat. Nos. 5,225,539, 5,585,089, and 5,585,089. 5,693,761, European Patent No. 0239400(B1), and British Patent No. 218 No. 8638. Humanized antibodies are also described in U.S. Pat. Nos. 5,639,641 and 5,639,641. Pedersen et al., J. Mol. Biol., 235, 959-973( They can also be generated using the antibody resurfacing technology described in (1994).

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

[0135] The term "antibody fragment" refers to an intact antibody or fragment thereof that retains the antigen-binding properties of the parent full-length antibody. refers to at least one part of the recombinant variant thereof. This may be, for example, recognition and binding. sufficient to confer specific binding (e.g., specific binding of an antibody fragment to a target such as an antigen). The term "antigen-binding domain" refers to the antigen-determining variable region of an intact antibody, for example. "Antigen-binding fragment" refers to a portion of an immunoglobulin molecule. Examples of antibody fragments include: Fab, Fab', F(ab')2, and Fv fragments, single-chain antibodies (scFv), linear antibodies , single domain antibodies (either VL or VH) such as sdAb, camelid VHH domains and multispecific antibodies formed from antibody fragments. .

[0136] The term "scFv" refers to an antibody fragment comprising at least one variable region of a light chain and at least one variable region of a heavy chain. and at least one antibody fragment containing the variable region of the antibody. In one embodiment, the light chain variable region and the heavy chain variable region are connected via a short, flexible polypeptide linker. The scFv can be expressed as a single polypeptide chain, with the scFv being the original Unless otherwise specified, as used herein, An scFv may have the VL and VH variable regions in any order, e.g., Regarding the N-terminal and C-terminal ends of the peptide, the scFv may comprise a VL-linker-VH. Alternatively, it may comprise VH-linker-VL.

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

[0138] In some embodiments, the antigen-binding fragment comprises a heavy chain complementarity determining region (CDR). complementarity determining regions (HCDRs) 1, 2, and / or 3, light chain complementarity determining regions (LCDRs) Light chain complementarity determining regions (LCDR) 1, 2, and / or 3, heavy chain variable region (VH), or light chain variable region (VL), Fab, F(ab' ) 2, Fd, and Fv fragments, and either one VH domain or one VL domain A domain antibody (dAb) is a domain antibody (dAb) comprising (for example consisting of) a VH domain and a VL domain. The domains may be linked together via a linker, for example a synthetic linker.

[0139] "Complementarity determining regions (CDRs)" are the regions in an antibody that CDRs are antigen-binding sites within VH. CDRs can be defined using various terms: (i) within VH Three in the VL (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR The complementarity-determining regions (CDRs) of the ribosomal DNA (LCDR2, LCDR3) are based on sequence diversity (Wu and Kabat,J Exp Med 132:211-50,1970;Kaba t et al.,Sequences of Proteins of Immuno logical Interest,5th Ed.Public Health Se rvice,National Institutes of Health,Beth (esda, Md., 1991). (ii) Three in VH (H1, H2, H3) and one in VL The three "hypervariable regions," "HVRs," or "HVs" (L1, L2, and L3) in thia and Lesk(Chothia and Lesk Mol Biol 196 :901-17, 1987), which are hypervariable in structure. This refers to the region of the antibody variable domain. The cs(IMGT) database (http: / / www_imgt_org) Standard numbering and definitions for binding sites are provided. CDR, HV, and IMGT Overview The correspondence of Lefranc et al.,Dev Comparative Immunol 27:55-77, 2003. As used herein, "CDR" , "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and the term "LCDR3" refers to the same as Ka listed above, unless otherwise specified herein. bat, Chothia, or as defined by IMGT The CDRs are

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

[0141] The CAR, polypeptide, or protein (including functional portions thereof) described herein Nucleic acids comprising nucleotide sequences encoding any of the nucleotide sequences (including variants) are disclosed herein. It is provided as follows.

[0142] The portion of the CAR that comprises an antibody or antibody fragment thereof may exist in various forms, in which case The antigen-binding domain may be, for example, a single domain antibody fragment (sdAb), scFv, or human It is expressed as part of a continuous polypeptide chain, such as a chimeric or humanized antibody (Ha rlow et al., 1999, In: Using Antibodies:AL aboratory Manual,Cold Spring Harbor Labo ratory Press, NY; Harlow et al., 1989, In: Antibodies:A Laboratory Manual,Cold Spri ng Harbor, NY; Houston et al., 1988, Proc. Natl.Acad.Sci.USA 85:5879-5883;Bird et a 1988, Science 242:423-426). In one embodiment, the C The antigen-binding domain of the AR composition comprises an antibody fragment. In one embodiment, the CAR comprises an scFv. It includes antibody fragments containing the antibody.

[0143] The term "recombinant antibody" refers to an antibody produced by, for example, a bacteriophage or yeast expression system. The term refers to antibodies produced using recombinant DNA technology, such as antibodies expressed in The antibody protein is produced by the synthesis of a DNA molecule that encodes the antibody. "antibody" should be construed to mean a protein or an antibody expressing an amino acid sequence that specifies the antibody. where the DNA or amino acid sequences are available and known in the art. It can be obtained using some recombinant DNA or amino acid sequencing techniques.

[0144] The term "antigen" refers to a molecule that provokes an immune response. This immune response can be antibody production. , or activation of specific immunologically competent cells, or both. It has been shown that virtually any macromolecule, including any protein or peptide, can function as an antigen. It will be understood that the antigen may be derived from recombinant or genomic DNA. Any DNA manufacturer can identify nucleotides that encode proteins that elicit an immune response. The term is used herein to include a nucleotide sequence or partial nucleotide sequence. It will be understood that the antigen encodes the "antigen" as follows. Furthermore, one skilled in the art will understand that the antigen It is understood that the gene need not be encoded solely by the full-length nucleotide sequence of the gene. While the present disclosure includes the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences may be arranged in various combinations. It is clear that the polypeptides that encode the desired immune response will be those that elicit the desired immune response.

[0145] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising an extracellular antigen-binding domain that binds to the hK2 antigen. Combine.

[0146] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising an extracellular antigen-binding domain, Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 1 3, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 4. and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:2 3, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 4. and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:3 3, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:6. and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:3 3, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 4. and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:2 3, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:6. and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 1 3, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:6. and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:3 3, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:5. and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 1 3, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:5. and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:2 3, and heavy chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:5. and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 74 R3, and heavy chain CDR1, CDR2 from the variable region comprising the amino acid sequence of SEQ ID NO: 76 , and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 75 R3, and heavy chain CDR1, CDR2 from the variable region comprising the amino acid sequence of SEQ ID NO: 77 , and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 147 heavy chain CDR1, CDR3, and a variable region comprising the amino acid sequence of SEQ ID NO: 140 R2, and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 75 R3, and a heavy chain CDR1, CDR2, CDR3, and a heavy chain CDR4 from a variable region comprising the amino acid sequence of SEQ ID NO: 141. 2, and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 75 R3, and a heavy chain CDR1, CDR2, CDR3, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 140 2, and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 148 heavy chain CDR1, CDR3, and a variable region comprising the amino acid sequence of SEQ ID NO: 142 R2, and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 149 heavy chain CDR1, CDR3, and a variable region comprising the amino acid sequence of SEQ ID NO: 143 R2, and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 150 heavy chain CDR1, CDR3, and a variable region comprising the amino acid sequence of SEQ ID NO: 144 R2, and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 151 heavy chain CDR1, CDR3, and a variable region comprising the amino acid sequence of SEQ ID NO: 145 R2, and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 152 heavy chain CDR1, CDR3, and a variable region comprising the amino acid sequence of SEQ ID NO: 146 R2, and CDR3; Light chain CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO: 318 heavy chain CDR1, CDR3, and a variable region comprising the amino acid sequence of SEQ ID NO: 317 R2 and CDR3 Including, The extracellular antigen-binding domain binds to the hK2 antigen.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0168] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising an extracellular antigen-binding domain, 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. The extracellular antigen-binding domain comprises a heavy chain CDR2 having a sequence of 3'-amino acids, and the extracellular antigen-binding domain binds to the hK2 antigen. Combine.

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

[0170] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising an extracellular antigen-binding domain, Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 4, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 66. CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 314, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 315, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 309, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 310, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 7 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:3, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66 CDR3; or heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 86, SEQ ID NO: 8 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 88 CDR3; heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 92, SEQ ID NO: 9 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:3, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:94 CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 103, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 106, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 107, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 108, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 110, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 111, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 112, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 114, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 115, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 116, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 118, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 119, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, and a heavy chain CDR3 comprising: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 123, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 124, and heavy chain CDR3, Including, The extracellular antigen-binding domain binds to the hK2 antigen.

[0171] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain having the amino acid sequence of SEQ ID NO: 63. heavy chain complementarity determining region 1 (CDR1), 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 has the amino acid sequence of SEQ ID NO: 314. Heavy chain complementarity determining region 1 (CDR1), heavy chain CDR having the amino acid sequence of SEQ ID NO: 315 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311.

[0173] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 309 Heavy chain complementarity determining region 1 (CDR1), a heavy chain CDR having the amino acid sequence of SEQ ID NO: 310 2, 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 having the amino acid sequence of SEQ ID NO: 63. heavy chain complementarity determining region 1 (CDR1), 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 having the amino acid sequence of SEQ ID NO: 72. heavy chain complementarity determining region 1 (CDR1), 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 having the amino acid sequence of SEQ ID NO: 86. heavy chain complementarity determining region 1 (CDR1), 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 having the amino acid sequence of SEQ ID NO: 92. heavy chain complementarity determining region 1 (CDR1), 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 has the amino acid sequence of SEQ ID NO: 102. Heavy chain complementarity determining region 1 (CDR1), a heavy chain CDR having the amino acid sequence of SEQ ID NO: 103 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104.

[0179] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 105. Heavy chain complementarity determining region 1 (CDR1), a heavy chain CDR having the amino acid sequence of SEQ ID NO: 106 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104.

[0180] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 107 Heavy chain complementarity determining region 1 (CDR1), heavy chain CDR having the amino acid sequence of SEQ ID NO: 108 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109.

[0181] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 110. Heavy chain complementarity determining region 1 (CDR1), heavy chain CDR having the amino acid sequence of SEQ ID NO: 111 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109.

[0182] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 102. Heavy chain complementarity determining region 1 (CDR1), a heavy chain CDR having the amino acid sequence of SEQ ID NO: 112 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:113.

[0183] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 105. Heavy chain complementarity determining region 1 (CDR1), a heavy chain CDR having the amino acid sequence of SEQ ID NO: 114 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:113.

[0184] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 115. Heavy chain complementarity determining region 1 (CDR1), heavy chain CDR having the amino acid sequence of SEQ ID NO: 116 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:117.

[0185] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 118 Heavy chain complementarity determining region 1 (CDR1), a heavy chain CDR having the amino acid sequence of SEQ ID NO: 119 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:117.

[0186] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 120 Heavy chain complementarity determining region 1 (CDR1), a heavy chain CDR having the amino acid sequence of SEQ ID NO: 121 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122.

[0187] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 123 Heavy chain complementarity determining region 1 (CDR1), a heavy chain CDR having the amino acid sequence of SEQ ID NO: 124 2, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122.

[0188] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising an extracellular antigen-binding domain, 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 and a light chain CDR1 having an amino acid sequence selected from the group consisting of these conservative modifications. The extracellular antigen-binding domain binds to the hK2 antigen.

[0189] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising an extracellular antigen-binding domain, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 90, SEQ ID NO: 96, SEQ ID NO: 126, SEQ ID NO: 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. The extracellular antigen-binding domain comprises a light chain CDR2 that binds to the hK2 antigen.

[0190] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising an extracellular antigen-binding domain, 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 a light chain CDR3 having an amino acid sequence corresponding to the hK2 antibody. Combine with Hara.

[0191] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising an extracellular antigen-binding domain, Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, SEQ ID NO: 6 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 9, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 7 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 71. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, SEQ ID NO: 9 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 91. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 95, SEQ ID NO: 9 6, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 71. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 312, SEQ ID NO: a light chain CDR2 having an amino acid sequence of SEQ ID NO: 313, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 91; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89, SEQ ID NO: 9 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 91. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, SEQ ID NO: 1 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 26, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 128, SEQ ID NO: 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; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 125, SEQ ID NO: 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; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 133, SEQ ID NO: 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; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 136, SEQ ID NO: 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 light chain CDR3 comprising: Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, SEQ ID NO: 1 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 139; and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 140. light chain CDR3, Including, The extracellular antigen-binding domain binds to the hK2 antigen.

[0192] In one embodiment, the extracellular antigen-binding domain comprises a light chain having the amino acid sequence of SEQ ID NO: 67. light chain complementarity-determining region 1 (CDR1), 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 having the amino acid sequence of SEQ ID NO: 68. light chain complementarity determining region 1 (CDR1), 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 having the amino acid sequence of SEQ ID NO: 89. light chain complementarity determining region 1 (CDR1), 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 having the amino acid sequence of SEQ ID NO: 95. light chain complementarity determining region 1 (CDR1), 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 has the amino acid sequence of SEQ ID NO: 312. Light chain complementarity determining region 1 (CDR1), a light chain CDR having the amino acid sequence of SEQ ID NO: 313 2, 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 having the amino acid sequence of SEQ ID NO: 89. light chain complementarity determining region 1 (CDR1), 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 having the amino acid sequence of SEQ ID NO: 67. Chain complementarity determining region 1 (CDR1), 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 has the amino acid sequence of SEQ ID NO: 128 Light chain complementarity determining region 1 (CDR1), a light chain CDR having the amino acid sequence of SEQ ID NO: 129 2, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 130.

[0200] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 125 Light chain complementarity determining region 1 (CDR1), a light chain CDR having the amino acid sequence of SEQ ID NO: 131 2, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 132.

[0201] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 133 Light chain complementarity determining region 1 (CDR1), a light chain CDR having the amino acid sequence of SEQ ID NO: 134 2, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 135.

[0202] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 136 Light chain complementarity determining region 1 (CDR1), a light chain CDR having the amino acid sequence of SEQ ID NO: 137 2, 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 having the amino acid sequence of SEQ ID NO: 97. Chain complementarity determining region 1 (CDR1), 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 method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising an extracellular antigen-binding domain, Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 4, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 314, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 315, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 312 1), a light chain CDR2 having the amino acid sequence of SEQ ID NO: 313, a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91 light chain CDR3 with sequence; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 103, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 128; 1), 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 CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 107, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 108, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 125; 1), 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 CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 112, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 133; 1), 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 CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 115, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 116, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 117 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 136; 1), 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 light chain CDR3 having the sequence Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122 and light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97. ), light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, light chain CDR3 having the amino acid sequence of SEQ ID NO: 138 a light chain CDR3 having the sequence Including, The extracellular antigen-binding domain binds to the hK2 antigen.

[0205] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising an extracellular antigen-binding domain, Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 4, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 314, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 315, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 312 1), a light chain CDR2 having the amino acid sequence of SEQ ID NO: 313, a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91 light chain CDR3 with sequence; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 103, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 128; 1), 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 CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 107, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 108, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 125; 1), 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 CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 112, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 133; 1), 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 CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 115, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 116, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 117 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 136; 1), 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 light chain CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122 and light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97. ), light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, light chain CDR3 having the amino acid sequence of SEQ ID NO: 138 light chain CDR3 with sequence; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 7 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 3, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 7 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 3, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 7 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 3, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 7 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 3, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 7 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 3, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 309, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 310, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 311 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 312 1), a light chain CDR2 having the amino acid sequence of SEQ ID NO: 313, a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91 light chain CDR3 with sequence; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 106, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 128; 1), 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 CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 110, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 111, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 125; 1), 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 CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 114, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 133; 1), 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 CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 118, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 119, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 117 a light chain CDR3, and a light chain complementarity determining region 1 (CDR) having the amino acid sequence of SEQ ID NO: 136; 1), 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 light chain CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 123, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 124, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122 and light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97. ), light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, light chain CDR3 having the amino acid sequence of SEQ ID NO: 139 light chain CDR3 with sequence; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122 and light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97. ), light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, light chain CDR3 having the amino acid sequence of SEQ ID NO: 139 a light chain CDR3 having the sequence; or heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 86, SEQ ID NO: 8 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 88 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89; light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 91 light chain CDR3, Including, The extracellular antigen-binding domain binds to the hK2 antigen.

[0206] In one embodiment, the extracellular antigen binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 4, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 Contains the light chain CDR3.

[0207] In one embodiment, the extracellular antigen binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 Contains the light chain CDR3.

[0208] In one embodiment, the extracellular antigen binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; It contains a light chain CDR3.

[0209] In one embodiment, the extracellular antigen binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 Contains the light chain CDR3.

[0210] In one embodiment, the extracellular antigen binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; It contains a light chain CDR3.

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

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

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

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

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

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

[0217] In one embodiment, the extracellular antigen binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 7 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 3, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 Contains the light chain CDR3.

[0218] In one embodiment, the extracellular antigen binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 7 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 3, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 68, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 70, light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 Contains the light chain CDR3.

[0219] In one embodiment, the extracellular antigen binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 7 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 3, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; It contains a light chain CDR3.

[0220] In one embodiment, the extracellular antigen binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 7 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 3, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, Light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 Contains the light chain CDR3.

[0221] In one embodiment, the extracellular antigen binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 7 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 3, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 66 R3, 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; It contains a light chain CDR3 having

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

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

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

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

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

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

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

[0229] In one embodiment, the extracellular antigen binding domain comprises: heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 86, SEQ ID NO: 8 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 88 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 89; light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 91 Contains the light chain CDR3.

[0230] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising an extracellular antigen-binding domain, 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 (H CVR), or LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 A combination of an HCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 6 wherein 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 chain variable region; 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 chain variable region; 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 chain variable region; 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 chain variable region; 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 chain variable region; 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 chain variable region; 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 chain variable region; 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 chain variable region; 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 chain variable region; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 74 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 76; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 75; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 77; a heavy chain variable region containing the amino acid sequence; A light chain variable region comprising the amino acid sequence of SEQ ID NO: 147 and the amino acid sequence of SEQ ID NO: 140 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and the amino acid sequence of SEQ ID NO: 141 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and the amino acid sequence of SEQ ID NO: 140 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 148 and the amino acid sequence of SEQ ID NO: 142 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 149 and the amino acid sequence of SEQ ID NO: 143 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 150 and the amino acid sequence of SEQ ID NO: 144 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 151 and the amino acid sequence of SEQ ID NO: 145 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 152 and the amino acid sequence of SEQ ID NO: 146 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 318 and the amino acid sequence of SEQ ID NO: 317 a heavy chain variable region comprising Includes:

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

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

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

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

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

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

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

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

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

[0241] In one embodiment, the extracellular antigen-binding domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 74. The heavy chain variable region comprises a variable region 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 comprising the amino acid sequence of SEQ ID NO: 75. The heavy chain variable region comprises a variable region 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 the amino acid sequence of SEQ ID NO: 147. The heavy chain variable region comprises a nucleotide sequence of SEQ ID NO: 140 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 comprising the amino acid sequence of SEQ ID NO: 75. The heavy chain variable region comprises a variable region 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 comprising the amino acid sequence of SEQ ID NO: 75. The heavy chain variable region comprises a variable region 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 the amino acid sequence of SEQ ID NO: 148. The heavy chain variable region comprises a nucleotide sequence of SEQ ID NO: 142 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 the amino acid sequence of SEQ ID NO: 149. The heavy chain variable region comprises a nucleotide sequence of SEQ ID NO: 143 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 the amino acid sequence of SEQ ID NO: 150. The heavy chain variable region comprises a nucleotide sequence of SEQ ID NO: 144 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 the amino acid sequence of SEQ ID NO: 151. The heavy chain variable region comprises a nucleotide sequence of SEQ ID NO: 145 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 the amino acid sequence of SEQ ID NO: 152. The heavy chain variable region comprises a nucleotide sequence of SEQ ID NO: 146 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 the amino acid sequence of SEQ ID NO: 318. The heavy chain variable region comprises a nucleotide sequence of SEQ ID NO: 317 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: SEQ ID NO: 1 and 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 At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity a light chain variable region comprising an amino acid sequence having SEQ ID NO: 4 and at least 50, At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least Also 93, at least 94, at least 95, at least 96, at least 97, at least a heavy chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; SEQ ID NO: 2 and 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 At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity a light chain variable region comprising an amino acid sequence having SEQ ID NO: 4 and at least 50, At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least Also 93, at least 94, at least 95, at least 96, at least 97, at least a heavy chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; SEQ ID NO: 3 and 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 At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity a light chain variable region comprising an amino acid sequence having SEQ ID NO: 6 and at least 50, At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least Also 93, at least 94, at least 95, at least 96, at least 97, at least a heavy chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; SEQ ID NO: 3 and 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 At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity a light chain variable region comprising an amino acid sequence having SEQ ID NO: 4 and at least 50, At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least Also 93, at least 94, at least 95, at least 96, at least 97, at least a heavy chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; SEQ ID NO: 2 and 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 At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity a light chain variable region comprising an amino acid sequence having SEQ ID NO: 6 and at least 50, At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least Also 93, at least 94, at least 95, at least 96, at least 97, at least a heavy chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; SEQ ID NO: 3 and 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 At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity a light chain variable region comprising an amino acid sequence having SEQ ID NO: 4 and at least 50, At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least Also 93, at least 94, at least 95, at least 96, at least 97, at least a heavy chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; SEQ ID NO: 1 and 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 At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity a light chain variable region comprising an amino acid sequence having SEQ ID NO: 6 and at least 50, At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least Also 93, at least 94, at least 95, at least 96, at least 97, at least a heavy chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; SEQ ID NO: 3 and 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 At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity a light chain variable region comprising an amino acid sequence having SEQ ID NO: 5 and at least 50, At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least Also 93, at least 94, at least 95, at least 96, at least 97, at least a heavy chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; SEQ ID NO: 1 and 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 At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity a light chain variable region comprising an amino acid sequence having SEQ ID NO: 5 and at least 50, At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least Also 93, at least 94, at least 95, at least 96, at least 97, at least a heavy chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; SEQ ID NO: 2 and 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 At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity a light chain variable region comprising an amino acid sequence having SEQ ID NO: 5 and at least 50, At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least Also 93, at least 94, at least 95, at least 96, at least 97, at least a heavy chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; SEQ ID NO: 74 and 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 a light chain variable region comprising an amino acid sequence having SEQ ID NO: 76 and at least 50, At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least heavy chain variable regions comprising amino acid sequences having at least 98, or at least 99% sequence identity; area; SEQ ID NO: 75 and 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 a light chain variable region comprising an amino acid sequence having SEQ ID NO: 77 and at least 50, At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least heavy chain variable regions comprising amino acid sequences having at least 98, or at least 99% sequence identity; area; SEQ ID NO: 147 and 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 a light chain variable region comprising an amino acid sequence having the sequence of SEQ ID NO: 140 and 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, heavy chains containing amino acid sequences with at least 98, or at least 99% sequence identity; variable region; SEQ ID NO: 75 and 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 a light chain variable region comprising an amino acid sequence having SEQ ID NO: 141 and at least 50, at least 1 ... At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least heavy chain variable fragments comprising an amino acid sequence having at least 98, or at least 99% sequence identity; region; SEQ ID NO: 75 and 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 a light chain variable region comprising an amino acid sequence having SEQ ID NO: 140 and at least 50, At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least heavy chain variable fragments comprising an amino acid sequence having at least 98, or at least 99% sequence identity; region; SEQ ID NO: 148 and 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 a light chain variable region comprising an amino acid sequence having the sequence of SEQ ID NO: 142 and 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, heavy chains containing amino acid sequences with at least 98, or at least 99% sequence identity; variable region; SEQ ID NO: 149 and 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 a light chain variable region comprising an amino acid sequence having the sequence of SEQ ID NO: 143 and 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, heavy chains containing amino acid sequences with at least 98, or at least 99% sequence identity; variable region; SEQ ID NO: 150 and 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 a light chain variable region comprising an amino acid sequence having the sequence of SEQ ID NO: 144 and 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, heavy chains containing amino acid sequences with at least 98, or at least 99% sequence identity; variable region; SEQ ID NO: 151 and 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 a light chain variable region comprising an amino acid sequence having the sequence of SEQ ID NO: 145 and 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, heavy chains containing amino acid sequences with at least 98, or at least 99% sequence identity; variable region; SEQ ID NO: 152 and 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 a light chain variable region comprising an amino acid sequence having the sequence of SEQ ID NO: 146 and 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, heavy chains containing amino acid sequences with at least 98, or at least 99% sequence identity; variable region; SEQ ID NO: 318 and 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 a light chain variable region comprising an amino acid sequence having the sequence of SEQ ID NO: 317 and 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, heavy chains containing amino acid sequences with at least 98, or at least 99% sequence identity; Variable domain Includes:

[0253] In one embodiment, the extracellular antigen binding domain has at least 50, at least 100, ... At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least Also 93, at least 94, at least 95, at least 96, at least 97, at least a light chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; and SEQ ID NO: 4 and 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 The heavy chain variable region comprises an amino acid sequence having the same structure as the heavy chain variable region.

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

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

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

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

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

[0259] In one embodiment, the extracellular antigen binding domain has at least 50, at least 100, ... At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least Also 93, at least 94, at least 95, at least 96, at least 97, at least a light chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; and SEQ ID NO: 6 and 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 The heavy chain variable region comprises an amino acid sequence having the same structure as the heavy chain variable region.

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

[0261] In one embodiment, the extracellular antigen binding domain has at least 50, at least 100, ... At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least Also 93, at least 94, at least 95, at least 96, at least 97, at least a light chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; and SEQ ID NO: 5 and 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 The heavy chain variable region comprises an amino acid sequence having the same structure as the heavy chain variable region.

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

[0263] In one embodiment, the extracellular antigen binding domain has at least 50 and at least 100 sequences identical to SEQ ID NO: 74. At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least a light chain variable region comprising an amino acid sequence having at least 98 or 99% sequence identity with , and SEQ ID NO: 76 and 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% of the sequence It contains a heavy chain variable region that contains identical amino acid sequences.

[0264] In one embodiment, the extracellular antigen binding domain has at least 50 and at least 100 amino acids selected from SEQ ID NO: 75. At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least a light chain variable region comprising an amino acid sequence having at least 98 or 99% sequence identity with , and SEQ ID NO: 77 and 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% of the sequence It contains a heavy chain variable region that contains identical amino acid sequences.

[0265] In one embodiment, the extracellular antigen binding domain has at least 50, at least 100 amino acids selected from SEQ ID NO: 147 and SEQ ID NO: 148. At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least a light chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; and SEQ ID NO: 140 and 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 At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% It contains a heavy chain variable region that contains an amino acid sequence having sequence identity.

[0266] In one embodiment, the extracellular antigen binding domain has at least 50 and at least 100 amino acids selected from SEQ ID NO: 75. At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least a light chain variable region comprising an amino acid sequence having at least 98 or 99% sequence identity with , and SEQ ID NO: 141 and at least 50, at least 55, at least 60, at least At least 65, at least 70, at least 75, at least 80, at least 85, at least At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% The heavy chain variable region contains an amino acid sequence having sequence identity.

[0267] In one embodiment, the extracellular antigen binding domain has at least 50 and at least 100 amino acids selected from SEQ ID NO: 75. At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least a light chain variable region comprising an amino acid sequence having at least 98 or 99% sequence identity with , and SEQ ID NO: 140 and at least 50, at least 55, at least 60, at least At least 65, at least 70, at least 75, at least 80, at least 85, at least At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% The heavy chain variable region contains an amino acid sequence having sequence identity.

[0268] In one embodiment, the extracellular antigen binding domain has at least 50, at least 100 amino acids selected from SEQ ID NO: 148 and SEQ ID NO: 149. At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least a light chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; and SEQ ID NO: 142 and 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 At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% It contains a heavy chain variable region that contains an amino acid sequence having sequence identity.

[0269] In one embodiment, the extracellular antigen binding domain has at least 50, at least 100 amino acids selected from SEQ ID NO: 149 and SEQ ID NO: 150. At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least a light chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; and SEQ ID NO: 143 and 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 At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% It contains a heavy chain variable region that contains an amino acid sequence having sequence identity.

[0270] In one embodiment, the extracellular antigen binding domain has at least 50, at least 100 amino acids selected from SEQ ID NO: 150. At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least a light chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; and SEQ ID NO: 144 and 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 At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% It contains a heavy chain variable region that contains an amino acid sequence having sequence identity.

[0271] In one embodiment, the extracellular antigen binding domain has at least 50, at least 100 amino acids selected from SEQ ID NO: 151 and SEQ ID NO: 152. At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least a light chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; and SEQ ID NO: 145 and 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 At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% It contains a heavy chain variable region that contains an amino acid sequence having sequence identity.

[0272] In one embodiment, the extracellular antigen binding domain has at least 50, at least 100 amino acids selected from SEQ ID NO: 152 and SEQ ID NO: 153. At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least a light chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; and SEQ ID NO: 146 and 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 At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% It contains a heavy chain variable region that contains an amino acid sequence having sequence identity.

[0273] In one embodiment, the extracellular antigen binding domain has at least 50, at least 100 amino acids selected from SEQ ID NO: 318 and SEQ ID NO: 319. At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least a light chain variable region comprising an amino acid sequence having at least 98, or at least 99% sequence identity; and SEQ ID NO: 317 and 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 At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% It contains a heavy chain variable region that contains an amino acid sequence having sequence identity.

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

[0275] In recombinant expression systems, the linker is a peptide linker and may be any naturally occurring Exemplary amino acids that can be included in the linker include Gly, Ser, Pr, o, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The CARs must be in the correct conformation relative to each other to retain the desired activity, such as binding to hK2. It must have a length suitable for linking VH and VL in such a way as to form a structure. be.

[0276] The linker can be about 5 to 50 amino acids in length. In some embodiments, the linker In some embodiments, the linker is about 10 to 40 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 15-20 amino acids in length. In 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 long. In some embodiments, the linker is 12 amino acids long. In some embodiments, the linker is 13 amino acids in length. In some embodiments, the linker is 14 amino acids long. In some embodiments, the linker is 16 amino acids long. In some embodiments, the linker is 17 amino acids in length. In some embodiments, the linker is 18 amino acids long. In some embodiments, the linker is 20 amino acids long. In some embodiments, the linker is 21 amino acids in length. In some embodiments, the linker is 22 amino acids long. In some embodiments, the linker is 24 amino acids long. In some embodiments, the linker is 25 amino acids in length. In some embodiments, the linker is 26 amino acids long. In some embodiments, the linker is 28 amino acids long. In some embodiments, the linker is 29 amino acids in length. In some embodiments, the linker is 30 amino acids long. In some embodiments, the linker is 32 amino acids long. In some embodiments, the linker is 33 amino acids in length. In some embodiments, the linker is 34 amino acids long. In some embodiments, the linker is 36 amino acids long. In some embodiments, the linker is 37 amino acids in length. In some embodiments, the linker is 38 amino acids long. In some embodiments, the linker is 40 amino acids long. Exemplary linkers that can be used include Gly-rich linkers, Gly- and Ser-containing linkers, and linkers, Gly- and Ala-containing linkers, Ala- and Ser-containing linkers, and other flexible linkers. It is a linker.

[0277] In one embodiment, the linker polypeptide comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the linker polypeptide has at least 50, at least 55, or at least 100 amino acids selected from SEQ ID NO:7 and SEQ ID NO:1. 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 At least 94, at least 95, at least 96, at least 97, at least 98, or In one embodiment, the linker comprises an amino acid sequence having at least 99% sequence identity. The polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 237 to 268. In embodiments, the linker polypeptide is at least one of SEQ ID NOs: 237-268. at least 50, at least 55, at least 60, at least 65, at least 70, at least At least 75, at least 80, at least 85, at least 90, at least 91, at least At least 92, at least 93, at least 94, at least 95, at least 96, at least and amino acid sequences having at least 97, at least 98, or at least 99% sequence identity with each other. include.

[0278] In one embodiment, the scFv is selected from the group consisting of SEQ ID NOs: 8 to 23, 169 to 184, and 340 to 34 3. In one embodiment, the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NO: No. 8 and 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 or the sequence of SEQ ID NO: 8. In one embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 9. 9 and at least 50, at least 55, at least 60, at least 65, at least 7 0, at least 75, at least 80, at least 85, at least 90, at least 9 1, at least 92, at least 93, at least 94, at least 95, at least 9 6. At least 97, at least 98, or at least 99% sequence identity In one embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 1. 0 and at least 50, at least 55, at least 60, at least 65, at least 7 0, at least 75, at least 80, at least 85, at least 90, at least 9 1, at least 92, at least 93, at least 94, at least 95, at least 9 6. At least 97, at least 98, or at least 99% sequence identity In one embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 10. 11 and 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 In one embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 11, or the sequence of SEQ ID NO: 12. No. 12 and at least 50, at least 55, at least 60, at least 65, at least At least 70, at least 75, at least 80, at least 85, at least 90, at least Also 91, at least 92, at least 93, at least 94, at least 95, at least have at least 96, at least 97, at least 98, or at least 99% sequence identity or the sequence of SEQ ID NO: 12. In one embodiment, the scFv comprises the amino acid sequence Number 13 and at least 50, at least 55, at least 60, at least 65, less At least 70, at least 75, at least 80, at least 85, at least 90, at least At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity or the sequence of SEQ ID NO: 13. In one embodiment, the scFv comprises an amino acid sequence having the sequence Column number 14 and at least 50, at least 55, at least 60, at least 65, at least At least 70, at least 75, at least 80, at least 85, at least 90, at least At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity or the sequence of SEQ ID NO: 14. In one embodiment, the scFv comprises an amino acid sequence having the following sequence: SEQ ID NO: 15 and 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 At least 91, at least 92, at least 93, at least 94, at least 95, at least At least 96, at least 97, at least 98, or at least 99% sequence identity In one embodiment, the scFv comprises an amino acid sequence having the following structure: , SEQ ID NO: 16 and 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 In one embodiment, the scFv comprises an amino acid sequence having the same identity as the scFv of SEQ ID NO: 16. SEQ ID NO: 17 and 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% of the sequence In one embodiment, the scF v is a sequence selected from SEQ ID NO: 18 and at least 50, at least 55, at least 60, at least 6 5, at least 70, at least 75, at least 80, at least 85, at least 9 0, at least 91, at least 92, at least 93, at least 94, at least 9 5. At least 96, at least 97, at least 98, or at least 99% In one embodiment, the sc Fv may be a sequence of SEQ ID NO: 19 and at least 50, at least 55, at least 60, 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% In one embodiment, s The cFv may have at least 50, at least 55, at least 60, or at least At least 65, at least 70, at least 75, at least 80, at least 85, at least At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% or the sequence of SEQ ID NO: 20. The scFv may have at least 50, at least 55, at least 60, or at least At least 65, at least 70, at least 75, at least 80, at least 85, at least At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99 % sequence identity, or the sequence of SEQ ID NO: 21. The scFv may comprise at least 50, at least 55, at least 60, or at least At least 65, at least 70, at least 75, at least 80, at least 85, at least At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 9 9% sequence identity, or the sequence of SEQ ID NO: 22. The scFv may have at least 50, at least 55, at least 60, or at least a few amino acids selected from SEQ ID NO: 23 and SEQ ID NO: 24. At least 65, at least 70, at least 75, at least 80, at least 85, at least At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least An amino acid sequence having 99% sequence identity, or the sequence of SEQ ID NO: 23. In the scFv, the scFv is a sequence of SEQ ID NO: 169 and at least 50, at least 55, or 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 less The amino acid sequence of SEQ ID NO: 169 has 99% sequence identity with the amino acid sequence of SEQ ID NO: 169. In an embodiment, the scFv has at least 50, at least 55, at least 100, or at least 150 amino acids selected from SEQ ID NO: 170 and SEQ ID NO: 171. At least 60, at least 65, at least 70, at least 75, at least 80, at least At least 85, at least 90, at least 91, at least 92, at least 93, at least Also 94, at least 95, at least 96, at least 97, at least 98, or an amino acid sequence having at least 99% sequence identity to, or comprising, the sequence of SEQ ID NO: 170 In one embodiment, the scFv has at least 50, at least 55, or at least 60 amino acids with SEQ ID NO: 171. 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 At least 94, at least 95, at least 96, at least 97, at least 98, or an amino acid sequence having at least 99% sequence identity, or the sequence of SEQ ID NO: 171 In one embodiment, the scFv comprises at least 50, at least 5 5, at least 60, at least 65, at least 70, at least 75, at least 8 0, at least 85, at least 90, at least 91, at least 92, at least 9 3, at least 94, at least 95, at least 96, at least 97, at least 9 8, or an amino acid sequence having at least 99% sequence identity, or SEQ ID NO: 172 In one embodiment, the scFv comprises a sequence of SEQ ID NO: 173 and at least 50 At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least an amino acid sequence having at least 98 or at least 99% sequence identity with SEQ ID NO: In one embodiment, the scFv comprises the sequence of SEQ ID NO: 174 and 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, Amino acid sequences with at least 98, or at least 99% sequence identity, or sequences In one embodiment, the scFv comprises the sequence of SEQ ID NO: 175 and 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 an amino acid sequence having 97, at least 98, or at least 99% sequence identity; or SEQ ID NO: 175. In one embodiment, the scFv comprises at least the sequence of SEQ ID NO: 176. at least 50, at least 55, at least 60, at least 65, at least 70, at least At least 75, at least 80, at least 85, at least 90, at least 91, at least At least 92, at least 93, at least 94, at least 95, at least 96, at least amino acids with at least 97, at least 98, or at least 99% sequence identity or the sequence of SEQ ID NO: 176. In one embodiment, the scFv comprises the sequence of SEQ ID NO: 177 and 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. In one embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 177. 1 78 and 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 or the sequence of SEQ ID NO: 178. In one embodiment, the scFv comprises the amino acid sequence Number 179 and at least 50, at least 55, at least 60, at least 65, at least At least 70, at least 75, at least 80, at least 85, at least 90, at least At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity or the sequence of SEQ ID NO: 179. In one embodiment, the scFv comprises an amino acid sequence having the sequence , SEQ ID NO: 180 and 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% of the sequence The amino acid sequence of SEQ ID NO: 180 is identical to the sequence of SEQ ID NO: 180. Fv may contain at least 50, at least 55, at least 60, or at least At least 65, at least 70, at least 75, at least 80, at least 85, at least At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% or the sequence of SEQ ID NO: 181. The scFv may comprise at least 50, at least 55, at least 60, or at least a few At least 65, at least 70, at least 75, at least 80, at least 85, at least At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity, or the sequence of SEQ ID NO: 182. In some embodiments, the scFv has at least 50, at least 55, at least 6 0, at least 65, at least 70, at least 75, at least 80, at least 8 5, at least 90, at least 91, at least 92, at least 93, at least 9 4, at least 95, at least 96, at least 97, at least 98, or less an amino acid sequence having at least 99% sequence identity with the sequence of SEQ ID NO: 183; In embodiments, the scFv has 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 95 ... At least 60, at least 65, at least 70, at least 75, at least 80, at least At least 85, at least 90, at least 91, at least 92, at least 93, at least At least 94, at least 95, at least 96, at least 97, at least 98, or an amino acid sequence having at least 99% sequence identity with the sequence of SEQ ID NO: 184. In one embodiment, the scFv has 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 an amino acid sequence having at least 99% sequence identity, or the sequence of SEQ ID NO: 340 In one embodiment, the scFv comprises at least 50 sequences of SEQ ID NO: 341 and 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 an amino acid sequence having at least 99% sequence identity, or SEQ ID NO: 34 In one embodiment, the scFv comprises the sequence of SEQ ID NO: 342 and at least 50, At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least an amino acid sequence having at least 98, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: In one embodiment, the scFv comprises the sequence of SEQ ID NO: 343 and 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 , an amino acid sequence having at least 98, or at least 99% sequence identity; or It comprises the sequence of SEQ ID NO: 343.

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

[0280] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising an extracellular antigen-binding domain, the extracellular antigen-binding domain being SEQ ID NO: 29. A compound selected from the group consisting of 44, 78 to 81, 221 to 236, and 273 to 308 SEQ ID NOs: 29-44, 78-81, 221-236, and 273-3 CA having an extracellular antigen-binding domain comprising an amino acid sequence selected from the group consisting of: Another feature of R is that the extracellular antigen-binding domain binds to the hK2 antigen.

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

[0282] In one embodiment, the CD137 component comprises the amino acid sequence of SEQ ID NO: 27. In the present specification, the CD137 component is a sequence of SEQ ID NO: 27 and 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 less Both contain amino acid sequences with 99% sequence identity.

[0283] In one embodiment, the CD3z component comprises the amino acid sequence of SEQ ID NO: 28. The CD3z component may comprise a sequence selected from the group consisting of SEQ ID NO: 28 and 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 It contains an amino acid sequence with 99% sequence identity.

[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 at least 50, at least 100, or at least 15 ... At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least It comprises an amino acid sequence with at least 98, or at least 99% sequence identity.

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

[0286] In one embodiment, the transmembrane domain is selected from the α, β, or ζ chain of a T cell receptor, CD28, C D3 epsilon, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, C D22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, In another embodiment, the transmembrane domain of the CD137 or CD154 is included. In is ζ, η, or FcεR1γ and -β, MB1 (Igα), B29, or CD3-γ In another embodiment, the transmembrane domains are The amino acids are synthetic, e.g., triplets of leucine and valine, phenylalanine, or contains primarily hydrophobic residues such as tryptophan.

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

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

[0289] h) In one such embodiment, the hinge region comprises a C CD8α hinge region and the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 26. The intracellular signaling domain is the CD3-zeta domain and It comprises 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 human having the amino acid sequence of SEQ ID NO: 25. The transmembrane domain is a CD8α-TM comprising the amino acid sequence of SEQ ID NO: 26. domain, and the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 27. -1BB / CD137 domain and CD3-zeta domain comprising the amino acid sequence of SEQ ID NO: 28 Including the main.

[0291] In one embodiment, the extracellular antigen-binding domain is selected from the group consisting of SEQ ID NOs: 29-44, 78-81, 22 In one embodiment, the amino acid sequence is selected from the group consisting of 1 to 236 and 273 to 308. In some embodiments, the extracellular antigen-binding domain comprises a sequence identical to SEQ ID NO: 29 and at least 50, at least 55, or , 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 an amino acid sequence having at least 99% sequence identity, or the sequence of SEQ ID NO: 29 In one embodiment, the extracellular antigen binding domain comprises a sequence identical to SEQ ID NO: 30 and 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 , an amino acid sequence having at least 98, or at least 99% sequence identity; or In one embodiment, the extracellular antigen-binding domain comprises the sequence of SEQ ID NO: 31. and 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. The extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 31. SEQ ID NO: 32 and 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% of the sequence or the sequence of SEQ ID NO: 32. The antigen-binding domain may be a sequence identical to SEQ ID NO: 33 and at least 50, at least 55, or 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 less The amino acid sequence of SEQ ID NO: 33 has 99% sequence identity with the amino acid sequence of SEQ ID NO: 33. In some embodiments, the extracellular antigen-binding domain comprises a sequence identical to SEQ ID NO: 34 and 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 an amino acid sequence having at least 99% sequence identity, or the sequence of SEQ ID NO: 34 In one embodiment, the extracellular antigen binding domain comprises a sequence identical to SEQ ID NO: 35 and 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 , an amino acid sequence having at least 98, or at least 99% sequence identity; or In one embodiment, the extracellular antigen-binding domain comprises the sequence of SEQ ID NO: 36. and 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. The extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 36. SEQ ID NO: 37 and 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% of the sequence or the sequence of SEQ ID NO: 37. The antigen-binding domain may be a sequence identical to SEQ ID NO: 38 and at least 50, at least 55, or 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 less The amino acid sequence of SEQ ID NO: 38 has 99% sequence identity with the amino acid sequence of SEQ ID NO: 38. In some embodiments, the extracellular antigen-binding domain comprises a sequence identical to SEQ ID NO: 39 and 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 an amino acid sequence having at least 99% sequence identity, or the sequence of SEQ ID NO: 39 In one embodiment, the extracellular antigen binding domain comprises a sequence identical to SEQ ID NO: 40 and 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 , an amino acid sequence having at least 98, or at least 99% sequence identity; or In one embodiment, the extracellular antigen-binding domain comprises the sequence of SEQ ID NO: 41. and 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. The extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 41. is SEQ ID NO: 42 and 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% of the sequence or the sequence of SEQ ID NO: 42. The antigen-binding domain may be a sequence identical to SEQ ID NO: 43 and at least 50, at least 55, or 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 less The amino acid sequence of SEQ ID NO: 43 has 99% sequence identity with the amino acid sequence of SEQ ID NO: 43. In some embodiments, the extracellular antigen-binding domain comprises a sequence identical to SEQ ID NO: 44 and 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 an amino acid sequence having at least 99% sequence identity, or the sequence of SEQ ID NO: 44 In one embodiment, the extracellular antigen binding domain comprises a sequence identical to SEQ ID NO: 78 and 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 , an amino acid sequence having at least 98, or at least 99% sequence identity; or In one embodiment, the extracellular antigen-binding domain comprises the sequence of SEQ ID NO: 79. and 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. The extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 79. SEQ ID NO: 80 and 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% of the sequence or the sequence of SEQ ID NO: 80. In one embodiment, the extracellular The antigen-binding domain may comprise at least 50, at least 55, or 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 less The amino acid sequence of SEQ ID NO: 81 has 99% sequence identity with the amino acid sequence of SEQ ID NO: 81. In one embodiment, the extracellular antigen-binding domain has at least 50, at least 5 5, at least 60, at least 65, at least 70, at least 75, at least 8 0, at least 85, at least 90, at least 91, at least 92, at least 9 3, at least 94, at least 95, at least 96, at least 97, at least 9 8, or an amino acid sequence having at least 99% sequence identity, or SEQ ID NO: 221 In one embodiment, the extracellular antigen binding domain comprises the sequence of at least SEQ ID NO: 222. At least 50, at least 55, at least 60, at least 65, at least 70, at least At least 75, at least 80, at least 85, at least 90, at least 91, at least At least 92, at least 93, at least 94, at least 95, at least 96, at least an amino acid sequence having at least 97, at least 98, or at least 99% sequence identity or SEQ ID NO: 222. In one embodiment, the extracellular antigen binding domain comprises the sequence Number 223 and 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 At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity or the sequence of SEQ ID NO: 223. In one embodiment, the extracellular antigen The binding domain may be a sequence identical to SEQ ID NO: 224 and 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 The amino acid sequence of SEQ ID NO: 224 has at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 224. In one embodiment, the extracellular antigen-binding domain has at least 50, at least 5 5 , 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 an amino acid sequence having at least 99% sequence identity, or SEQ ID NO: 225 In one embodiment, the extracellular antigen-binding domain comprises the sequence SEQ ID NO: 226 and 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 an amino acid sequence having 97, at least 98, or at least 99% sequence identity; or SEQ ID NO: 226. In one embodiment, the extracellular antigen binding domain comprises the sequence of SEQ ID NO: No. 227 and at least 50, at least 55, at least 60, at least 65, less At least 70, at least 75, at least 80, at least 85, at least 90, at least At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity or the sequence of SEQ ID NO: 227. In one embodiment, the extracellular antigen binding The binding domain may have at least 50, at least 55, at least 60, or at least At least 65, at least 70, at least 75, at least 80, at least 85, at least At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity, or the sequence of SEQ ID NO: 228. In some embodiments, the extracellular antigen-binding domain comprises at least 50, at least 55, or both of SEQ ID NO: 229 and SEQ ID NO: 230. , 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 an amino acid sequence having at least 99% sequence identity, or SEQ ID NO: 229 In one embodiment, the extracellular antigen binding domain comprises the sequence SEQ ID NO: 230 and 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 an amino acid sequence having 97, at least 98, or at least 99% sequence identity; or SEQ ID NO: 230. In one embodiment, the extracellular antigen binding domain comprises the sequence of SEQ ID NO: No. 231 and at least 50, at least 55, at least 60, at least 65, less At least 70, at least 75, at least 80, at least 85, at least 90, at least At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity or the sequence of SEQ ID NO: 231. In one embodiment, the extracellular antigen binding The binding domain may be at least 50, at least 55, at least 60, or at least At least 65, at least 70, at least 75, at least 80, at least 85, at least At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity, or the sequence of SEQ ID NO: 232. In some embodiments, the extracellular antigen-binding domain comprises at least 50, at least 55, or both of SEQ ID NO: 233 and SEQ ID NO: 234. , 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 an amino acid sequence having at least 99% sequence identity, or SEQ ID NO: 233 In one embodiment, the extracellular antigen-binding domain comprises the sequence SEQ ID NO: 234 and 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 an amino acid sequence having 97, at least 98, or at least 99% sequence identity; or SEQ ID NO: 234. In one embodiment, the extracellular antigen binding domain comprises the sequence of SEQ ID NO: No. 235 and at least 50, at least 55, at least 60, at least 65, less At least 70, at least 75, at least 80, at least 85, at least 90, at least At least 91, at least 92, at least 93, at least 94, at least 95, at least at least 96, at least 97, at least 98, or at least 99% sequence identity or the sequence of SEQ ID NO: 235. In one embodiment, the extracellular antigen binding The binding domain may be at least 50, at least 55, at least 60, or at least At least 65, at least 70, at least 75, at least 80, at least 85, at least At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least It comprises an amino acid sequence with 99% sequence identity or the sequence of SEQ ID NO: 236.

[0292] CAR constructs and CAR-expressing immunoresponsive cells The term "encoding" refers to the encoding of a defined sequence of nucleotides (e.g., rR an organism with either a defined sequence of amino acids (RNA, tRNA, and mRNA) or Serve as templates for the synthesis of other polymers and macromolecules in biological processes Identification of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, It refers to the inherent properties of the sequence of a cell, as well as the biological properties that result from it. In other biological systems, transcription and translation of mRNA corresponding to a gene produces a protein. In this case, the gene, cDNA, or RNA encodes a protein. the coding strand, whose sequence is identical to the mRNA sequence, and the template for transcription of the gene or cDNA Any non-coding strand used as a coding sequence may be used to encode a protein or other product of that gene or cDNA. It may be referred to as encoding an entity.

[0293] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" are degenerate sequences of each other. All nucleotide sequences encoding the same amino acid sequence are The phrase "nucleotide sequence encoding a protein or RNA" also includes In some cases, the nucleotide sequence encoding the protein may contain introns. It may include introns to the extent necessary.

[0294] The term "expression vector" refers to a vector that contains a nucleotide sequence operably linked to it to be expressed. An expression vector refers to a vector containing a recombinant polynucleotide that includes an expression control sequence. , containing sufficient cis-acting elements for expression, and other elements for expression are present in the host cell. The expression vector can be provided in a recombinant form, such as a recombinant vector, or in an in vitro expression system. Cosmids, plasmids (e.g., naked or liposomal) incorporating polynucleotides containing), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, any vector known in the art, including vectors such as avian flu, flu virus, and adeno-associated virus. Examples include:

[0295] In one embodiment, the present disclosure provides a cell that expresses a nucleic acid molecule encoding a CAR. In one embodiment, the CAR of the present disclosure is selected from the group consisting of SEQ ID NOs: 46-61, 82-85, and 20 In some embodiments, the present invention comprises an amino acid sequence selected from the group consisting of 5 to 220. The disclosed CARs may comprise at least 50, at least 55, at least 60, or at least 10 ... At least 65, at least 70, at least 75, at least 80, at least 85, at least At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity, or the sequence of SEQ ID NO: 46. In embodiments, a CAR of the disclosure comprises at least 50, at least 55, or at least 60 ... 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 At least 94, at least 95, at least 96, at least 97, at least 98, or an amino acid sequence having at least 99% sequence identity, or the sequence of SEQ ID NO: 47 In some embodiments, a CAR of the present disclosure comprises at least 50, at least 100, or at least 15 ... At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least an amino acid sequence having at least 98, or at least 99% sequence identity with the sequence In some embodiments, the CAR of the present disclosure comprises the sequence of SEQ ID NO: 49 and at least one of SEQ ID NO: 48. At least 50, at least 55, at least 60, at least 65, at least 70, at least 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 Amino acids with at least 97, at least 98, or at least 99% sequence identity or the sequence of SEQ ID NO: 49. In some embodiments, the CAR of the present disclosure comprises SEQ ID NO: 50 and 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 At least 91, at least 92, at least 93, at least 94, at least 95, at least At least 96, at least 97, at least 98, or at least 99% sequence identity In some embodiments, the present invention comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 50. The disclosed CARs may comprise at least 50, at least 55, at least 60, or at least 10 ... At least 65, at least 70, at least 75, at least 80, at least 85, at least At least 90, at least 91, at least 92, at least 93, at least 94, at least At least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity, or the sequence of SEQ ID NO: 51. In embodiments, a CAR of the present disclosure comprises at least 50, at least 55, or at least 60, or at least 65 ... 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 At least 94, at least 95, at least 96, at least 97, at least 98, or an amino acid sequence having at least 99% sequence identity, or the sequence of SEQ ID NO: 52 In some embodiments, a CAR of the present disclosure comprises at least 50, at least 100, or ... At least 55, at least 60, at least 65, at least 70, at least 75, at least At least 80, at least 85, at least 90, at least 91, at least 92, at least At least 93, at least 94, at least 95, at least 96, at least 97, at least an amino acid sequence having at least 98, or at least 99% sequence identity with the sequence In some embodiments, the CAR of the present disclosure comprises the sequence of SEQ ID NO: 54. At least 50, at least 55, at least 60, at least 65, at least 70, at least 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 Amino acids with at least 97, at least 98, or at least 99% sequence identity In some embodiments, the CAR of the present disclosure comprises the sequence of SEQ ID NO: 54. SEQ ID NO: 55 and 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 At least 91, at least 92, at least 93, at least 94, at least 95, at least At least 96, at least 97, at least 98, or at least 99% sequence identity In some embodiments, the present invention comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 55. The disclosed CARs may comprise at least 50, at least 55, at least 60, or at least 10 ... At least 65, at least 70, at least 75, at least 80, at least 85, at least At least 90, at least 91,...

Claims

1. A chimeric antigen receptor (CAR), (a) an extracellular domain that specifically binds to the human kallikrein-2 (hK2) antigen; (b) a transmembrane domain; and (c) an intracellular signaling domain that optionally includes at least one costimulatory domain; and, Including, CAR.

2. The CAR according to claim 1, (d) CD8a-hinge region Further comprising: the transmembrane domain comprises a CD8a transmembrane region (CD8a-TM) polypeptide; The intracellular signaling domain is a TNF receptor superfamily member 9 (C D137) component and the T cell surface glycoprotein CD3 zeta chain (CD 3z) A CAR comprising a primary signaling domain comprising a moiety.

3. The CAR according to claim 2, The CD8a-hinge region is selected from SEQ ID NOs: 25, 269, 270, 271, or 27 2, comprising an amino acid sequence that is at least 90% identical to 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 is an amino acid sequence that is at least 90% identical to SEQ ID NO:

27. a costimulatory domain having an amino acid sequence at least 90% identical to SEQ ID NO:28; A CAR comprising a primary signaling domain having an amino acid sequence.

4. An extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. A chimeric antigen receptor (CAR), wherein the extracellular antigen-binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, SEQ ID NO:6 4, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:

66. CDR3; 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; heavy chain CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 309, SEQ ID NO: heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 310, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, SEQ ID NO:6 5, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:

66. CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, SEQ ID NO:7 3, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:

66. CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 86, SEQ ID NO: 8 7, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:

88. CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:92, SEQ ID NO:9 3, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:

94. CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 103, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, SEQ ID NO: heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 106, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 107, SEQ ID NO: heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 108, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 110, SEQ ID NO: heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 111, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 112, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 114, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 115, SEQ ID NO: heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 116, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 118, SEQ ID NO: heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 119, and heavy chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, SEQ ID NO: heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 121, and a heavy chain CDR3 comprising: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 123, SEQ ID NO: heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 124, and heavy chain CDR3, Including, The extracellular antigen-binding domain binds to an hK2 antigen.

5. An extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. A chimeric antigen receptor (CAR), wherein the extracellular antigen-binding domain comprises: Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, SEQ ID NO:6 9, and a light chain CDR2 having the amino acid sequence of SEQ ID NO:

71. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68, SEQ ID NO:7 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO:

71. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, SEQ ID NO:9 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO:

91. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:95, SEQ ID NO:9 6, and a light chain CDR2 having the amino acid sequence of SEQ ID NO:

71. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 312, SEQ ID NO: a light chain CDR2 having an amino acid sequence of SEQ ID NO: 313, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 91; Light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, SEQ ID NO:9 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO:

91. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, SEQ ID NO:1 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 26, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 128, SEQ ID NO: a light chain CDR2 having an amino acid sequence of SEQ ID NO: 129, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 130; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 125, SEQ ID NO: a light chain CDR2 having an amino acid sequence of SEQ ID NO: 131, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 132; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 133, SEQ ID NO: a light chain CDR2 having an amino acid sequence of SEQ ID NO: 134, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 135; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 136, SEQ ID NO: a light chain CDR2 having an amino acid sequence of SEQ ID NO: 137, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 138; a light chain CDR3 comprising: Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:97, SEQ ID NO:1 and a light chain CDR2 having the amino acid sequence of SEQ ID NO:

139. Light chain CDR3, Including, The CAR, wherein the extracellular antigen-binding domain binds to an hK2 antigen.

6. 5. The CAR of claim 4, wherein the extracellular antigen-binding domain comprises: Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, SEQ ID NO:6 9, and a light chain CDR2 having the amino acid sequence of SEQ ID NO:

71. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68, SEQ ID NO:7 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO:

71. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, SEQ ID NO:9 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO:

91. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:95, SEQ ID NO:9 6, and a light chain CDR2 having the amino acid sequence of SEQ ID NO:

71. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, SEQ ID NO:9 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO:

91. CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, SEQ ID NO:1 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 26, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 128, SEQ ID NO: a light chain CDR2 having an amino acid sequence of SEQ ID NO: 129, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 130; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 125, SEQ ID NO: a light chain CDR2 having an amino acid sequence of SEQ ID NO: 131, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 132; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 133, SEQ ID NO: a light chain CDR2 having an amino acid sequence of SEQ ID NO: 134, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 135; light chain CDR3; Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 136, SEQ ID NO: a light chain CDR2 having an amino acid sequence of SEQ ID NO: 137, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 138; a light chain CDR3 comprising: Light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:97, SEQ ID NO:1 and a light chain CDR2 having the amino acid sequence of SEQ ID NO:

139. Light chain CDR3, The CAR further comprises:

7. 7. The CAR of claim 6, wherein the extracellular antigen-binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, SEQ ID NO:6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO:4, heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67; Light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, Light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, SEQ ID NO:6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO:5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68; Light chain CDR2 having the amino acid sequence of SEQ ID NO:70, light chain CDR3 having the amino acid sequence of SEQ ID NO:71 Light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, SEQ ID NO:6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO:5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67; Light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, SEQ ID NO:6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO:5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67; Light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, Light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, SEQ ID NO:6 heavy chain CDR2 having the amino acid sequence of SEQ ID NO:5, heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66 R3, and a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67; Light chain CDR2 having the amino acid sequence of SEQ ID NO: 126, light chain CDR3 having the amino acid sequence of SEQ ID NO: 71 light chain CDR3; Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 314, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO:311; and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

312. 1), a light chain CDR2 having the amino acid sequence of SEQ ID NO:313, a light chain CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 103, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104 and light chain complementarity determining region 1 (CDR2) having the amino acid sequence of SEQ ID NO:

128. 1), 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 CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 107, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 109; and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

125. 1), a light chain CDR2 having the amino acid sequence of SEQ ID NO: 131, and an amino acid sequence of SEQ ID NO: 132 a light chain CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO:113; and light chain complementarity determining region 1 (CDR2) having the amino acid sequence of SEQ ID NO:

133. 1), a light chain CDR2 having the amino acid sequence of SEQ ID NO: 134, and an amino acid sequence of SEQ ID NO: 135 a light chain CDR3 having the sequence: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 115, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO:117; and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

136. 1), a light chain CDR2 having the amino acid sequence of SEQ ID NO: 137, and an amino acid sequence of SEQ ID NO: 138 a light chain CDR3 having the sequence Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, SEQ ID NO: heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 122; and light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:

97. 127; light chain CDR2 having the amino acid sequence of SEQ ID NO: 138; A CAR comprising a light chain CDR3 having the sequence:

8. The extracellular antigen-binding domain has the amino acid sequence of SEQ ID NO:

86. Region 1 (CDR1), heavy chain having the amino acid sequence of SEQ ID NO: 87 CDR2, SEQ ID NO: 88 and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

89. Complementarity determining region 1 (CDR1), light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, sequence The CAR of claim 6, comprising a light chain CDR3 having an amino acid sequence of number 91.

9. An extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. A chimeric antigen receptor (CAR), wherein the extracellular antigen-binding domain comprises: SEQ ID NOs: 1, 2, 3, 74, 75, 147, 148, 149, 150, 151, 152 and / or a light chain variable domain (VL) comprising an amino acid sequence selected from teeth SEQ ID NOs: 4, 5, 6, 76, 77, 140, 141, 142, 143, 144, 145 a heavy chain variable domain (VH) comprising an amino acid sequence selected from: Including, The extracellular antigen-binding domain binds to the human kallikrein-2 (hK2) antigen. A.R.

10. 10. The CAR of claim 9, wherein 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. Chain variable region; 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 Chain variable region; 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. Chain variable region; 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. Chain variable region; 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. Chain variable region; 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. Chain variable region; 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. Chain variable region; 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. Chain variable region; 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. Chain variable region; 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. Chain variable region; 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. Chain variable region; A light chain variable region comprising the amino acid sequence of SEQ ID NO:74 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

76. A heavy chain variable region; A light chain variable region comprising the amino acid sequence of SEQ ID NO:75 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

77. A heavy chain variable region; A light chain variable region comprising the amino acid sequence of SEQ ID NO: 147 and the amino acid sequence of SEQ ID NO: 140 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and the amino acid sequence of SEQ ID NO: 141 A heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 75 and the amino acid sequence of SEQ ID NO: 140 A heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 148 and the amino acid sequence of SEQ ID NO: 142 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 149 and the amino acid sequence of SEQ ID NO: 143 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 150 and the amino acid sequence of SEQ ID NO: 144 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 151 and the amino acid sequence of SEQ ID NO: 145 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO: 152 and the amino acid sequence of SEQ ID NO: 146 a heavy chain variable region comprising: A light chain variable region comprising the amino acid sequence of SEQ ID NO:318 and the amino acid sequence of SEQ ID NO:317 a heavy chain variable region comprising Including, CAR.

11. the extracellular antigen-binding domain comprises a single chain variable fragment (scFv), 11. The method according to claim 1, comprising a light chain variable region (VL) and a heavy chain variable region (VH). The CAR described herein.

12. The scFv has a linker between the light chain variable region (VL) and the heavy chain variable region (VH). The CAR of claim 11, comprising a CAR polypeptide.

13. The linker polypeptide of claim 12 comprises the amino acid sequence of SEQ ID NO:

7. C.A.R.

14. The scFv is selected from the group consisting of SEQ ID NOs: 8-23, 169-184, and 340-343. The CAR according to any one of claims 11 to 13, comprising an amino acid sequence selected from the group consisting of:

15. The method according to any one of claims 1 to 14, wherein the extracellular antigen-binding domain comprises a signal polypeptide. The CAR described in any one of the above.

16. The method of claim 15, wherein the signal polypeptide comprises the amino acid sequence of SEQ ID NO:

24. CAR.

17. The intracellular signaling domain is a TNF receptor superfamily member 9 (C D137) component, T cell surface glycoprotein CD3 zeta chain (CD3z) component, surface antigen component Class (CD27) components, cluster of differentiation superfamily member components, and combinations thereof The method according to any one of claims 4 to 16, comprising administering to a patient a polypeptide component selected from the group consisting of The CAR described herein.

18. The CA of claim 17, wherein the CD137 component comprises the amino acid sequence of SEQ ID NO:

27. R.

19. 19. The method of claim 17 or 18, wherein the CD3z component comprises the amino acid sequence of SEQ ID NO:

28. CAR.

20. The intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 45; or Optionally, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

27. domain and a primary signaling domain comprising the amino acid sequence of SEQ ID NO:

28. The CAR according to any one of items 17 to 19.

21. the transmembrane domain comprises a CD8a transmembrane region (CD8a-TM) polypeptide; The CAR according to any one of claims 4 to 20.

22. 21. The CD8a-TM polypeptide comprising the amino acid sequence of SEQ ID NO:

26. A CAR described in.

23. further comprising a hinge region connecting the transmembrane domain to the extracellular antigen binding domain. The CAR according to any one of claims 4 to 22.

24. The CAR of claim 23, wherein the hinge region is a CD8a-hinge region.

25. The CD8a-hinge region is selected from the group consisting of SEQ ID NOs: 25, 269, 270, 271, and 272. The CAR of claim 24, comprising an amino acid sequence.

26. The extracellular antigen-binding domain is selected from the group consisting of SEQ ID NOs: 29-44, 78-81, 221-236, 273-308, The CAR described in any one of the above.

27. An aminium selected from the group consisting of SEQ ID NOs: 46-61, 82-85, and 205-220. The CAR according to any one of claims 1 to 26, comprising a nucleotide sequence.

28. An isolated lymphocyte expressing the CAR according to any one of claims 1 to 27.

29. 29. The isolated lymphocyte of claim 28, which is a T lymphocyte.

30. 29. The isolated lymphocyte of claim 28, which is a natural killer (NK) cell.

31. An isolated nucleic acid molecule encoding the CAR of any one of claims 1 to 27.

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

33. A cell expressing the nucleic acid molecule of claim 31.

34. A pharmaceutical composition comprising an effective amount of lymphocytes according to any one of claims 28 to 30.

35. An effective amount of lymphocytes according to any one of claims 28 to 30 and a pharma- ceutical acceptable excipient. and a pharmaceutical composition comprising:

36. A CAR according to any one of claims 1 to 27, or claim 1 for use in therapy.

36. The pharmaceutical composition according to claim 34 or 35.

37. A method for treating a subject with cancer according to any one of claims 1 to 27. CAR, or the pharmaceutical composition of claim 34 or 35.

38. 1. A method of treating a subject having cancer, comprising: A therapeutically effective amount of the lymphocytes according to any one of claims 28 to 30 is administered to a patient in need thereof. administering the compound to a subject, thereby inducing said lymphocytes to kill cancer cells in said subject. and

39. 39. The method of claim 38, wherein the cancer is prostate cancer or androgen receptor expressing breast cancer. or a CAR or pharmaceutical composition for use in the method of claim 37.

40. 1. A method for targeted killing of cancer cells, comprising: The cancer cells are contacted with lymphocytes according to any one of claims 28 to 30, thereby wherein said lymphocytes induce killing of said cancer cells.

41. 41. The method of claim 40, wherein the cancer cell is a prostate cancer cell or an androgen receptor-expressing breast cancer cell. The method described above.

42. 1. A method for detecting the presence of cancer in a subject, comprising: Contacting a cell sample obtained from the subject with the CAR according to any one of claims 1 to 27. thereby forming a CAR-cell complex; and detecting said complex, wherein detecting said complex indicates the presence of cancer in said subject. Indicates presence, A method comprising:

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