Anti-HK2 chimeric antigen receptor (CAR)

Abstract

The present disclosure provides chimeric antigen receptors (CARs) that specifically target human kallikrein 2 (hK2), and immune responsive cells comprising such CARs, for use in the treatment of cancer.

Description

[0001] This application is a divisional application of the invention application filed on July 23, 2020, with Chinese national application number 202080054021.5 and invention title "Anti-HK2 Chimeric Antigen Receptor (CAR)". Technical Field

[0002] This invention relates to a chimeric antigen receptor (CAR) targeting hK2 comprising a single-stranded variable fragment targeting hK2, and engineered hK2-targeting immune cells expressing the CAR. It also provides a nucleic acid encoding the CAR and an expression vector, recombinant cells comprising the vector, and compositions comprising engineered immune cells expressing the hK2-targeting CAR. Methods for preparing the CAR and engineered immune cells are also provided, as well as methods for treating diseases, including cancer, using the engineered immune cells. Background Technology

[0003] For men, prostate cancer is the second most frequently diagnosed cancer and the sixth leading cause of cancer death, accounting for 14% (903,500) of all new cancer cases and 6% (258,400) of all cancer deaths worldwide. Based on disease severity, hormonal status, and the presence of detectable metastases, the progression of prostate cancer from diagnosis to death is most appropriately classified into the following series of clinical stages: localized disease, elevated prostate-specific antigen (PSA) levels after radiation therapy or surgery without detectable metastases, and clinical metastases in the non-castration or castration phase. While surgery, radiation, or a combination of both may be effective for patients with localized disease, a significant proportion of these patients experience recurrence, as evidenced by elevated PSA levels. Recurrence can lead to further metastasis, particularly in high-risk groups, progressing to the lethal stage of the disease.

[0004] Androgen depletion therapy (ADT) is the standard treatment with the following generally predictable outcomes: decreased PSA levels, a stable period of tumor non-proliferation, followed by elevated PSA levels and regrowth into castration-resistant disease. Historically, ADT has been the standard of care for patients with metastatic prostate cancer.

[0005] Kallikrein-associated peptidase 2 (hK2, HK2) is a trypsin-like enzyme with androgen receptor (AR)-driven expression specific to prostate tissue and prostate cancer. hK2 is activated by the transmembrane protease serine 2 (TMPRSS2) and secreted into the prostatic ducts. In the prostatic ducts, hK2 triggers a cascade reaction that cleaves seminal vesicle proteins (the extracellular matrix in seminal fluid) to enhance sperm motility. hK2 expression is restricted to prostate and prostate cancer tissues; however, it has recently been demonstrated that hK2 can be detected in breast cancer cell lines and primary patient samples after appropriate activation of the AR pathway by steroid hormones (US Patent Publication 2018 / 0326102). Similar to PSA, when the highly structured tissue of the prostate is damaged due to hypertrophy or malignant transformation, catalytically inactive hK2 is retrogradely released into the bloodstream.

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

[0007] This article discloses chimeric antigen receptor (CAR) (e.g., CAR targeting human kallikrein-2 (hK2)), CAR-containing cells, CAR-encoding vectors (e.g., recombinant expression vectors), and nucleic acid molecules encoding CAR, methods for preparing CAR, compositions, peptides, proteins, nucleic acids, host cells, cell populations, and methods for treating diseases (e.g., cancer) using the disclosed CAR.

[0008] In one aspect, a chimeric antigen receptor (CAR) is provided, the chimeric antigen receptor comprising:

[0009] (a) An extracellular domain containing scFv and specifically binding to the human kallikrein-2 (hK2) antigen.

[0010] (b) Transmembrane domains; and

[0011] (c) An intracellular signal transduction domain, which optionally includes at least one co-stimulatory domain.

[0012] In some implementations, the CAR also includes:

[0013] (d)CD8a - Hinge area,

[0014] The transmembrane domain contains the CD8a transmembrane region (CD8a-TM) polypeptide; and

[0015] The intracellular signal transduction domain includes a co-stimulatory domain and a primary signal transduction domain. The co-stimulatory domain contains the TNF receptor superfamily member 9 (CD137) component, and the primary signal transduction domain contains the T cell surface glycoprotein CD3ζ chain (CD3z) component.

[0016] In some embodiments, the CD8a hinge region contains an amino acid sequence that is at least 90% identical to SEQ ID NO:25, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271 or SEQ ID NO:272;

[0017] The transmembrane domain contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:26; and / or

[0018] The intracellular signal transduction domain includes a costimulatory domain and a primary signal transduction domain. The costimulatory domain has an amino acid sequence that is at least 90% identical to that of SEQ ID NO:27, and the primary signal transduction domain has an amino acid sequence that is at least 90% identical to that of SEQ ID NO:28.

[0019] On the other hand, a chimeric antigen receptor (CAR) is provided, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0020] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:64, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66.

[0021] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:314, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:315, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:311.

[0022] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:309, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:310, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:311.

[0023] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66.

[0024] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:73, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66.

[0025] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:86, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:87, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:88.

[0026] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:92, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:93, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:94;

[0027] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:103, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:104.

[0028] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:105, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:106, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:104.

[0029] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:107, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:108, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:109.

[0030] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:110, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:111, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:109.

[0031] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:112, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:113.

[0032] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:105, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:114, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:113.

[0033] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:115, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:116, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:117.

[0034] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:118, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:119, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:117.

[0035] Heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:120, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:121, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:122; or

[0036] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:123, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:124, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:122.

[0037] The extracellular antigen-binding domain binds to the hK2 antigen.

[0038] On the other hand, a chimeric antigen receptor (CAR) is provided, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0039] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0040] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68, the light chain CDR2 having the amino acid sequence of SEQ ID NO:70, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0041] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, the light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91;

[0042] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:95, the light chain CDR2 having the amino acid sequence of SEQ ID NO:96, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0043] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:312, the light chain CDR2 having the amino acid sequence of SEQ ID NO:313, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91.

[0044] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, the light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91;

[0045] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0046] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:128, the light chain CDR2 having the amino acid sequence of SEQ ID NO:129, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:130;

[0047] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:125, the light chain CDR2 having the amino acid sequence of SEQ ID NO:131, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:132;

[0048] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:133, the light chain CDR2 having the amino acid sequence of SEQ ID NO:134, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:135.

[0049] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:136, the light chain CDR2 having the amino acid sequence of SEQ ID NO:137, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:138; or

[0050] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, the light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, and the light chain CDR3 having the amino acid sequence of SEQ ID NO: 139.

[0051] The extracellular antigen-binding domain binds to the hK2 antigen.

[0052] In some implementations, the extracellular antigen-binding domain further includes:

[0053] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71; or

[0054] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68, the light chain CDR2 having the amino acid sequence of SEQ ID NO:70, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0055] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, the light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91;

[0056] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:95, the light chain CDR2 having the amino acid sequence of SEQ ID NO:96, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0057] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:312, the light chain CDR2 having the amino acid sequence of SEQ ID NO:313, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91.

[0058] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, the light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91;

[0059] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0060] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:128, the light chain CDR2 having the amino acid sequence of SEQ ID NO:129, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:130;

[0061] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:125, the light chain CDR2 having the amino acid sequence of SEQ ID NO:131, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:132;

[0062] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:133, the light chain CDR2 having the amino acid sequence of SEQ ID NO:134, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:135.

[0063] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:136, the light chain CDR2 having the amino acid sequence of SEQ ID NO:137, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:138; or

[0064] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:97, the light chain CDR2 having the amino acid sequence of SEQ ID NO:127, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:139.

[0065] On the other hand, a chimeric antigen receptor (CAR) is provided, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0066] A light chain variable domain (VL) comprising an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152 and SEQ ID NO:318; and / or

[0067] A heavy chain variable domain (VH) comprising an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146 and SEQ ID NO:317;

[0068] The extracellular antigen-binding domain binds to the human kallikrein-2 (hK2) antigen.

[0069] In some implementations, the extracellular antigen-binding domain includes:

[0070] The light chain variable region containing the amino acid sequence of SEQ ID NO:1 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:4;

[0071] The light chain variable region containing the amino acid sequence of SEQ ID NO:2 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:4;

[0072] The light chain variable region containing the amino acid sequence of SEQ ID NO:3 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:6;

[0073] The light chain variable region containing the amino acid sequence of SEQ ID NO:3 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:4;

[0074] The light chain variable region containing the amino acid sequence of SEQ ID NO:2 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:6;

[0075] The light chain variable region containing the amino acid sequence of SEQ ID NO:3 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:4;

[0076] The light chain variable region containing the amino acid sequence of SEQ ID NO:1 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:6;

[0077] The light chain variable region containing the amino acid sequence of SEQ ID NO:3 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:5;

[0078] The light chain variable region containing the amino acid sequence of SEQ ID NO:1 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:5;

[0079] The light chain variable region containing the amino acid sequence of SEQ ID NO:2 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:5;

[0080] The light chain variable region containing the amino acid sequence of SEQ ID NO:3 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:5;

[0081] The light chain variable region containing the amino acid sequence of SEQ ID NO:74 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:76;

[0082] The light chain variable region containing the amino acid sequence of SEQ ID NO:75 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:77;

[0083] The light chain variable region containing the amino acid sequence of SEQ ID NO:147 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:140;

[0084] The light chain variable region containing the amino acid sequence of SEQ ID NO:75 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:141;

[0085] The light chain variable region containing the amino acid sequence of SEQ ID NO:75 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:140;

[0086] The light chain variable region containing the amino acid sequence of SEQ ID NO:148 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:142;

[0087] The light chain variable region containing the amino acid sequence of SEQ ID NO:149 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:143;

[0088] The light chain variable region containing the amino acid sequence of SEQ ID NO:150 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:144;

[0089] The light chain variable region containing the amino acid sequence of SEQ ID NO:151 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:145;

[0090] The light chain variable region containing the amino acid sequence of SEQ ID NO:152 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:146;

[0091] The light chain variable region containing the amino acid sequence of SEQ ID NO:318 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:317.

[0092] In some embodiments, the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv comprises a linker polypeptide located between the light chain variable region (VL) and the heavy chain variable region (VH). In some embodiments, the linker polypeptide comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, the scFv comprises an amino acid sequence selected from SEQ ID NO:8-23, SEQ ID NO:169-184, and SEQ ID NO:340-343. In some embodiments, the extracellular antigen-binding domain comprises a signaling polypeptide. In some embodiments, the signaling polypeptide comprises the amino acid sequence of SEQ ID NO:24.

[0093] In some embodiments, the intracellular signal transduction domain comprises a polypeptide component selected from: a TNF receptor superfamily member 9 (CD137) component, a T cell surface glycoprotein CD3ζ chain (CD3z) component, a differentiation cluster (CD27) component, a differentiation cluster superfamily member component, and combinations thereof. In some embodiments, the CD137 component comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, the CD3z component comprises the amino acid sequence of SEQ ID NO:28. In some embodiments, the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:45. In some embodiments, the transmembrane domain comprises the CD8a transmembrane region (CD8a-TM) polypeptide. In some embodiments, the CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO:26.

[0094] In various embodiments, the CAR further includes a hinge region connecting the transmembrane domain to the extracellular antigen-binding domain. In some embodiments, the hinge region is a CD8a hinge region. In some embodiments, the CD8a hinge region includes an amino acid sequence of SEQ ID NO:25, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271, or SEQ ID NO:272. In some embodiments, the extracellular antigen-binding domain includes an amino acid sequence selected from SEQ ID NO:29-44, SEQ ID NO:78-81, SEQ ID NO:221-236, and SEQ ID NO:273-308. In some embodiments, the CAR includes an amino acid sequence selected from SEQ ID NO:46-61, SEQ ID NO:82-85, and SEQ ID NO:205-220.

[0095] On the other hand, isolated lymphocytes expressing any of the aforementioned CARs are provided. In some embodiments, the lymphocytes are T lymphocytes. In some embodiments, the lymphocytes are natural killer (NK) cells.

[0096] Also provided are isolated nucleic acid molecules encoding any of the aforementioned CARs. A vector containing the nucleic acid molecules is also provided. Furthermore, cells expressing the nucleic acid molecules are also provided.

[0097] Also provided is a pharmaceutical composition comprising an effective amount of any one of the above lymphocytes and a pharmaceutically acceptable excipient.

[0098] In one aspect, this disclosure provides a CAR according to this disclosure for use in treatment methods.

[0099] In one aspect, this disclosure provides lymphocytes according to this disclosure for use in treatment methods.

[0100] In one aspect, this disclosure provides compositions according to this disclosure, such as pharmaceutical compositions, for use in treatment methods.

[0101] In one aspect, this disclosure provides a CAR according to this disclosure for use in methods of treating cancer.

[0102] In one aspect, this disclosure provides lymphocytes according to this disclosure for use in methods of treating cancer.

[0103] In one aspect, this disclosure provides compositions according to this disclosure, such as pharmaceutical compositions, for use in methods of treating cancer.

[0104] In one implementation, the cancer is prostate cancer.

[0105] In one implementation, the cancer is the expression of the androgen receptor (AR) in breast cancer.

[0106] On the other hand, a method for treating a subject with cancer is provided. This method involves administering a therapeutically effective amount of any one of the above-mentioned lymphocytes to the subject in need, thereby inducing the lymphocytes to kill cancer cells in the subject. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is an androgen receptor (AR) expressing breast cancer.

[0107] On the other hand, a method for targeted killing of cancer cells is provided, comprising contacting the cancer cells with any of the lymphocytes mentioned above, 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 an androgen receptor (AR) expressing breast cancer.

[0108] On the other hand, a method for detecting the presence of cancer in a subject is provided, the method comprising:

[0109] (a) Expose a cell sample obtained from the subject to any of the above-mentioned CARs.

[0110] This leads to the formation of the CAR-cell complex, and

[0111] (b) Detection of the complex, wherein detection of the complex indicates the presence of cancer in the subject. Attached Figure Description

[0112] The foregoing will become apparent from the following more specific description of the exemplary embodiments shown in the accompanying drawings.

[0113] This patent application contains at least one color drawing. Upon request and payment of the necessary fees, the U.S. Patent and Trademark Office will provide a color copy of this patent application.

[0114] Figure 1A-1C The construction and expression of hK2 CAR on human T cells are shown. Figure 1A A schematic diagram of the construct used for hK2 CAR is shown; TM: transmembrane. Figure 1B and Figure 1C This illustrates hK2 CAR expression on the surface of T cells. Primary human T cells were electroporated using either no mRNA (MOCK) or 10 μg of mRNA expressing hK2 scFv CAR or an unrelated control CAR. The cells were then electroporated with PE (penicillin-conjugated PE) containing 2 μg / mL biotinylated L protein and streptavidin. Figure 1B ) or biotinylated hK2 (1 μg / mL) and streptavidin-conjugated PE ( Figure 1C After staining, CAR surface expression was measured by flow cytometry 24 hours after electroporation.

[0115] Figure 2 This study demonstrates the cytotoxicity of CAR-T cells against hK2-expressing tumor cells in a 20-hour flow-based assay at a specified effector-to-target (E / T) ratio. Target cells (Vcap and DU145) were labeled with Cell Trace Violet (CTV) fluorescent dye 24 hours after transient transfection and then co-cultured with hK2 CAR-T cells. Mock T cells served as a negative effector control. The kill percentage 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 not co-cultured with CAR-T cells.

[0116] Figures 3A-3B This demonstrates real-time hK2-specific CAR-T cell-mediated cytotoxicity. Target cells were incubated for approximately 72 hours with Mock, 10 μg mRNA electroporated (24 hours post-transfection), hK211B6CAR LH, or control CAR-T cells at different E:T ratios. Figure 3A VCap (5E4 cells) and ( Figure 3BNormalized Cell Index (CI) curves of DU145 (5E3 cells). When seeded alone, target cells adhere to the plate and proliferate, thus increasing the CI reading. When T cells were added to the target cells, hK2 CAR and control CAR-T cells caused hK2(+) Vcap cell lysis and a subsequent decrease in CI at E / T ratios ranging from 5:1 to 0.156:1. The decrease in CI value after the addition of effector cells reflects the loss of viability of the target cells. Parallel experiments using hK2(-) ​​DU145 target cells were also performed, as shown in (…). Figure 3B As shown in the figure; at the E:T ratio tested in this experiment, hK2 and control CART cells did not exhibit cell lysis activity and did not decrease CI after addition. The Y-axis is the normalized CI generated and displayed in real time by RTCA software. The X-axis is the cell culture time and treatment time in hours. The mean ± standard deviation of CI is plotted.

[0117] Figure 4 This demonstrates the production of the Th1 cytokine interferon-γ (IFN-γ) in antigen-stimulated CAR-T cells. Supernatants were collected from approximately 70 hours of co-culture (4 cells of VCap#5E and 3 cells of DU145#5E) using an xCELLigence-based kill assay (see [link to assay]). Figures 3A-3B hK2CAR LH and control CAR-modified T cells secreted IFN-γ during co-culture with hK2-expressing VCap cells (but non-hK2-negative DU145 cells). Mean IFN-γ concentration ± standard deviation (pg / mL) of replicate cultures is shown.

[0118] Figures 5A-5B This demonstrates the evaluation of the antigen-dependent activity of the 11B6 thermostable scFv CAR clone in the JNL reporter gene assay. Figure 5A As shown, Jurkat cells (referred to as JNL cells) containing a luciferase gene driven by a signal transduction-responsive NFAT promoter were transduced using various hK2CAR constructs. Expression was confirmed via biotinylated hK2 followed by streptavidin-conjugated PE. Figure 5B This study demonstrates that binding between the CAR construct and its homologous cellular antigen (hK2 on target cells) leads to luciferase expression in JNL cells. JNL cells containing the specified CAR clone and untransduced JNL cells (UTDs) were co-cultured with target cell lines (VCap or DU145 cells), and luciferase activity was measured by luminescence intensity. In the presence of cells expressing the antigen, a clone was considered active when the luminescence intensity exceeded 1.5 times the level in UTD cells.

[0119] Figure 6hK2 CAR expression on the surface of T cells is shown. Primary human T cells were transduced with a thermostable 11B6 lentivirus based on parental scFv (multiple of infection (MOI): 3), and CAR expression was determined 14 days post-transduction by biotinylated hK2 (1 μg / mL), followed by streptavidin-conjugated PE.

[0120] Figures 7A-7B This study demonstrates the evaluation of antigen-dependent cytotoxicity of 11B6 thermostable scFv CAR-T cells using a real-time IncuCyte kill assay. Thermostable hK2 CAR-T cells and parental 11B6 CAR-T cells were compared with VCaP cells (…). Figure 7A ) and PC3 cells ( Figure 7B Incubate at room temperature for 88 hours. Calculate the effector to target cell (E / T) ratio based on CAR expression data (see...). Figure 6 The target cells stably expressed the red nuclear dye, which was measured in real time using the 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 (%).

[0121] Figure 7C This shows the percentage of tumor cell growth inhibition against hK2-positive VCaP cells by T cells transduced with CAR1-10 or parental 11B6_HL or 11B6_LH at a 1:1 or 0.5:1 effector:target cell ratio in a real-time IncuCyte killing assay for assessing antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (Initial number of viable target cells - Current number of viable target cells) / Initial number of viable cells * 100 (%).

[0122] Figure 7D This shows the percentage of tumor cell growth inhibition on PC3 cells by T cells transduced with CAR1-10 or parental 11B6_HL or 11B6_LH at a 1:1 effector:target cell ratio in a real-time IncuCyte killing assay for assessing antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (Initial number of viable target cells - Current number of viable target cells) / Initial number of viable cells * 100 (%).

[0123] Figure 7EThe figure shows the percentage of tumor cell growth inhibition against 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 for assessing antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (initial number of viable target cells - current number of viable target cells) / initial number of viable cells * 100 (%).

[0124] Figure 7F The figure shows the percentage of 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 the real-time IncuCyte killing assay for assessing antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (initial number of viable target cells - current number of viable target cells) / initial number of viable cells * 100 (%).

[0125] Figure 8 Cytokine release from hK2 lenti-CAR-T cells is shown. Supernatants collected from overnight (approximately 20 hours) co-cultures of hK2 CAR-T cells and 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 secreted cytokines during co-culture with hK2-expressing VCap cells, but secreted the least for untransduced T cells (UTDs). Mean cytokine concentrations ± standard deviation (pg / mL) of replicate cultures are shown.

[0126] Figure 9 This illustrates the release of IFN-γ from hK2 lenti-CAR-T cells. Supernatants were collected from overnight (approximately 20 hours) co-cultures of hK2 CAR-T cells with VCap and DU145 (5E4 cells) cells at a 1:1 E / T ratio. hK2 CAR-modified T cells secreted IFN-γ during co-culture with hK2-expressing VCap cells (but not hK2-negative DU145 cells). Mean IFN-γ concentration ± standard deviation (pg / mL) of replicate cultures is shown. CD3 / 28 beads stimulated T cells, and T cells alone served as positive and negative controls, respectively.

[0127] Figures 10A-10E The results of the hK2 CAR-T cell proliferation assay are shown. Figure 10A , Figure 10ChK2 CAR and untransduced (UTD) T cells were labeled with CellTrace Violet (CTV; 5 μM) and co-cultured with hK2(+) VCap cells and hK2(-) ​​DU145 cells. Five days after co-culture, cells were harvested and stained with CD3, CD25, Live / dead near-infrared 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 with CTV dye dilution and the activation marker CD25 was determined. Figure 10B , 10D CD3 / 28 beads stimulated T cells, and T cells alone were used as positive and negative controls, respectively. Figure 10E Data showed that hK2 CAR+ T cells proliferated more robustly than the CD3 / 28 mitochondrial-positive control after 5 days of co-culture with VCap cells. Different CAR constructs engineered T cells exhibited different proliferative activities and displayed different CAR+ T cell counts. CAR+ T cell counts were based on mean absolute cell counts from three parallel assays using SEM.

[0128] Figures 11A-11B The results show the determination of hK2 CAR expression levels in primary T cells using biotin-labeled hK2 protein. Figure 11A This is a schematic diagram of the construct used for hK2 CAR; TM: transmembrane. In Figure 11B In this study, primary human T cells were transduced with novel hK2 scFv-based KL2B413HL&LH and heavily humanized 11B6 KL2B359 HL&LH CAR lentiviruses (MOI 3), and CAR expression was determined 14 days post-transduction by biotinylated hK2 (1 μg / mL) followed by streptavidin-conjugated PE. Figure 11B A summary of the percentage of hK2 CAR+ T cells (positive%) detected by the novel KL2B413 HL&LH and heavily humanized 11B6 KL2B359HL&LH CARs analyzed is provided. As shown in the figure, different clones had different CAR expression levels, ranging from 45.1% to 59.9%. All CAR T cells were normalized to be equivalent CAR+ T cells for subsequent functional assays.

[0129] Figures 12A-12B The results show the evaluation of the antigen-dependent activity of novel hK2 KL2B413 and rehumanized 11B6 CAR clones in the JNL reporter gene assay. Figure 12AIn this study, Jurkat cells (referred to as JNL cells) containing a luciferase gene driven by a signal transduction-responsive NFAT promoter were transduced using various hK2 CAR constructs. Expression was confirmed via biotinylated hK2 followed by streptavidin-conjugated PE. CAR expression in transduced JNL cells was confirmed for selected clones in which 46%–50% of cells expressed the rehumanized 11B6KL2B359 CAR and 73.7%–96% of cells expressed the KL2B413 CAR. Figure 12B The data shown indicate that binding between the hK2 CAR construct and its homologous cellular antigen (hK2 on target cells) leads to luciferase expression in JNL cells. JNL cells containing the specified CAR clone and JNL cells (UTDs) were co-cultured with target cell lines (VCap, LNCap / Hk2, LNCap, C4-2B, 22Rv1, or DU145 cells), and luciferase activity was measured by luminescence intensity. In the presence of cells expressing the antigen, the clone was considered active when the luminescence intensity exceeded 1.5 times the level of UTD cells. No antigen-dependent activation was observed in the novel KL2B413 and rehumanized 11B6 HL&LH CARs.

[0130] Figures 13A-13B The results of evaluating the antigen-dependent cytotoxicity of HK2 CAR-T cells using a real-time IncuCyte kill assay are shown. HK2 CAR-T cells were compared with HK2+VCaP cells (… Figure 13A ) and HK2-DU145 cells ( Figure 13B Incubate at room temperature for 96 hours. Calculate the effector to target cell (ET) ratio based on CAR expression data. Figure 11B The target cells stably expressed the red nuclear dye, which was measured in real time using the 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 (%)

[0131] Figure 14This figure shows data indicating Th1 cytokine IFN-γ production by CAR-T cells stimulated by antigens. IFN-γ produced by cytotoxic T cells is crucial for administering immune surveillance of tumors, which can directly inhibit proliferation and induce apoptosis in some malignancies both in vivo and in vitro. To determine whether hK2 CAR-modified human T cells can recognize and be activated by hK2(+) tumor cells, primary T cells containing the specified CAR clone and control untransduced T cells (UTDs) were co-cultured with target cell lines (LNCap / Hk2, LNCap, C4-2B, 22Rv1, or DU145 cells), and the supernatant was collected for IFN-γ concentration measurement. As shown in the figure, hK2 CAR-modified T cells secreted IFN-γ during co-culture with hK2-expressing LNCap / hK2 cells (and even very few Hk2-expressing C4-2B and LNCap cells, but not non-hK2-negative DU145 cells). The undisclosed control CAR secreted significantly more IFN-γ due to much higher antigen expression levels than hK2. Mean IFN-γ concentrations ± SD (pg / mL) from replicate cultures are shown.

[0132] Figure 15 The results of the CD107a degranulation assay are shown to evaluate the antitumor activity of hK2 CAR T cells. Target cells (5 × 10⁻⁶) were used. 4 (Number of cells) were co-cultured in 96-well plates with an equal number of effector cells in 0.1 mL per well. Control wells contained only T cells. Anti-CD107a (5 μL per well) was added in addition to 1 μL / monensin sample (BD Biosciences), and the cells were incubated at 37°C for 4 h. Cells were washed twice with PBS, stained for hK2 CAR, CD3, and CD8 expression, and analyzed on a BD Fortessa flow cytometer. As shown in the figure, the presence of hK2 antigen on Vcap cells led to an increase in CD107A+hK2 CAR(+) T cells, but not in hK2 CAR(-) and UTD T cells, as measured by flow cytometry, while only background staining (<2%) was observed in CAR T cells that were not stimulated by tumor cells. Phorbolol 12-tetradecanoate 13-acetate (PMA) and iomycin (eBiosciences) were used. TM The cell stimulation mixture (500X) was a consistent inducer of CD107a cell surface expression in this 4-hour cell culture model and was used as a positive control. Results from representative donor T cells are shown, and these results are reproducible in multiple donors.

[0133] Figures 16A-16DResults 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 cells and HK2(-) ​​DU145 cells. Five days after co-culture, cells were harvested and stained with CD3, CD25, Live / dead near-infrared dye, and hK2 CAR. 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 with CTV dye dilution and the activation marker CD25 was determined. Gating was performed on CD3+ T cells, such as... Figures 16A-16B As shown, hK2(+) Vcap cells but non-hK2(-) ​​DU145 cells promote the proliferation of all CAR construct engineered T cells and upregulate the activation marker CD25, such as Figures 16C-16D As shown. CD3 / 28 beads stimulated T cells, and T cells alone served as positive and negative controls, respectively. Only unstimulated T cells did not proliferate, and CD3 / 28 bead-stimulated T cells exhibited an equivalent proliferation pattern. As shown in the figure, after 5 days of co-culture with VCap cells, hK2 CAR+ T cells proliferated more robustly than the CD3 / 28 bead-positive control. Different CAR constructs-engineered T cells exhibited different proliferative activities and showed different CAR+ T cell counts. A summary of the percentage of proliferating T cells and CD25-expressing T cells based on mean absolute cell count + / - SEM of replicate cultures is shown in the figure. Figure 16B and Figure 16D .

[0134] Figure 17A The sequence comparison of the VH domains of mu11B6, hu11B6, KL2B357, KL2B358, KL2B359, KL2B360, HCF3, and HCG5 is shown. Figure 17A SEQ ID NO:317, SEQ ID NO:5, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:77, SEQ ID NO:140, SEQ ID NO:6, SEQ ID NO:4 and SEQ ID NO:345 are disclosed in appearance order. Figure 17B The protected fragment is mapped onto the sequence of the hK2 antigen to visualize the binding epitopes of the hK2 antibody identified by HDX-MS. Figure 17BSEQ ID NO:351, SEQ ID NO:351, SEQ ID NO:351, SEQ ID NO:351, SEQ ID NO:351 and SEQ ID NO:351 are disclosed in appearance order respectively. Detailed Implementation

[0135] An exemplary implementation scheme is described below:

[0136] This disclosure provides chimeric antigen receptors (CARs) that target human kallikrein-2 (hK2), cells containing such CARs, and methods for treating cancers (e.g., prostate cancer or breast cancer expressing AR) using the CARs described herein.

[0137] The CAR of this invention is antigen-specific to hK2. As used herein, the phrases "antigen-specific" and "provides an antigen-specific response" mean that the CAR can specifically bind to and immunely recognize the antigen, such that the binding of the CAR to the hK2 antigen triggers an immune response. Methods for testing the antigen specificity of the CAR and its ability to recognize target cells are known in the art.

[0138] This disclosure also provides related nucleic acids, recombinant expression vectors, host cells, cell populations, antibodies or antigen-binding portions thereof, and pharmaceutical compositions related to the CAR of the present invention.

[0139] Several aspects of the invention are described below with reference to embodiments for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are set forth to provide a comprehensive understanding of the invention. However, those skilled in the art will readily recognize that the invention can be practiced without one or more specific details, or can be practiced using other methods, schemes, reagents, cell lines, and animals. The invention is not limited to the order in which actions or events are shown, as some actions may occur in a different order and / or simultaneously with other actions or events. Furthermore, not all shown actions, steps, or events are necessary for implementing the method according to the invention. Many techniques and procedures described or referenced herein are well known to those skilled in the art and are generally adopted by those skilled in the art using conventional methods.

[0140] Unless otherwise specified, all technical terms, symbols, and other scientific terms or specialized expressions used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, for clarity and / or ease of reference, terms are defined herein with their commonly understood meanings, and these definitions should not necessarily be construed as indicating a significant difference from the meanings commonly understood in the art. It should also be understood that terms (such as those defined in common dictionaries) should be interpreted as having meanings consistent with their meanings in the relevant field context and / or as otherwise defined herein.

[0141] When a list is provided, unless otherwise indicated, it should be understood that each individual element in the list and each combination of the list is a separate implementation. For example, a list of implementations presented as “A, B or C” will be understood to include implementations “A”, “B”, “C”, “A or B”, “A or C”, “B or C”, or “A, B or C”.

[0142] definition

[0143] As used in this specification and the appended claims, unless otherwise expressly stated, the singular forms “a,” “an,” and “described” include plural references. Thus, for example, a reference to “a cell” includes a combination of two or more cells, and so on.

[0144] The transitional terms “comprising,” “substantially consisting of,” and “consisting of” are intended to imply their accepted meaning in patent terminology; that is, (i) “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open at the end, and does not exclude additional, unlisted elements or method steps; (ii) “consisting of” excludes any element, step, or component not specified in the claims; and (iii) “substantially consisting of” limits the scope of the claims to the specified material or step “and material or step that does not substantially affect the essential and novel features of the invention protected by the claims.” Embodiments described with the phrase “comprising” (or its equivalents) are also provided, as are those described independently with “consisting of” and “substantially consisting of”.

[0145] "Activation" or "stimulation" refers to the induction of changes in the biological state of cells, leading to the expression of activation markers, cytokine production, proliferation, or mediating cytotoxicity of target cells. Cells can be activated by primary stimuli. Co-stimulatory signals can amplify the magnitude of primary signals and inhibit cell death following initial stimulation, resulting in a more persistent state of activation and therefore greater cytotoxic capacity. "Co-stimulatory signals" are signals that, in combination with primary signals (such as TCR / CD3 linkages), lead to the upregulation or downregulation of T cell and / or NK cell proliferation and / or key molecules.

[0146] An "antigen-binding fragment" or "antigen-binding domain" refers to the portion of a protein that binds to an antigen. Antigen-binding fragments can be synthetic, enzymatically obtained, or genetically engineered polypeptides, and include antigen-binding portions of immunoglobulins such as VH, VL, VH, and VL; Fab, Fab', F(ab')2, Fd, and Fv fragments; domain antibodies (dAbs) consisting of a VH domain or a VL domain; shark variable IgNAR domains; humped VH domains; VHH domains; minimal recognition units consisting of amino acid residues of CDRs of antibody mimics such as FR3-CDR3-FR4, HCDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3; alternative scaffolds for antigen binding; and multispecific proteins containing antigen-binding fragments. Antigen-binding fragments (such as VH and VL) can be linked together via synthetic linkers to form various types of monoclonal antibody designs. Where the VH and VL domains are expressed as separate single chains, the VH / VL domains can be paired intramolecularly or intermolecularly to form monovalent antigen-binding domains, such as single-chain Fv (scFv) or bivalent antibodies. Antigen-binding fragments can also be conjugated to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds, which can be monospecific or multispecific, engineered to be bispecific or multispecific proteins.

[0147] "Cancer" refers to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" can include tumors.

[0148] A full-length antibody consists of two heavy chains (HC) and two light chains (LC) linked by disulfide bonds and their polymers (e.g., IgM). Each heavy chain comprises a variable domain (VH) and a constant domain, the constant domain consisting of subdomains CH1, hinge, CH2, and CH3. Each light chain comprises a variable domain (VL) and a constant domain (CL). VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL consists of three CDRs and four FR segments, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0149] "Human antibody" refers to an antibody optimized to produce a minimal immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. If a human antibody contains a constant region or a portion of a constant region, that constant region is also derived from a human immunoglobulin sequence. If the variable region of a human antibody is obtained using a system that uses human germline immunoglobulins or rearranged immunoglobulin genes, the human antibody contains both heavy-chain and light-chain variable regions "derived" from human-origin sequences. Exemplary systems of this kind include human immunoglobulin gene libraries displayed on bacteriophages and transgenic nonhuman animals, such as mice or rats carrying human immunoglobulin loci. Due to differences between the systems used to obtain human antibodies and human immunoglobulin loci, the introduction of somatic mutations, or the intentional introduction of substitutions into the frame or CDR, or both, "human antibodies" typically contain amino acid differences compared to immunoglobulins expressed in humans. Typically, the amino acid sequence of a “human antibody” has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence encoded by a human germline immunoglobulin gene or rearranged immunoglobulin gene. In some cases, a “human antibody” may contain a common frame sequence derived from human frame sequence analysis (e.g., as described in Knappik et al., (2000) J Mol Biol 296:57-86), or be bound to a synthetic HCDR3 in a human immunoglobulin gene library displayed on a bacteriophage (e.g., as described in Shi et al., (2010) J Mol Biol 397:385-96 and International Patent Publication WO2009 / 085462). The definition of "human antibody" does not include antibodies with at least one CDR derived from a non-human species.

[0150] "Humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one frame is derived from a human immunoglobulin sequence. Humanized antibodies may contain substitutions in the frame such that the frame may not be an exact copy of the expressed human immunoglobulin or the germline gene sequence of a human immunoglobulin.

[0151] "Isolated" refers to a homogeneous group of molecules (such as synthetic polynucleotides or polypeptides) that have been substantially isolated and / or purified from other components of the system that produced the molecules (such as recombinant cells), as well as proteins that have undergone at least one purification or isolation step. "Isolated" also refers to molecules that are substantially free of other cellular material and / or chemicals, and encompasses molecules isolated to higher purities (such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity).

[0152] "Modulation" refers to the enhancement or reduction mediated by the test molecule compared to a response mediated by a control or mediator. The ability to enhance or reduce the response (i.e., downstream effect).

[0153] The terms "natural killer cells" and "NK cells" are used interchangeably and synonymously in this article. NK cells refer to cells with CD16+. + CD56 + and / or CD57 + TCR - Differentiated lymphocytes are phenotypes. NK cells are characterized by their ability to bind to and kill cells that do not express "self" MHC / HLA antigens by activating specific lysinases, to kill tumor cells or other diseased cells that express ligands of NK activation receptors, and to release protein molecules called cytokines that stimulate or inhibit immune responses.

[0154] "Specific binding" or "binding" refers to the binding of protein molecules to an antigen or an epitope within an antigen with a greater affinity than to other antigens. Typically, protein molecules bind with an affinity of approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 5×10) -8 M or smaller, approximately 1×10 -8 M or smaller, approximately 1×10 -9 M or smaller, approximately 1×10 -10 M or smaller, approximately 1×10 -11 M or smaller, or approximately 1×10 -12 The equilibrium dissociation constant (K) of (or less) D ) binds to antigens or epitopes within antigens, usually K D Compared to its binding to non-specific antigens (such as BSA, casein), K DAt least one hundred times smaller. In the context of prostate neoantigen as described in this article, "specific binding" refers to the binding of protein molecules to the prostate neoantigen, but not to detectable binding to wild-type proteins that are variants of the neoantigen.

[0155] "Tumor cells" or "cancer cells" refer to cancerous, precancerous, or transformed cells in vivo, in vitro, or in tissue cultures that exhibit spontaneous or induced phenotypic changes. These changes do not necessarily involve the uptake of new genetic material. However, transformation can occur due to infection with transforming viruses and the binding of new genomic nucleic acids, the uptake of exogenous nucleic acids, or it can occur spontaneously or after exposure to carcinogens, thereby causing mutations in endogenous genes. Examples of transformation / cancer include morphological changes in vitro, in vivo, and in vitro in suitable animal hosts (such as nude mice), cell immortalization, abnormal growth control, lesion formation, proliferation, malignancy, regulation of tumor-specific marker levels, invasion, and tumor growth.

[0156] As used herein, the term "chimeric antigen receptor" or "CAR" is defined as a cell surface receptor comprising an extracellular target-binding domain, a transmembrane domain, and an intracellular signaling domain, all of which are present together non-naturally on a single protein. This specifically includes receptors in which the extracellular domain and the intracellular signaling domain are present together non-naturally on a single receptor protein. The chimeric antigen receptor of the present invention is primarily intended for use with lymphocytes such as T cells and natural killer (NK) cells.

[0157] The terms “T cell” and “T lymphocyte” are interchangeable and used synonymously herein. As used herein, T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+CD8+ T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for a particular implementation include naive T cells and memory T cells. Also included is “NKT cells,” which refers to a specialized population of T cells that expresses the semi-invariant αβ T cell receptor but also expresses multiple molecular markers commonly associated with NK cells, such as NK1.1. NKT cells include NK1.1+ and NK1.1- cells, as well as CD4+, CD4-, CD8+, and CD8- cells. The unique feature of the TCR on NKT cells is that it recognizes glycolipid antigens presented by the MHC I-like molecule CD1d. Because NKT cells can produce cytokines that promote inflammation or immune tolerance, they can have protective or destructive effects. Also included are "γ-δT cells (γδT cells)," a specialized subset of T cells with unique TCRs on their surface. Unlike most T cells, where the TCR consists of two glycoprotein chains named α-TCR and β-TCR, the TCR in γδT cells consists of both γ- and δ-chains. γδT cells can play a role in immune surveillance and immune regulation and have been found to be an important source of IL-17 and induce strong CD8+ cytotoxic T cell responses. Also included are "regulatory T cells" or "Tregs," which are T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Tregs are typically Foxp3-positive CD4+ T cells, and may also include Foxp3-negative regulatory T cells, which are IL-10-producing CD4+ T cells.

[0158] The terms “natural killer cell” and “NK cell” are interchangeable and used synonymously herein. As used herein, NK cells refer to differentiated lymphocytes with a CD16+CD56+ and / or CD57+TCR- phenotype. NK cells are characterized by their ability to bind to and kill cells that do not express “self” MHC / HLA antigens by activating specific cytolytic enzymes, to kill tumor cells or other diseased cells expressing ligands of the NK activation receptor, and to release protein molecules called cytokines that stimulate or suppress immune responses.

[0159] As used herein, the term "antigen" refers to any reagent molecule (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, parts thereof, or combinations thereof) capable of being bound by T-cell receptors. Antigens can also elicit an immune response. Examples of an immune response may involve, but are not limited to, antibody production, or activation of specific immune-active cells, or both. Those skilled in the art will understand that antigens do not necessarily need to be encoded by "genes." It will be apparent that antigens can be generated synthetically or derived from biological samples, or can be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or fluids containing other biological components, organisms, protein / antigen subunits, killed or inactivated whole cells, or lysates.

[0160] The term "antibody" refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), intracellular antibodies, microantibodies, biantibodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against antigen-specific TCRs), and epitope-binding fragments of any of the above antibodies. The term "antibody" also refers to covalent biantibodies, such as those disclosed in U.S. Patent Application Publication 2007 / 0004909; and Ig-DARTS, such as those disclosed in U.S. Patent Application Publication 2009 / 0060910. Antibodies that can be used as TCR-binding molecules include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, and IgA2), or subclass.

[0161] The term "host cell" refers to any cell containing heterologous nucleic acid. Heterologous nucleic acid can be a vector (e.g., an expression vector). For example, a host cell can be a cell from any organism that is selected, modified, transformed, grown, used, or manipulated in any way for the cellular expression of substances produced by the cell, such as genes, DNA or RNA sequences, proteins, or enzymes. An appropriate host can be determined. For example, a host cell can be selected based on the vector backbone and the desired outcome. By way of example, plasmids or granules can be introduced into prokaryotic host cells to replicate several types of vectors. Bacterial cells (such as, but not limited to, DH5α, JM109, and KCB) Competent cells and SOLOPACK Gold cells can be used as host cells for vector replication and / or expression. Additionally, bacterial cells such as *Escherichia coli* LE392 can be used as host cells against bacteriophage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to, yeast (e.g., YPH499, YPH500, and YPH501), insects, and mammals. Examples of mammalian eukaryotic host cells used for vector replication and / or expression include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12.

[0162] The host cells disclosed herein include T cells and natural killer cells, which contain DNA or RNA sequences encoding CARs and express CARs on their cell surface. These host cells can be used to enhance T cell activity, natural killer cell activity, and to treat cancer and autoimmune diseases.

[0163] "Activation" or "stimulation" refers to the induction of a change in the biological state of a cell, through which cells (e.g., T cells and NK cells) express activation markers, produce cytokines, proliferate, and / or become cytotoxic to target cells. All of these changes can be generated by primary stimulus signals. Co-stimulatory signals can amplify the magnitude of primary signals and inhibit cell death following initial stimulation, resulting in a more persistent state of activation and therefore greater cytotoxic capacity. "Co-stimulatory signals" refer to signals that, in combination with primary signals (such as TCR / CD3 linkages), lead to the upregulation or downregulation of T cell and / or NK cell proliferation and / or key molecules.

[0164] The term "proliferation" refers to an increase in cell division (symmetrical or asymmetrical cell division). The term "expansion" refers to the result of cell division and cell death.

[0165] The term "differentiation" refers to methods that reduce the efficiency or proliferation of cells or bring cells into a more restricted state of development.

[0166] The term "expression" refers to the permission or initiation of the production of information from a gene or DNA sequence, such as the production of proteins by activating cellular functions involved in the transcription and translation of the corresponding gene or DNA sequence. DNA sequences are expressed in or through cells to form "expression products," such as proteins. It is also said that the expression products themselves (e.g., the resulting proteins) can be "expressed" by cells. Expression products can be characterized as intracellular, extracellular, or transmembrane.

[0167] The term "transfection" refers to the introduction of "foreign" (i.e., external or extracellular) nucleic acids into a cell using recombinant DNA technology. The term "genetic modification" refers to the introduction of a "foreign" (i.e., external or extracellular) gene, DNA, or RNA sequence into a host cell, causing the host cell to express the introduced gene or sequence to produce a desired substance (typically a protein or enzyme encoded by the introduced gene or sequence). The introduced gene or sequence may also be referred to as a "cloned" or "foreign" gene or sequence and may include regulatory or control sequences operatively linked to a multinucleotide encoding a chimeric antigen receptor, such as a promoter sequence, termination sequence, promoter, signaling sequence, secretory sequence, or other sequence used by the cell's genetic mechanisms. Genes or sequences may include non-functional sequences that do not have a known function. The host cell that receives and expresses the introduced DNA or RNA has been "genetically engineered." The DNA or RNA introduced into the host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.

[0168] The term "transduction" refers to the introduction of foreign nucleic acids into cells using a viral vector.

[0169] The term "regulatory element" refers to any cis-acting genetic factor that controls some aspect of nucleic acid sequence expression. In some embodiments, the term "promoter" essentially includes the minimum sequence required to initiate transcription. In some embodiments, the term "promoter" includes the sequence that initiates transcription, and additionally includes sequences that can upregulate or downregulate transcription, often referred to as "enhancer elements" and "repressor elements," respectively.

[0170] As used herein, when referring to nucleic acids or amino acids, the term "operably linked" and similar phrases refer to the operational linking of nucleic acid or amino acid sequences, respectively placed in a functional relationship with each other. For example, operably linked promoter, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements result in transcription of open reading frames and ultimately the production of peptides (i.e., expression of open reading frames). As another example, an operably linked peptide is a peptide in which functional domains are placed at appropriate distances from each other to confer the intended function of each domain.

[0171] "Enhanced," "promoted," "increased," "amplified," or "improved" generally refers to the ability of the composition contemplated herein to produce, initiate, or induce a greater physiological response (i.e., downstream effect) compared to the response induced by the mediator or control molecule / composition. Measurable physiological responses may include increased T-cell expansion, activation, effector function, persistence, and / or increased cancer cell killing capacity, as well as responses readily apparent from the understanding in the art and the description herein. In some embodiments, the amount of "increased" or "enhanced" may be a "statistically significant" amount and may include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more (e.g., 500, 1000) in response compared to the mediator or control composition (including all integers and decimals between and above 1, such as 1.5, 1.6, 1.7, 1.8, etc.).

[0172] "Reduced," "decreased," "less," "less," or "weakened" generally refers to the ability of the composition contemplated herein to produce, initiate, or induce a smaller physiological response (i.e., downstream effect) compared to the response induced by the mediator or control molecule / composition. In some embodiments, the amount of "reduced" or "less" can be a "statistically significant" amount and can include a reduction of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more times (e.g., 500, 1000 times) compared to the response induced by the mediator, control composition (reference response), or response in a particular cell lineage (e.g., 500, 1000 times) (including all integers and decimals in between and above 1, such as 1.5, 1.6, 1.7, 1.8, etc.).

[0173] The term "effective" when applied to dosage or amount refers to the amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered alone. The exact amount required will vary from subject to subject, depending on the subject's species, age and general condition, the severity of the condition being treated, the specific medication used, the mode of administration, etc.

[0174] The phrase “pharmaceuticalally acceptable” used in conjunction with the compositions described herein refers to the molecular entity and other components of such compositions that are physiologically tolerable and generally do not produce adverse effects when administered to mammals (e.g., humans). Preferably, the term “pharmaceuticalally acceptable” means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in mammals, and more specifically in humans.

[0175] The term “protein” as used herein encompasses all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fused proteins, and modified proteins, including but not limited to glycoproteins and all other types of modified proteins (e.g., proteins obtained by phosphorylation, acetylation, myristylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation, etc.).

[0176] Unless otherwise stated, the terms “nucleic acid,” “nucleotide,” and “polynucleotide” cover both DNA and RNA. “Nucleic acid sequence” or “nucleotide sequence” means a nucleic acid sequence that encodes an amino acid; these terms may also refer to a nucleic acid sequence that includes portions encoding any amino acids added as a clone, including any amino acids encoded by a linker.

[0177] The term "carrier" refers to a diluent, excipient, or medium that is applied together with a compound. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water or aqueous solutions, saline solutions, as well as dextrose solutions and glycerol solutions, are preferred as carriers, especially for injectable solutions. Alternatively, carriers can be solid dosage form carriers, including, but not limited to, one or more of binders (for compressed pellets), gliding agents, encapsulating agents, flavoring agents, and coloring agents. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, written by EW Martin.

[0178] The term “about” or “approximately” includes a range of values ​​that are statistically significant. Such a range may be within the order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5%. The permissible deviation covered by the term “about” or “approximately” depends on the specific system studied and is readily understood by those skilled in the art.

[0179] "Kallikrein-associated peptidase 2", "hK2", or "klk2" refers to a known protein, also known as kallikrein 2, adenosine kallikrein 2, or HK2. hK2 is produced as a proprotein and cleaved during proteolysis to generate the active protease. All hK2 isoforms and variants are covered under the term "hK2". The amino acid sequences of the various isoforms can be retrieved from GenBank accessions NP_005542.1, NP_001002231.1, and NP_001243009. The full-length amino acid sequence of hK2 is shown in SEQ ID NO:62. The sequence includes a signal peptide (residues 1-18) and a propeptide region (residues 19-24).

[0180] It should be understood that the terminology used herein is for the purpose of describing specific implementations only and is not intended to be restrictive. As used herein, unless the context clearly indicates otherwise, the indefinite articles “a,” “an,” and “the” should be understood to include plural references.

[0181] Chimeric antigen receptor

[0182] This invention relates generally to the use of genetically modified T cells to stably express desired chimeric antigen receptors. Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides containing an antigen-binding domain of an antibody (scFv) linked to a T cell signaling domain. CARs can be characterized by their ability to specifically and reactivity redirect T cells to selected targets in a non-MHC-restricted manner, thereby utilizing the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition confers the ability of CAR-expressing T cells to recognize antigens independently of antigen processing, thus bypassing major mechanisms of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the α and β chains of the endogenous T cell receptor (TCR).

[0183] The CAR described herein provides a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain (also referred to herein as a "cytoplasmic signaling domain"), the intracellular signaling domain including functional signaling domains derived from stimulatory molecules as defined below. T cells expressing CAR are referred to herein as CAR T cells, CAR-T cells, or CAR-modified T cells, and these terms are used interchangeably herein. These cells can be genetically modified to stably express antibody-binding domains on their surface, thereby conferring novel MHC-independent antigen specificity.

[0184] In some cases, T cells are genetically modified to stably express a CAR that combines the antigen-recognition domain of a specific antibody with the intracellular domain of a CD3-ζ chain or an FcγRI protein into a single chimeric protein. In one embodiment, the stimulating molecule is a ζ chain associated with a T cell receptor complex.

[0185] As used herein, the terms "intracellular signaling domain" or "cytoplasmic signaling domain" refer to the intracellular portion of a molecule. Functional portions of proteins act by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers, or to act as effectors in response to such messengers. Intracellular signaling domains generate signals that promote the function of immune effectors in CAR-containing cells (e.g., CAR-T cells). Examples of immune effector functions (e.g., in CAR-T cells) include cytolytic activity and cofactor activities, including the secretion of cytokines.

[0186] In one embodiment, the intracellular signaling domain may include a primary intracellular signaling domain. Example primary intracellular signaling domains include those derived from molecules responsible for primary or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may include a co-stimulatory intracellular domain. Example co-stimulatory intracellular signaling domains include those derived from molecules responsible for co-stimulatory signals or antigen-independent stimulation. For example, in the case of CAR-T, the primary intracellular signaling domain may contain a cytoplasmic sequence of a T cell receptor, and the co-stimulatory intracellular signaling domain may contain a cytoplasmic sequence derived from a co-receptor or co-stimulatory molecule.

[0187] Primary intracellular signal transduction domains may contain signal transduction motifs, referred to as immune receptor tyrosine-based activation motifs or ITAMs. Examples of primary cytoplasmic signal transduction sequences containing ITAMs include, but are not limited to, those derived from CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d, as well as DAP10 and DAP12.

[0188] The term "ζ" or alternatively "ζ chain," "CD3-ζ," or "TCR-ζ" is defined as a protein provided with GenBank accession number BAG36664.1, or equivalent residues from non-human species (e.g., mice, rabbits, primates, rodents, monkeys, apes, etc.), and "ζ-stimulatory domain" or alternatively "CD3-ζ-stimulatory domain" or "TCR-ζ-stimulatory domain" is defined as amino acid residues from the cytoplasmic domain of the ζ chain sufficient to functionally transmit the initial signal necessary for T cell activation. Sometimes, for example, in aspects and / or embodiments of this disclosure, "ζ-stimulatory domain" or "CD3-ζ-stimulatory domain" is also referred to as a "primary signal transduction domain." In one aspect, the cytoplasmic domain of the CD3-ζ-stimulatory domain comprises residues 52 to 164 of GenBank accession number BAG36664.1, or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.) of its functional ortholog. In a preferred embodiment, the intracellular signal transduction domain comprises a CD3-ζ stimulation domain. In one aspect, the “ζ stimulation domain” or “CD3-ζ stimulation domain” is a sequence provided as SEQ ID NO:28, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:28.

[0189] The term "co-stimulatory molecule" refers to a homologous binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response in the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137).

[0190] The intracellular signal transduction domain of a costimulatory molecule can be the intracellular portion of that molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte-activating molecules (SLAM proteins), and activated NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, MyD88, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and ligands that specifically bind to CD83, etc.

[0191] Intracellular signal transduction domains can contain the entire intracellular portion of their source molecule or the entire natural intracellular signal transduction domain, or a functional fragment thereof.

[0192] The term "4-1BB" or alternatively "CD137" refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank accession number AAA62478.2, or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.); and the "4-1BB co-stimulatory domain" is defined as amino acid residues 214 to 255 of GenBank accession number AAA62478.2, or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.). Sometimes, for example, in aspects and / or embodiments of this disclosure, the "4-1BB co-stimulatory domain" is also referred to as a "co-stimulatory domain".

[0193] In a preferred embodiment, the intracellular signal transduction domain includes a co-stimulatory intracellular signal transduction domain, wherein the co-stimulatory intracellular signal transduction domain is a 4-1BB co-stimulatory domain or a "CD137 co-stimulatory domain". In one aspect, the "4-1BB co-stimulatory domain" or "CD137 co-stimulatory domain" is the sequence provided as SEQ ID NO:27, or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.), or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:27. In a preferred embodiment, the 4-1BB co-stimulatory domain is the sequence provided as SEQ ID NO:27.

[0194] In one embodiment, a transmembrane domain that naturally associates with a domain of the CAR is used. In another embodiment, the transmembrane domain may be selected or modified by amino acid substitution to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. In an example embodiment, the transmembrane domain includes a CD8α hinge domain. In one embodiment, the CAR comprises a CD8α hinge domain and a CD8α transmembrane domain.

[0195] In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined herein. In one embodiment, the co-stimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, CD3-ζ, and / or CD28. CD28 is a T cell marker important in T cell co-stimulation. CD27 is a member of the tumor necrosis factor receptor superfamily and acts as a co-stimulatory immune checkpoint molecule. 4-1BB delivers a potent co-stimulatory signal to T cells, thereby promoting T lymphocyte differentiation and enhancing long-term T lymphocyte survival. CD3-ζ associates with the TCR to generate a signal and contains an immune receptor tyrosine-based activation motif (ITAM). In another embodiment, the co-stimulatory molecule is MyD88 or CD40.

[0196] In one embodiment, the CAR comprises an intracellular hinge domain and an intracellular T-cell receptor signaling domain, wherein the intracellular hinge domain includes CD8, and the intracellular T-cell receptor signaling domain includes CD28, 4-1BB, and CD3-ζ. In another embodiment, the CAR comprises an intracellular hinge domain and an intracellular T-cell receptor signaling domain, the intracellular T-cell receptor signaling domain including CD28, 4-1BB, and CD3-ζ, wherein the hinge domain comprises all or part of the extracellular region of CD8, CD4, or CD28; all or part of the antibody constant region; and all or part of the FcyRIIIa receptor, IgG hinge, IgM hinge, IgA hinge, IgD hinge, IgE hinge, or Ig hinge. The IgG hinge may be derived from IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof.

[0197] The CAR described herein provides a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain (also referred to herein as a "cytoplasmic signal transduction domain"), the intracellular signal transduction domain including, for example, functional signal transduction domains derived from stimulatory molecules as defined below.

[0198] In one embodiment, the CAR comprises a chimeric fusion protein including an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain includes a functional signal transduction domain derived from a stimulating molecule. In another embodiment, the CAR comprises a chimeric fusion protein including an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain includes a functional signal transduction domain derived from a co-stimulating molecule and a functional signal transduction domain derived from a stimulating molecule. In yet another embodiment, the CAR comprises a chimeric fusion protein including an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain includes at least two functional signal transduction domains derived from one or more co-stimulating molecules and a functional signal transduction domain derived from a stimulating molecule.

[0199] The CAR of the present invention can be designed to include a CD28 signaling domain and / or a 4-1BB signaling domain, or in combination with any other desired cytoplasmic domains in the context of the CAR of the present invention. In one embodiment, the cytoplasmic domain of the CAR may further include the CD3-ζ signaling domain. For example, the cytoplasmic domain of the CAR may include, but is not limited to, CD3-ζ, 4-1BB, and CD28 signaling modules, and combinations thereof.

[0200] In one embodiment, the CAR includes a hinge domain and an intracellular signal transduction domain, wherein the hinge domain includes a CD8α hinge domain and a CD8α transmembrane domain, and the intracellular signal transduction domain includes CD28, 4-1BB, and CD3-ζ. In another embodiment, the CAR includes a hinge domain and an intracellular signal transduction domain, wherein the hinge domain includes a CD8α hinge domain and a CD8α transmembrane domain, and the intracellular signal transduction domain includes CD28, 4-1BB, and CD3-ζ.

[0201] In another embodiment, the CAR includes a CD8α hinge domain, a CD8α transmembrane domain, and an intracellular signal transduction domain, the intracellular signal transduction domain including the CD3-ζ and 4-1BB co-stimulatory domains.

[0202] 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 an intracellular signal transduction domain comprising a co-stimulatory domain and a primary signal transduction domain, the co-stimulatory domain comprising the amino acid sequence of SEQ ID NO:27 and the primary signal transduction domain comprising the amino acid sequence of SEQ ID NO:28.

[0203] Therefore, the present invention provides CAR T cells and methods for using them in adoptive therapy.

[0204] This disclosure also provides variants of the CARs, nucleic acids, peptides, and proteins described herein, such as functional variants. A “variant” is a peptide or polynucleotide that differs from a reference peptide or reference polynucleotide due to one or more modifications (e.g., substitution, insertion, or deletion). As used herein, the term “functional variant” refers to a CAR, peptide, or protein that has substantial or significant sequence identity or similarity to a parental CAR, peptide, or protein, and that retains the biological activity of the CAR, peptide, or protein that is a variant of it. Functional variants encompass those variants of, for example, the CARs, peptides, or proteins (parental CARs, peptides, or proteins) described herein, which retain the ability to recognize target cells to a degree, the same degree, or greater than that of the parental CAR, peptide, or protein. Regarding the parental CAR, polypeptide, or protein, the functional variant may, for example, have at least about 30%, about 40%, about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher identity with the amino acid sequence of the parental CAR, polypeptide, or protein.

[0205] In this document, the structure of a polypeptide is defined based on the % sequence identity with the reference sequence (with a given SEQ ID NO). In this context, the % sequence identity between two amino acid sequences can be determined by comparing the two sequences in an optimal alignment, wherein the amino acid sequences to be compared may contain additions or deletions relative to the reference sequence for optimal alignment between the two sequences. The identity percentage is calculated by determining the number of identical positions of amino acid residues between the two sequences, dividing the number of identical positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the identity percentage between the two sequences. Typically, the comparison window corresponds to the full length of the sequences being compared. For example, the BLAST program, “BLAST 2 sequences” (Tatusova et al., “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250), can be used. It is available at http: / / www.ncbi.nlm.nih.gov / gorf / bl2.html. The parameters used are those given by default (specifically for the parameters “open gap penalty”: 5 and “extended gap penalty”: 2; the selected matrix is, for example, the matrix “BLOSUM 62” recommended by the program). The percentage of identity between the two sequences to be compared is calculated directly by the program. Determining the sequence identity between the query sequence and the reference sequence is within the capabilities of a person skilled in the art and can be done using commercially available analytical software (such as BLAST). TM )conduct.

[0206] The functional variant may, for example, comprise the amino acid sequence of a parent CAR, peptide, or protein having at least one conserved amino acid substitution. In another embodiment, the functional variant may comprise the amino acid sequence of a parent CAR, peptide, or protein having at least one non-conserved amino acid substitution. In this case, the non-conserved amino acid substitution may not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent CAR, peptide, or protein.

[0207] The amino acid substitutions in the CAR of the present invention can be conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include substitutions in which one amino acid having specific physical and / or chemical properties is replaced by another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be the substitution of an acidic amino acid with another acidic amino acid (e.g., Asp or Glu), the substitution of an amino acid with a nonpolar side chain with another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), the substitution of a basic amino acid with another basic amino acid (Lys, Arg, etc.), the substitution of an amino acid with a polar side chain with another amino acid with a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), and so on.

[0208] The CAR, polypeptide, or protein may consist substantially of one or more specified amino acid sequences as described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0209] The CARs, peptides, and proteins (including functional moieties and functional variants) of the embodiments disclosed herein can have any length, i.e., can contain any number of amino acids, provided that the CAR, peptide, or protein (or its functional moieties or functional variants) retains its biological activity, such as the ability to specifically bind antigens, detect diseased cells (e.g., cancer cells) in the host, or treat or prevent diseases in the host. For example, the length of the peptide can be from about 50 to about 5000 amino acids, such as about 50, about 70, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 47 ... The polypeptides of this invention contain 5, approximately 500, approximately 525, approximately 550, approximately 575, approximately 600, approximately 625, approximately 650, approximately 675, approximately 700, approximately 725, approximately 750, approximately 775, approximately 800, approximately 825, approximately 850, approximately 875, approximately 900, approximately 925, approximately 950, approximately 975, approximately 1000, or more amino acids. The polypeptides of this invention also include oligopeptides.

[0210] The CARs, peptides, and proteins (including the functional portions and functional variants of the invention) of embodiments of the present invention may comprise synthetic amino acids that replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, leucine, α-aminodecanoic acid, homoserine, S-acetaminomethylcysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, α-(2-amino-2-norbornene)carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenyl Serine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, dihydroindole-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, and α-tert-butylglycine.

[0211] The CARs, peptides, and proteins (including functional moieties and functional variants) of embodiments of the present invention can undergo post-translational modifications. They can be glycosylated, esterified, N-acylated, amidated, carboxylated, phosphorylated, esterified, via, for example, disulfide cyclization, or converted into acid addition salts. In some embodiments, they are dimerized or polymerized, or conjugated.

[0212] The CARs, peptides, and / or proteins (including their functional moieties and functional variants) of embodiments of the present invention can be obtained by methods known in the art. Suitable methods for de novo synthesis of peptides and proteins are described in references such as: Chan et al., “Fmoc Solid Phase Peptide Synthesis,” Oxford University Press, Oxford, United Kingdom, 2000; “Peptide and Protein Drug Analysis,” edited by Reid, R., Marcel Dekker, Inc., 2000; and Epitope Mapping, edited by Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001. Alternatively, standard recombinant methods can be used to recombinantly generate peptides and proteins using the nucleic acids described herein. See, for example, Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001; and Ausubel et al., “Current Protocols in Molecular Biology,” Greene Publishing Associates and John Wiley & Sons, NY, 1994. Additionally, some of the CARs, peptides, and proteins of this invention (including their functional moieties and functional variants) can be isolated and / or purified from sources such as plants, bacteria, insects, mammals, etc. Methods for isolation and purification are known in the art. Alternatively, the CARs, peptides, and / or proteins described herein (including their functional moieties and functional variants) can be commercially synthesized. In this respect, the CARs, peptides, and proteins can be synthetic, recombinant, isolated, and / or purified.

[0213] Examples of modified nucleotides that can be used to generate recombinant nucleic acids for producing the polypeptides described herein include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, N... 6 -Substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylquinoline, 5″-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6 -Isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, piracetamidine, β-D-galactosylpiracetamidine, inosine, N 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl 5-oxyuracil uracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

[0214] The nucleic acid may comprise any isolated or purified nucleotide sequence that encodes a CAR, a polypeptide, or a protein, or a functional portion or variant thereof. Alternatively, the nucleotide sequence may comprise a nucleotide sequence degenerate from or a combination of degenerate sequences.

[0215] Some embodiments of the present invention also provide isolated or purified nucleic acids comprising a nucleotide sequence complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence hybridizing with the nucleotide sequence of any nucleic acid described herein under stringent conditions.

[0216] Nucleotide sequences that hybridize under stringent conditions can hybridize under highly stringent conditions. "Highly stringent conditions" means that the nucleotide sequence specifically hybridizes with the target sequence (the nucleotide sequence of any nucleic acid described herein) in a detectable amount stronger than nonspecific hybridization. Highly stringent conditions include conditions that distinguish polynucleotides with precisely complementary sequences, or polynucleotides containing only a few scattered mismatches, from random sequences containing a small region (e.g., 3 to 12 bases) that happens to match that nucleotide sequence. These small complementary regions are easier to unwind than full-length complementary sequences of 14 to 17 or more bases, and highly stringent hybridization makes them easily distinguishable. Relatively highly stringent conditions would include, for example, low-salt and / or high-temperature conditions, such as those provided by about 0.02 M NaCl to 0.1 M NaCl or equivalents at temperatures of about 50°C to 70°C. Such highly stringent conditions allow for very few (if any) mismatches between the nucleotide sequence and the template or target strand and are particularly suitable for detecting the expression of any CAR described herein. It is generally believed that conditions can be made more stringent by adding an incremental amount of formamide.

[0217] In one embodiment, the nucleic acids of the present invention may be incorporated into a recombinant expression vector. This disclosure provides recombinant expression vectors comprising any nucleic acid of the present invention. As used herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, a protein, a polypeptide, or a peptide, allows the host cell to express the mRNA, protein, polypeptide, or peptide when the vector is contacted with a host cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell. The vectors described herein are not naturally occurring as a whole; however, portions of these vectors may be naturally occurring. The recombinant expression vectors may contain any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially derived from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide bonds, or both. Non-naturally occurring or modified nucleotides or internucleotide bonds do not impede transcription or replication of the vector.

[0218] In one embodiment, the recombinant expression vector of the present invention can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for proliferation and amplification or for expression, or both, such as plasmids and viruses. Vectors can be selected from the group consisting of: pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λEMBL4, λNM1149, and λZapII (Stratagene) can be used. Examples of plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Recombinant expression vectors can be viral vectors, such as retroviral vectors, like gamma retroviral vectors.

[0219] In one embodiment, the recombinant expression vector of the present invention is prepared using standard recombinant DNA techniques, such as those described in Sambrook et al. (ibid.) and Ausubel et al. (ibid.). Circular or linear expression vector constructs can be prepared to contain a functional replication system in a prokaryotic or eukaryotic host cell. The replication system may be derived from, for example, ColE1, SV40, 2μ plasmid, λ, bovine papillomavirus, etc.

[0220] Recombinant expression vectors may contain regulatory sequences, such as transcription and translation start and stop codons, which are specific to the host type (e.g., bacteria, plants, fungi, or animals) in which the vector will be appropriately introduced, and take into account whether the vector is DNA-based or RNA-based.

[0221] Recombinant expression vectors may contain one or more marker genes, which allow selection of transformed or transfected hosts. Marker genes include biocidal resistance (e.g., resistance to antibiotics, heavy metals, etc.), prototrophic complementation in auxotrophic hosts, and so on. Suitable marker genes for said expression vectors include, for example, neomycin / G418 resistance genes, histidine x resistance genes, histidine resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0222] The recombinant expression vector may contain a nucleotide sequence operably linked to encoding the CAR, polypeptide, or protein (including its functional portion and functional variants), or a natural or standard promoter operably linked to a nucleotide sequence complementary to or hybridizing with the nucleotide sequence encoding the CAR, polypeptide, or protein. The selection of the promoter (e.g., a strong promoter, a weak promoter, a tissue-specific promoter, an inducible promoter, and a development-specific promoter) is within the skill of a person skilled in the art. Similarly, the combination of the nucleotide sequence with the promoter is also within the skill of a person skilled in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an RSV promoter, an SV40 promoter, or a promoter found in the long terminal repeat sequence of a mouse stem cell virus.

[0223] Recombinant expression vectors can be designed for transient expression, stable expression, or both. Furthermore, they can be prepared for constitutive or inducible expression.

[0224] Additionally, recombinant expression vectors can be prepared to include suicide genes. As used herein, the term "suicide gene" refers to a gene that causes cell death in cells expressing the suicide gene. A suicide gene can be a gene that confers sensitivity to a reagent, such as a drug, to cells expressing the gene and causes cell death upon contact with or exposure to the reagent. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0225] Included within the scope of this invention are conjugates (e.g., biological conjugates) comprising any of the following: CAR, polypeptide or protein (including any of its functional moieties or functional variants), host cell, nucleic acid, recombinant expression vector, host cell population, or antibody or its antigen-binding moiety. Generally, conjugates and methods for synthesizing conjugates are known in the art (see, for example, Hudecz, F., Methods Mol. Biol., Vol. 298, pp. 209-223, 2005 and Kirin et al., Inorg Chem., Vol. 44, No. 15, pp. 5405-5415, 2005).

[0226] One embodiment of the present invention also provides an antibody or an antigen-binding portion thereof that binds (e.g., specifically binds) to an epitope of the CAR of the present invention.

[0227] The antibody can be any type of immunoglobulin known in the art. Immunoglobulins can be classified into five major classes: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further classified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of vertebrate species can be designated as one of two types, κ and λ, based on the amino acid sequence of its constant domain. The antibody can be any type or isotype.

[0228] Antibodies include immunoglobulin molecules, specifically including monoclonal antibodies (including mouse monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies, and chimeric monoclonal antibodies), polyclonal antibodies, antigen-binding fragments, bispecific or multispecific antibodies, monomeric antibodies, dimeric antibodies, tetrameric antibodies, or polymeric antibodies, single-chain antibodies, domain antibodies, and any other modified conformation of an immunoglobulin molecule containing an antigen-binding site with desired specificity. Antibodies can be naturally occurring antibodies, such as antibodies isolated and / or purified from mammals (e.g., mice, primates, rats, rabbits, goats, horses, chickens, hamsters, humans, etc.). Alternatively, antibodies can be engineered (e.g., genetically engineered) antibodies.

[0229] Humanized antibodies possess antigen-binding sites derived from non-human species, and their variable region framework is derived from human immunoglobulin sequences. Human antibodies have heavy chain variable regions and light chain variable regions, where both the framework and antigen-binding sites are derived from human-derived sequences.

[0230] Furthermore, the antibody can possess any level of affinity or affinity for the functional portion of the CAR. In some embodiments, the antibody can have a range of affinities (K... D The antibody binds to the hK2 antigen. In one embodiment of the invention, and in some embodiments of each of the numbered embodiments listed below, the antibody binds to the hK2 antigen with high affinity, for example, as determined by surface plasmon resonance or the Kinexa method, with a KD equal to or less than about 10. -7 M, such as, but not limited to, 1 to 9.9 (or any range or value therein, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9) × 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 thereof, as practiced by those skilled in the art. An example affinity is equal to or less than 1 × 10⁻⁶. -8 M. Another example has an affinity equal to or less than 1 × 10⁻⁶. -9 M.

[0231] Methods for testing the ability of an antibody to bind to any functional part of a CAR are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and competitive inhibition assay.

[0232] Suitable methods for preparing antibodies are known in the art. For example, the standard hybridoma method is described in the following literature: And Milstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA Janeway et al. (eds.), Immunobiology, 5th edition, Garland Publishing, New York, NY (2001)). Alternatively, other methods such as the EBV hybridoma method (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984) and Roder et al., Methods Enzymol., 121, 140-67 (1986)) and phage vector expression systems (see, for example, Huse et al., Science, 246, 1275-81 (1989)) are known in the art. In addition, methods for generating antibodies in non-human animals are described, for example, in U.S. Patents 5,545,806, 5,569,825 and 5,714,352 and U.S. Patent Application Publication 2002 / 0197266A1.

[0233] Phage display can also be used to generate antibodies. In this regard, phage libraries encoding antigen-binding variable (V) domains of antibodies can be generated using standard molecular biology techniques and recombinant DNA techniques (see, for example, Sambrook et al., ibid. and Ausubel et al., ibid.)). A phage encoding a variable region with desired specificity is selected for specific binding to a desired antigen (i.e., hK2), and an antibody containing the selected variable domain, either fully or partially, is reconstructed. The nucleic acid sequence encoding the reconstructed antibody is introduced into a suitable cell line (such as myeloma cells used to generate hybridomas) so that an antibody with monoclonal antibody properties is secreted by that cell (see, for example, Janeway et al., ibid., Huse et al., and U.S. Patent No. 6,265,150).

[0234] Antibodies can be produced by transgenic mice that are transgenic for specific heavy chain immunoglobulin genes and light chain immunoglobulin genes. Such methods are known in the art and are described, for example, in U.S. Patents 5,545,806 and 5,569,825 and in Janeway et al. (ibid.).

[0235] Methods for generating humanized antibodies are known in the art and described, for example, in Janeway et al. (ibid.), U.S. Patents 5,225,539, 5,585,089, and 5,693,761, European Patent 0239400B1, and British Patent 2,188,638. Humanized antibodies can also be generated using antibody surface reconstruction techniques described in U.S. Patent 5,639,641 and Pedersen et al., J. Mol. Biol., 235,959-973 (1994).

[0236] As used herein, antibodies can be multi-chain or single-chain, or complete immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[0237] The term "antibody fragment" refers to at least a portion of a complete antibody or a recombinant variant thereof that retains the antigen-binding properties of the full-length parent antibody. It refers, for example, an antigen-binding domain, such as the antigen-determining variable region of a complete antibody, sufficient to confer recognition and binding, for example, the specific binding of an antibody fragment to a target (such as an antigen). "Antigen-binding fragment" refers to a portion of an immunoglobulin molecule. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, single-chain antibodies (scFv), linear antibodies, single-domain antibodies such as sdAb (VL or VH), the Camelidae VHH domain, and multispecific antibodies formed from antibody fragments.

[0238] The term "scFv" refers to a protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region. In some embodiments, the light chain variable region and the heavy chain variable region are sequentially linked via a short, flexible peptide linker and are capable of being expressed as a single-chain polypeptide, wherein the scFv retains the specificity of its derived intact antibody. Unless otherwise specified, as used herein, the scFv may have VL and VH variable regions in either order; for example, the scFv may contain VL-linker-VH or VH-linker-VL relative to the N-terminus and C-terminus of the polypeptide.

[0239] One embodiment of the invention also provides an antigen-binding portion of any of the antibodies described herein. This antigen-binding portion can be any portion having at least one antigen-binding site, such as Fab, F(ab')2, dsFv, sFv, biantibodies, and triantibodies.

[0240] In some embodiments, the antigen-binding fragment is a heavy chain complementarity-determining region (HCDR) 1, 2, and / or 3, a light chain complementarity-determining region (LCDR) 1, 2, and / or 3, a heavy chain variable region (VH) or a light chain variable region (VL), a Fab, F(ab')2, Fd, and Fv fragment, and a domain antibody (dAb) comprising (e.g., any of the following) a VH domain or a VL domain. The VH and VL domains may be linked together via a linker (e.g., a synthetic linker).

[0241] The complementarity-determining region (CDR) is the antigen-binding site in an antibody. CDRs can be defined using various terms: (i) a complementarity-determining region (CDR) with three regions in the VH (HCDR1, HCDR2, HCDR3) and three regions in the VL (LCDR1, LCDR2, LCDR3) based on sequence variability (Wu and Kabat, J Exp Med 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) The “hypervariant region,” “HVR,” or “HV” with three in VH (H1, H2, H3) and three in VL (L1, L2, L3) refers to a structurally hypervariable region of the antibody variable domain, as defined by Chothia and Lesk (Chothia and Lesk, Mol Biol 196:901-17, 1987). The International Immunogenetics (IMGT) database (http: / / www_imgt_org) provides standardized numbers and definitions for antigen-binding sites. The correspondence between CDR, HV, and IMGT descriptions is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003. Unless otherwise expressly stated, the terms “CDR,” “HCDR1,” “HCDR2,” “HCDR3,” “LCDR1,” “LCDR2,” and “LCDR3” as used herein include the CDR as defined by any of the above methods (Kabat, Chothia, or IMGT).

[0242] In addition, antibodies or their antigen-binding portions can be modified to include detectable tags, such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0243] This disclosure also provides nucleic acids comprising nucleotide sequences encoding any of the CARs, peptides, or proteins (including their functional portions and functional variants) described herein.

[0244] The portion of the CAR containing an antibody or an antibody fragment thereof can exist in various forms, wherein the antigen-binding domain is represented as part of a continuous polypeptide chain, including, for example, single-domain antibody fragments (sdAbs), scFvs, and human chimeric or humanized antibodies (Harlow et al., 1999, in: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, in: Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science, Vol. 242, pp. 423-426). In one aspect, the antigen-binding domain of the CAR composition of the present invention comprises an antibody fragment. In another aspect, the CAR comprises an antibody fragment having an scFv.

[0245] The term "recombinant antibody" refers to antibodies produced using recombinant DNA technology, such as antibodies expressed by phage or yeast expression systems. This term should also be understood to mean antibodies produced by synthesizing a DNA molecule encoding the antibody and expressing the antibody protein, or specifying the amino acid sequence of the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequencing technologies available and known in the art.

[0246] The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve antibody production, or activation of specific immune-active cells, or both. Those skilled in the art will understand that any macromolecule (including virtually all proteins or peptides) can be used as an antigen. Furthermore, antigens can be derived from recombinant DNA or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or a portion of a protein encoding an immune response therefore encodes the term "antigen" as used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be apparent that this disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response.

[0247] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain binds to the hK2 antigen.

[0248] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0249] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:1, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:4;

[0250] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:2, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:4;

[0251] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:3, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:6;

[0252] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:3, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:4;

[0253] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:2, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:6;

[0254] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:1, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:6;

[0255] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:3, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:5;

[0256] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:1, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:5;

[0257] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:2, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:5;

[0258] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:74, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:76;

[0259] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:75, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:77;

[0260] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:147, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:140;

[0261] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:75, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:141;

[0262] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:75, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:140;

[0263] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:148, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:142;

[0264] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:149, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:143;

[0265] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:150, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:144;

[0266] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:151, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:145;

[0267] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:152, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:146;

[0268] Light chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:318, and heavy chains CDR1, CDR2, and CDR3 from the variable region containing the amino acid sequence of SEQ ID NO:317;

[0269] The extracellular antigen-binding domain binds to the hK2 antigen.

[0270] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:1, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:4.

[0271] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:2, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:4.

[0272] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:3, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:6.

[0273] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:3, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:4.

[0274] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:2, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:6.

[0275] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:1, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:6.

[0276] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:3, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:5.

[0277] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:1, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:5.

[0278] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:2, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:5.

[0279] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:74, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:76.

[0280] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:75, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:77.

[0281] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:147, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:140.

[0282] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:75, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:141.

[0283] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:75, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:140.

[0284] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:148, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:142.

[0285] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:149, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:143.

[0286] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:150, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:144.

[0287] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:151, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:145.

[0288] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:152, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:146.

[0289] In one embodiment, the extracellular antigen-binding domain comprises light chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:318, and heavy chains CDR1, CDR2, and CDR3 from the variable region comprising the amino acid sequence of SEQ ID NO:317.

[0290] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0291] Heavy chain CDR1 having an amino acid sequence selected from SEQ ID NO:63, SEQ ID NO:72, SEQ ID NO:86, SEQ ID NO:92, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:110, SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:123, SEQ ID NO:309 and SEQ ID NO:314 and their conserved modifications, wherein the extracellular antigen-binding domain binds to the hK2 antigen.

[0292] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0293] Heavy chain CDR2 having an amino acid sequence selected from 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 their conserved modifications, wherein the extracellular antigen-binding domain binds to the hK2 antigen.

[0294] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0295] Heavy chain CDR3 having an amino acid sequence selected from SEQ ID NO:66, SEQ ID NO:88, SEQ ID NO:94, SEQ ID NO:104, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:117, SEQ ID NO:122 and SEQ ID NO:311 and their conserved modifications, wherein the extracellular antigen-binding domain binds to the hK2 antigen.

[0296] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0297] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:64, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66.

[0298] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:314, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:315, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:311.

[0299] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:309, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:310, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:311.

[0300] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66.

[0301] Heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:73, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66; or

[0302] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:86, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:87, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:88.

[0303] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:92, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:93, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:94;

[0304] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:103, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:104.

[0305] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:105, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:106, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:104.

[0306] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:107, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:108, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:109.

[0307] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:110, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:111, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:109.

[0308] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:112, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:113.

[0309] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:105, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:114, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:113.

[0310] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:115, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:116, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:117.

[0311] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:118, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:119, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:117.

[0312] Heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:120, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:121, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:122; or

[0313] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:123, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:124, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:122.

[0314] The extracellular antigen-binding domain binds to the hK2 antigen.

[0315] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:64, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66.

[0316] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:314, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:315, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:311.

[0317] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:309, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:310, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:311.

[0318] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66.

[0319] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:73, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66.

[0320] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:86, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:87, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:88.

[0321] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:92, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:93, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:94.

[0322] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:103, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:104.

[0323] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 106, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 104.

[0324] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:107, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:108, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:109.

[0325] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 110, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 111, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 109.

[0326] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 102, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 112, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113.

[0327] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 105, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 114, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 113.

[0328] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:115, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:116, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:117.

[0329] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:118, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:119, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:117.

[0330] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:120, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:121, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:122.

[0331] In one embodiment, the extracellular antigen-binding domain comprises a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:123, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:124, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:122.

[0332] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0333] The light chain CDR1 has an amino acid sequence selected from 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 their conserved modifications, wherein the extracellular antigen-binding domain binds to the hK2 antigen.

[0334] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0335] The light chain CDR2 has an amino acid sequence selected from SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:90, SEQ ID NO:96, SEQ ID NO:126, SEQ ID NO:137, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:134, SEQ ID NO:137 and SEQ ID NO:313 and their conserved modifications, wherein the extracellular antigen-binding domain binds to the hK2 antigen.

[0336] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0337] The light chain CDR3 has an amino acid sequence selected from 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 their conserved modifications, wherein the extracellular antigen-binding domain binds to the hK2 antigen.

[0338] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0339] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0340] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68, the light chain CDR2 having the amino acid sequence of SEQ ID NO:70, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0341] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, the light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91;

[0342] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:95, the light chain CDR2 having the amino acid sequence of SEQ ID NO:96, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0343] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:312, the light chain CDR2 having the amino acid sequence of SEQ ID NO:313, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91.

[0344] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, the light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91;

[0345] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0346] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:128, the light chain CDR2 having the amino acid sequence of SEQ ID NO:129, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:130;

[0347] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:125, the light chain CDR2 having the amino acid sequence of SEQ ID NO:131, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:132;

[0348] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:133, the light chain CDR2 having the amino acid sequence of SEQ ID NO:134, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:135.

[0349] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:136, the light chain CDR2 having the amino acid sequence of SEQ ID NO:137, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:138; or

[0350] The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, the light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, and the light chain CDR3 having the amino acid sequence of SEQ ID NO: 139.

[0351] The extracellular antigen-binding domain binds to the hK2 antigen.

[0352] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, a light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0353] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68, a light chain CDR2 having the amino acid sequence of SEQ ID NO:70, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0354] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, a light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:91.

[0355] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:95, a light chain CDR2 having the amino acid sequence of SEQ ID NO:96, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0356] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:312, a light chain CDR2 having the amino acid sequence of SEQ ID NO:313, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:91.

[0357] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, a light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:91.

[0358] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, a light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0359] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:128, a light chain CDR2 having the amino acid sequence of SEQ ID NO:129, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:130.

[0360] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:125, a light chain CDR2 having the amino acid sequence of SEQ ID NO:131, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:132.

[0361] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:133, a light chain CDR2 having the amino acid sequence of SEQ ID NO:134, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:135.

[0362] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:136, a light chain CDR2 having the amino acid sequence of SEQ ID NO:137, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:138.

[0363] In one embodiment, the extracellular antigen-binding domain comprises a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:97, a light chain CDR2 having the amino acid sequence of SEQ ID NO:127, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:139.

[0364] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0365] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:64, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0366] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68, the light chain CDR2 having the amino acid sequence of SEQ ID NO:70, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0367] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0368] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0369] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0370] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:314, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:315, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:311, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:312, the light chain CDR2 having the amino acid sequence of SEQ ID NO:313, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91;

[0371] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:103, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:104, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:128, the light chain CDR2 having the amino acid sequence of SEQ ID NO:129, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:130;

[0372] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:107, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:108, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:109, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:125, the light chain CDR2 having the amino acid sequence of SEQ ID NO:131, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:132;

[0373] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:112, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:113, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:133, the light chain CDR2 having the amino acid sequence of SEQ ID NO:134, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:135;

[0374] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:115, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:116, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:117, and the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:136, the light chain CDR2 having the amino acid sequence of SEQ ID NO:137, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:138; or

[0375] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:120, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:121, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:122, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:97, the light chain CDR2 having the amino acid sequence of SEQ ID NO:127, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:138;

[0376] The extracellular antigen-binding domain binds to the hK2 antigen.

[0377] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0378] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:64, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0379] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68, the light chain CDR2 having the amino acid sequence of SEQ ID NO:70, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0380] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0381] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0382] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0383] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:314, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:315, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:311, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:312, the light chain CDR2 having the amino acid sequence of SEQ ID NO:313, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91;

[0384] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:103, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:104, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:128, the light chain CDR2 having the amino acid sequence of SEQ ID NO:129, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:130;

[0385] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:107, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:108, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:109, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:125, the light chain CDR2 having the amino acid sequence of SEQ ID NO:131, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:132;

[0386] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:112, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:113, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:133, the light chain CDR2 having the amino acid sequence of SEQ ID NO:134, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:135;

[0387] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:115, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:116, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:117, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:136, the light chain CDR2 having the amino acid sequence of SEQ ID NO:137, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:138;

[0388] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:120, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:121, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:122, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:97, the light chain CDR2 having the amino acid sequence of SEQ ID NO:127, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:138;

[0389] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:73, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0390] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:73, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68, the light chain CDR2 having the amino acid sequence of SEQ ID NO:70, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0391] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:73, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0392] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:73, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0393] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:73, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71;

[0394] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:309, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:310, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:311, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:312, the light chain CDR2 having the amino acid sequence of SEQ ID NO:313, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91;

[0395] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:105, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:106, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:104, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:128, the light chain CDR2 having the amino acid sequence of SEQ ID NO:129, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:130;

[0396] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:110, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:111, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:109, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:125, the light chain CDR2 having the amino acid sequence of SEQ ID NO:131, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:132;

[0397] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:105, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:114, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:113, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:133, the light chain CDR2 having the amino acid sequence of SEQ ID NO:134, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:135;

[0398] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:118, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:119, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:117, and the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:136, the light chain CDR2 having the amino acid sequence of SEQ ID NO:137, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:138;

[0399] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:123, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:124, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:122, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:97, the light chain CDR2 having the amino acid sequence of SEQ ID NO:127, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:139;

[0400] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:120, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:121, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:122, and the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:97, the light chain CDR2 having the amino acid sequence of SEQ ID NO:127, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:139; or

[0401] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:86, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:87, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:88, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, the light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91;

[0402] The extracellular antigen-binding domain binds to the hK2 antigen.

[0403] In one embodiment, the extracellular antigen-binding domain includes:

[0404] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:64, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0405] In one embodiment, the extracellular antigen-binding domain includes:

[0406] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68, the light chain CDR2 having the amino acid sequence of SEQ ID NO:70, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0407] In one embodiment, the extracellular antigen-binding domain includes:

[0408] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0409] In one embodiment, the extracellular antigen-binding domain includes:

[0410] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0411] In one embodiment, the extracellular antigen-binding domain includes:

[0412] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0413] In one embodiment, the extracellular antigen-binding domain includes:

[0414] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:314, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:315, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:311, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:312, the light chain CDR2 having the amino acid sequence of SEQ ID NO:313, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91.

[0415] In one embodiment, the extracellular antigen-binding domain includes:

[0416] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:103, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:104, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:128, the light chain CDR2 having the amino acid sequence of SEQ ID NO:129, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:130.

[0417] In one embodiment, the extracellular antigen-binding domain includes:

[0418] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:107, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:108, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:109, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:125, the light chain CDR2 having the amino acid sequence of SEQ ID NO:131, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:132.

[0419] In one embodiment, the extracellular antigen-binding domain includes:

[0420] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:112, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:113, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:133, the light chain CDR2 having the amino acid sequence of SEQ ID NO:134, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:135.

[0421] In one embodiment, the extracellular antigen-binding domain includes:

[0422] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:115, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:116, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:117, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:136, the light chain CDR2 having the amino acid sequence of SEQ ID NO:137, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:138.

[0423] In one embodiment, the extracellular antigen-binding domain includes:

[0424] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:120, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:121, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:122, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:97, the light chain CDR2 having the amino acid sequence of SEQ ID NO:127, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:138.

[0425] In one embodiment, the extracellular antigen-binding domain includes:

[0426] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:73, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0427] In one embodiment, the extracellular antigen-binding domain includes:

[0428] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:73, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68, the light chain CDR2 having the amino acid sequence of SEQ ID NO:70, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0429] In one embodiment, the extracellular antigen-binding domain includes:

[0430] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:73, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0431] In one embodiment, the extracellular antigen-binding domain includes:

[0432] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:73, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0433] In one embodiment, the extracellular antigen-binding domain includes:

[0434] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:73, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0435] In one embodiment, the extracellular antigen-binding domain includes:

[0436] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:309, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:310, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:311, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:312, the light chain CDR2 having the amino acid sequence of SEQ ID NO:313, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91.

[0437] In one embodiment, the extracellular antigen-binding domain includes:

[0438] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:105, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:106, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:104, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:128, the light chain CDR2 having the amino acid sequence of SEQ ID NO:129, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:130.

[0439] In one embodiment, the extracellular antigen-binding domain includes:

[0440] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:110, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:111, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:109, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:125, the light chain CDR2 having the amino acid sequence of SEQ ID NO:131, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:132.

[0441] In one embodiment, the extracellular antigen-binding domain includes:

[0442] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:105, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:114, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:113, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:133, the light chain CDR2 having the amino acid sequence of SEQ ID NO:134, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:135.

[0443] In one embodiment, the extracellular antigen-binding domain includes:

[0444] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:118, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:119, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:117, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:136, the light chain CDR2 having the amino acid sequence of SEQ ID NO:137, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:138.

[0445] In one embodiment, the extracellular antigen-binding domain includes:

[0446] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:123, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:124, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:122, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:97, the light chain CDR2 having the amino acid sequence of SEQ ID NO:127, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:139.

[0447] In one embodiment, the extracellular antigen-binding domain includes:

[0448] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:120, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:121, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:122, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:97, the light chain CDR2 having the amino acid sequence of SEQ ID NO:127, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:139.

[0449] In one embodiment, the extracellular antigen-binding domain includes:

[0450] The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:86, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:87, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:88, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, the light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91.

[0451] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises:

[0452] The extracellular antigen-binding domain binds to the hK2 antigen. The region comprises a light chain variable region (LCVR) containing amino acid sequences selected from SEQ ID NO:1-3, or a heavy chain variable region (HCVR) containing amino acid sequences selected from SEQ ID NO:4-6, or a combination of an LCVR containing amino acid sequences selected from SEQ ID NO:1-3 and an HCVR containing amino acid sequences selected from SEQ ID NO:4-6.

[0453] In one embodiment, the extracellular antigen-binding domain includes:

[0454] The light chain variable region containing the amino acid sequence of SEQ ID NO:1 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:4;

[0455] The light chain variable region containing the amino acid sequence of SEQ ID NO:2 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:4;

[0456] The light chain variable region containing the amino acid sequence of SEQ ID NO:3 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:6;

[0457] The light chain variable region containing the amino acid sequence of SEQ ID NO:3 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:4;

[0458] The light chain variable region containing the amino acid sequence of SEQ ID NO:2 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:6;

[0459] The light chain variable region containing the amino acid sequence of SEQ ID NO:1 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:6;

[0460] The light chain variable region containing the amino acid sequence of SEQ ID NO:1 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:5;

[0461] The light chain variable region containing the amino acid sequence of SEQ ID NO:2 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:5;

[0462] The light chain variable region containing the amino acid sequence of SEQ ID NO:3 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:5;

[0463] The light chain variable region containing the amino acid sequence of SEQ ID NO:74 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:76; the light chain variable region containing the amino acid sequence of SEQ ID NO:75 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:77;

[0464] The light chain variable region containing the amino acid sequence of SEQ ID NO:147 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:140;

[0465] The light chain variable region containing the amino acid sequence of SEQ ID NO:75 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:141;

[0466] The light chain variable region containing the amino acid sequence of SEQ ID NO:75 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:140;

[0467] The light chain variable region containing the amino acid sequence of SEQ ID NO:148 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:142;

[0468] The light chain variable region containing the amino acid sequence of SEQ ID NO:149 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:143;

[0469] The light chain variable region containing the amino acid sequence of SEQ ID NO:150 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:144;

[0470] The light chain variable region containing the amino acid sequence of SEQ ID NO:151 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:145;

[0471] The light chain variable region containing the amino acid sequence of SEQ ID NO:152 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:146;

[0472] The light chain variable region containing the amino acid sequence of SEQ ID NO:318 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:317.

[0473] 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.

[0474] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4.

[0475] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6.

[0476] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4.

[0477] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6.

[0478] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6.

[0479] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5.

[0480] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5.

[0481] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5.

[0482] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO:74 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:76.

[0483] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO:75 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:77.

[0484] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:147 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:140.

[0485] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO:75 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:141.

[0486] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO:75 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:140.

[0487] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:148 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:142.

[0488] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:149 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:143.

[0489] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:150 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:144.

[0490] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:151 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:145.

[0491] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:152 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:146.

[0492] In one embodiment, the extracellular antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:318 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:317.

[0493] In one implementation, the extracellular antigen-binding domain includes:

[0494] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:4;

[0495] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:2; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:4;

[0496] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:3; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:6;

[0497] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:3; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:4;

[0498] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:2; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:6;

[0499] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:3; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:4;

[0500] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:6;

[0501] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:3; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:5;

[0502] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:5;

[0503] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:2; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:5;

[0504] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:74; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:76;

[0505] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:75; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:77;

[0506] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:147; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:140;

[0507] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:75; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:141;

[0508] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:75; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:140;

[0509] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:148; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:142;

[0510] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:149; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:143;

[0511] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:150; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:144;

[0512] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:151; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:145;

[0513] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:152; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:146;

[0514] The light chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:318; and the heavy chain variable region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:317.

[0515] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:4.

[0516] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:2; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:4.

[0517] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:3; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:6.

[0518] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:3; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:4.

[0519] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:2; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:6.

[0520] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:3; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:4.

[0521] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:6.

[0522] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:3; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:5.

[0523] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:5.

[0524] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:2; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:5.

[0525] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:74; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:76.

[0526] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:77; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:77.

[0527] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:147; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:140.

[0528] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 75; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 141.

[0529] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 75; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 140.

[0530] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:142; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:142.

[0531] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:149; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:143.

[0532] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:150; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:144.

[0533] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:151; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:145.

[0534] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:152; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:146.

[0535] In one embodiment, the extracellular antigen-binding domain comprises: a light chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:318; and a heavy chain variable region comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:317.

[0536] In one embodiment, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the scFv comprises a linker polypeptide located between a light chain variable region and a heavy chain variable region. In some embodiments, the extracellular antigen-binding domain is an scFv comprising an amino acid sequence selected from SEQ ID NO:8-23, SEQ ID NO:169-184, and SEQ ID NO:340-343, and specifically binds to an hK2 polypeptide (e.g., a human hK2 polypeptide having the amino acid sequence SEQ ID NO:62, or a fragment thereof).

[0537] In recombinant expression systems, the linker is a peptide linker and can contain any naturally occurring amino acid. Exemplary amino acids that can be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker should be long enough to allow VH and VL to connect in the correct conformation relative to each other, so that they retain their desired activity (such as binding to hK2).

[0538] The linker length can be approximately 5 to 50 amino acids. In some embodiments, the linker length is approximately 10 to 40 amino acids. In some embodiments, the linker length is approximately 10 to 35 amino acids. In some embodiments, the linker length is approximately 10 to 30 amino acids. In some embodiments, the linker length is approximately 10 to 25 amino acids. In some embodiments, the linker length is approximately 10 to 20 amino acids. In some embodiments, the linker length is approximately 15 to 20 amino acids. In some embodiments, the linker length is 6 amino acids. In some embodiments, the linker length is 7 amino acids. In some embodiments, the linker length is 8 amino acids. In some embodiments, the linker length is 9 amino acids. In some embodiments, the linker length is 10 amino acids. In some embodiments, the linker length is 11 amino acids. In some embodiments, the linker length is 12 amino acids. In some embodiments, the linker length is 13 amino acids. In some embodiments, the linker length is 14 amino acids. In some embodiments, the linker length is 15 amino acids. In some embodiments, the linker length is 16 amino acids. In some embodiments, the linker is 17 amino acids long. In some embodiments, the linker is 18 amino acids long. In some embodiments, the linker is 19 amino acids long. In some embodiments, the linker is 20 amino acids long. In some embodiments, the linker is 21 amino acids long. In some embodiments, the linker is 22 amino acids long. In some embodiments, the linker is 23 amino acids long. In some embodiments, the linker is 24 amino acids long. In some embodiments, the linker is 25 amino acids long. In some embodiments, the linker is 26 amino acids long. In some embodiments, the linker is 27 amino acids long. In some embodiments, the linker is 28 amino acids long. In some embodiments, the linker is 29 amino acids long. In some embodiments, the linker is 30 amino acids long. In some embodiments, the linker is 31 amino acids long. In some embodiments, the linker is 32 amino acids long. In some embodiments, the linker is 33 amino acids long. In some embodiments, the linker is 34 amino acids long. In some embodiments, the linker is 35 amino acids long. In some embodiments, the linker is 36 amino acids long. In some embodiments, the linker is 37 amino acids long. In some embodiments, the linker is 38 amino acids long. In some embodiments, the linker is 39 amino acids long. In some embodiments, the linker is 40 amino acids long.Exemplary connectors that can be used include Gly-rich connectors, connectors containing Gly and Ser, connectors containing Gly and Ala, connectors containing Ala and Ser, and other flexible connectors.

[0539] In one embodiment, the adapter polypeptide comprises the amino acid sequence of SEQ ID NO:7. In one embodiment, the adapter polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:7. In one embodiment, the adapter polypeptide comprises the amino acid sequence of any one of SEQ ID NO:237 to SEQ ID NO:268. In one embodiment, the adapter polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NO:237 to SEQ ID NO:268.

[0540] In one embodiment, the scFv comprises an amino acid sequence selected from SEQ ID NO:8-23, SEQ ID NO:169-184, and SEQ ID NO:340-343. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:8. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:10 or SEQ ID NO:10. In another embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:10. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:12 or SEQ ID NO:12. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:12 or SEQ ID NO:12.In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:14 or SEQ ID NO:14. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:14 or SEQ ID NO:14. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:16 or SEQ ID NO:16. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:16 or SEQ ID NO:16. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:18 or SEQ ID NO:18. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:18 or SEQ ID NO:18.In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:20 or SEQ ID NO:20. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:20 or SEQ ID NO:20. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:22 or SEQ ID NO:22. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:22 or SEQ ID NO:22. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:169 or SEQ ID NO:169. In another embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:169.In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:171 or SEQ ID NO:171. In another embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:171. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:173 or SEQ ID NO:173. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:173 or SEQ ID NO:173. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:175 or SEQ ID NO:175. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:175.In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:177 or SEQ ID NO:177. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:177. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:179 or SEQ ID NO:179. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:179. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:181 or SEQ ID NO:181. In another embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:181.In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:183 or SEQ ID NO:183. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:183 or SEQ ID NO:183. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 340 or SEQ ID NO: 340. In another embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 340. In one embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:342 or SEQ ID NO:342. In another embodiment, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:342 or SEQ ID NO:342.In one embodiment, scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:343 or SEQ ID NO:343.

[0541] In one embodiment, the extracellular antigen-binding domain comprises a signaling polypeptide. The signaling polypeptide may be localized at the N-terminus of the hK2-binding extracellular antigen-binding domain. The signaling polypeptide may optionally be cleaved from the extracellular antigen-binding domain during cell processing and CAR localization to the cell membrane. Any of a variety of signaling polypeptides known to those skilled in the art may be used as the signaling polypeptide. Non-limiting examples of peptides from which the signaling polypeptide can be derived include FcεR, the variable region of the human immunoglobulin (IgG) heavy chain (HC), CD8α, or any of a variety of other proteins secreted by T cells. In various embodiments, the signaling polypeptide is compatible with the secretory pathway of T cells. In some embodiments, the signaling polypeptide comprises the amino acid sequence of SEQ ID NO:24. In one embodiment, the signaling polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:24.

[0542] In one aspect, this disclosure provides a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises an amino acid sequence selected from SEQ ID NO:29-44, SEQ ID NO:78-81, SEQ ID NO:221-236, and SEQ ID NO:273-308. Another feature of the CAR having an extracellular antigen-binding domain comprising an amino acid sequence selected from SEQ ID NO:29-44, SEQ ID NO:78-81, SEQ ID NO:221-236, and SEQ ID NO:273-308 is that the extracellular antigen-binding domain binds to the hK2 antigen.

[0543] In one embodiment, the intracellular signal transduction domain comprises a polypeptide component selected from the group consisting of: TNF receptor superfamily member 9 (CD137) component, T cell surface glycoprotein CD3ζ chain (CD3z) component, differentiation cluster (CD27) component, differentiation cluster superfamily member (such as CD28 or inducible T cell costimulator (ICOS)) component, and combinations thereof.

[0544] In one embodiment, the CD137 component comprises the amino acid sequence of SEQ ID NO:27. In another embodiment, the CD137 component comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:27.

[0545] In one embodiment, the CD3z component comprises the amino acid sequence of SEQ ID NO:28. In another embodiment, the CD3z component comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:28.

[0546] In one embodiment, the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:45. In another embodiment, the intracellular signal transduction domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:45.

[0547] In one embodiment, the transmembrane domain comprises a CD8a transmembrane region (CD8a-TM) polypeptide. In some embodiments, the CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO:26. In some embodiments, the CD8a-TM polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:26.

[0548] In one embodiment, the transmembrane domain comprises at least the transmembrane region of the following: the α, β, or ζ chain of the T-cell receptor; CD28, CD3ε, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154. In another embodiment, the transmembrane domain comprises at least the following: ζ, η, or FcεR1γ and -β, MB1 (Igα.), B29 or CD3-γ, ζ, or η. In yet another embodiment, the transmembrane domain is synthetic, for example, primarily comprising hydrophobic residues such as leucine and valine, a phenylalanine triplet, or tryptophan.

[0549] In one embodiment, the CAR further includes a hinge region linking the transmembrane domain to the extracellular antigen-binding domain. In some embodiments, the hinge region is a CD8a hinge region. In some embodiments, the CD8a hinge region includes the amino acid sequence of SEQ ID NO:25. In some embodiments, the CD8a hinge region includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:25. In some embodiments, the hinge region comprises the sequence EPKSCDKTHTCPPCP (SEQ ID NO:269), or comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with EPKSCDKTHTCPPCP (SEQ ID NO:269). In some embodiments, the hinge region comprises the sequence ERKCCVECPPCP (SEQ ID NO:270), or comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with ERKCCVECPPCP (SEQ ID NO:270). In some embodiments, the hinge region comprises the sequence ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO:271), or comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO:271).In some embodiments, the hinge region comprises the sequence ESKYGPPCPSCP (SEQ ID NO:272), or comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with ESKYGPPCPSCP (SEQ ID NO:272).

[0550] In one embodiment, the CAR includes an extracellular antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signal transduction domain. In such an embodiment, the hinge region is a CD8α hinge region, the transmembrane domain is a CD8α-TM domain, and the intracellular signal transduction domain includes a CD3-ζ domain and a 4-1BB / CD137 domain.

[0551] In one such embodiment, the hinge region is a CD8α hinge region containing the amino acid sequence of SEQ ID NO:25, the transmembrane domain is a CD8α-TM domain containing the amino acid sequence of SEQ ID NO:26, and the intracellular signal transduction domain includes a CD3-ζ domain and a 4-1BB / CD137 domain containing the amino acid sequence of SEQ ID NO:45.

[0552] In another embodiment, the hinge region is a CD8α hinge region containing the amino acid sequence of SEQ ID NO:25, the transmembrane domain is a CD8α-TM domain containing the amino acid sequence of SEQ ID NO:26, and the intracellular signal transduction domain includes a 4-1BB / CD137 domain containing the amino acid sequence of SEQ ID NO:27 and a CD3-ζ domain containing the amino acid sequence of SEQ ID NO:28.

[0553] In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence selected from SEQ ID NO:29-44, SEQ ID NO:78-81, SEQ ID NO:221-236, and SEQ ID NO:273-308. In another embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:29. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:31 or SEQ ID NO:31. In another embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:31. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:32 or SEQ ID NO:33. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:33 or SEQ ID NO:33.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:34 or SEQ ID NO:35. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:35 or SEQ ID NO:35. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:37. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:37. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:38 or SEQ ID NO:38.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:39. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:40. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:42 or SEQ ID NO:42. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:42 or SEQ ID NO:42. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:43 or SEQ ID NO:43.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:44 or SEQ ID NO:44. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:78 or SEQ ID NO:78. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:80 or SEQ ID NO:80. In another embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:80. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:81 or SEQ ID NO:81.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:221 or SEQ ID NO:221. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:222 or SEQ ID NO:222. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:223 or SEQ ID NO:223. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:224 or SEQ ID NO:224. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:225 or SEQ ID NO:225.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:226 or SEQ ID NO:226. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:227 or SEQ ID NO:227. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:228 or SEQ ID NO:228. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:229 or SEQ ID NO:229. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:230 or SEQ ID NO:230.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:231 or SEQ ID NO:231. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:232 or SEQ ID NO:232. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:233 or SEQ ID NO:233. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:234 ​​or SEQ ID NO:234. In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:235 or SEQ ID NO:235.In one embodiment, the extracellular antigen-binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:236 or SEQ ID NO:236.

[0554] CAR constructs and CAR-expressing immune response cells

[0555] The term "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having defined nucleotide sequences (e.g., rRNA, tRNA, and mRNA) or defined amino acid sequences and the resulting biological properties. Therefore, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, then the gene, cDNA, or RNA encodes that protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence) and the non-coding strand (which serves as a template for gene or cDNA transcription) can be said to encode that protein, or other products of that gene or cDNA.

[0556] Unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. The phrase "nucleotide sequence" encoding a protein or RNA may also include introns to such an extent that the nucleotide sequence encoding that protein may contain introns in some forms.

[0557] The term "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or by an in vitro expression system. Expression vectors include all those known in the art, including clomiphene citrates, plasmids (e.g., naked plasmids or plasmids contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) bound to the recombinant polynucleotide.

[0558] In one embodiment, this disclosure provides a cell expressing a nucleic acid molecule encoding a CAR. In one embodiment, the CAR of this disclosure comprises an amino acid sequence selected from SEQ ID NO:46-61, SEQ ID NO:82-85, and SEQ ID NO:205-220. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:46 or SEQ ID NO:46. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:48 or SEQ ID NO:48. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:48 or SEQ ID NO:48. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 50. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 50.In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:52 or SEQ ID NO:52. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:52 or SEQ ID NO:52. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:54 or SEQ ID NO:54. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:54 or SEQ ID NO:54. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:55 or SEQ ID NO:55.In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:57. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:57. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:59. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:59. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:60 or SEQ ID NO:60.In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 82 or SEQ ID NO: 82. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 82 or SEQ ID NO: 82. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:84 or SEQ ID NO:84. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:84 or SEQ ID NO:84. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:85 or SEQ ID NO:85.In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:206 or SEQ ID NO:206. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:206 or SEQ ID NO:206. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:208 or SEQ ID NO:208. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:208 or SEQ ID NO:208. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:209.In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:211 or SEQ ID NO:211. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:211 or SEQ ID NO:211. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:213 or SEQ ID NO:213. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:213 or SEQ ID NO:213. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:214 or SEQ ID NO:214.In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:215 or SEQ ID NO:216. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:216 or SEQ ID NO:216. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:217 or SEQ ID NO:218. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:218 or SEQ ID NO:218. In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:219 or SEQ ID NO:219.In some embodiments, the CAR of this disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:220 or SEQ ID NO:220.

[0559] In some embodiments, the CAR may comprise a signal peptide. In some embodiments, the signal peptide is located at the N-terminus of the CAR sequence. In some embodiments, the signal peptide is located at the C-terminus of the CAR sequence. In some embodiments, the signal peptide comprises or consists of the amino acid sequence of SEQ ID NO:24.

[0560] In this document, the structure of a nucleic acid molecule is defined based on the % sequence identity with the reference sequence (with a given SEQ ID NO). In this context, % sequence identity of a nucleic acid molecule refers to the similarity between at least two different nucleic acid sequences. Two compared sequences are identical when positions in both sequences are occupied by the same bases; for example, if a position in each of two DNA molecules is occupied by adenine, then the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions and multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% identical (or homologous). For instance, DNA sequences ATTGCC and TATGGC share 50% identity (or homology). Generally, two sequences are compared when the maximum homology is given. The corresponding percentage of identity can be determined using standard alignment algorithms, such as the Basic Local Alignment Search Tool (BLAST) described by Altshul et al. ((1990) J. MoI. Biol. Vol. 215, pp. 403-410); the algorithm of Needleman et al. (1970, J. MoI. Biol. 48: 444-53); or the algorithm of Meyers et al. ((1988) Comput. Appl. Biosci. 4: 11-17). A set of parameters can be the Blosum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5. The percentage identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller ((1989) CABIOS 4: 11-17) (which has been incorporated into the ALIGN program (version 2.0)) using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Percentage identity is typically calculated by comparing sequences of similar length.

[0561] In various embodiments, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence of any one of SEQ ID NO:153 to SEQ ID NO:160. In various embodiments, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO:153 to SEQ ID NO:160.

[0562] In various embodiments, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence of any one of SEQ ID NO:161 to SEQ ID NO:168. In various embodiments, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO:161 to SEQ ID NO:168.

[0563] In various embodiments, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence of any one of SEQ ID NO:195 to SEQ ID NO:204. In various embodiments, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO:195 to SEQ ID NO:204.

[0564] In various embodiments, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence of any one of SEQ ID NO:320 to SEQ ID NO:325. In various embodiments, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO:320 to SEQ ID NO:325.

[0565] In various embodiments, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence of any one of SEQ ID NO:326 to SEQ ID NO:331. In various embodiments, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO:326 to SEQ ID NO:331.

[0566] In various embodiments, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence of any one of SEQ ID NO:336 to SEQ ID NO:339. In various embodiments, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO:336 to SEQ ID NO:339.

[0567] In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:153 or SEQ ID NO:153. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:154 or SEQ ID NO:154. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:155 or SEQ ID NO:155. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:156 or SEQ ID NO:156. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:157 or SEQ ID NO:157.In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:158 or SEQ ID NO:158. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:159 or SEQ ID NO:159. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:160 or SEQ ID NO:160. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:161 or SEQ ID NO:161. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:162 or SEQ ID NO:162.In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:163 or SEQ ID NO:163. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:164 or SEQ ID NO:164. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:165 or SEQ ID NO:165. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:166 or SEQ ID NO:166. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:167 or SEQ ID NO:167.In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:168 or SEQ ID NO:168. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:195 or SEQ ID NO:195. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO:196 or SEQ ID NO:196. In one embodiment, the nucleic acid molecule encoding a CAR comprises a nucleic acid sequence including a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence id...

Claims

1. A chimeric antigen receptor (CAR) comprising: (a) An extracellular domain that specifically binds to the human kallikrein-2 (hK2) antigen. (b) Transmembrane domains; and (c) Intracellular signal transduction domain, wherein the intracellular signal transduction domain optionally includes at least one co-stimulatory domain.

2. The CAR according to claim 1, wherein the CAR further comprises: (d)CD8a - Hinge area, The transmembrane domain comprises a CD8a transmembrane region (CD8a-TM) polypeptide; and The intracellular signal transduction domains include a co-stimulatory domain and a primary signal transduction domain. The co-stimulatory domain contains a component of TNF receptor superfamily member 9 (CD137), and the primary signal transduction domain contains a component of the T cell surface glycoprotein CD3ζ chain (CD3z).

3. The CAR according to claim 2, wherein The CD8a hinge region contains an amino acid sequence that is at least 90% identical to SEQ ID NO:25, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271 or SEQ ID NO:272; The transmembrane domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:26; and / or The intracellular signal transduction domain includes a co-stimulatory domain and a primary signal transduction domain. The co-stimulatory domain has an amino acid sequence that is at least 90% identical to that of SEQ ID NO:27, and the primary signal transduction domain has an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

28.

4. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction 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, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:64, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66; heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:314, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:315, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:311; The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:309, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:310, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

311. The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

66. The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:72, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:73, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

66. The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:86, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:87, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

88. The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:92, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:93, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:94; The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:103, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

104. The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:105, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:106, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

104. The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:107, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:108, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

109. The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:110, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:111, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

109. The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:112, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

113. The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:105, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:114, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

113. The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:115, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:116, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

117. The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:118, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:119, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

117. Heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:120, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:121, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:122; or The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:123, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:124, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

122. The extracellular antigen-binding domain thereunder binds to the hK2 antigen.

5. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises: The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68, the light chain CDR2 having the amino acid sequence of SEQ ID NO:70, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, the light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:95, the light chain CDR2 having the amino acid sequence of SEQ ID NO:96, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:312, the light chain CDR2 having the amino acid sequence of SEQ ID NO:313, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:

91. The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, the light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:128, the light chain CDR2 having the amino acid sequence of SEQ ID NO:129, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:130; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:125, the light chain CDR2 having the amino acid sequence of SEQ ID NO:131, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:132; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:133, the light chain CDR2 having the amino acid sequence of SEQ ID NO:134, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:

135. The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:136, the light chain CDR2 having the amino acid sequence of SEQ ID NO:137, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:138; or The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 97, the light chain CDR2 having the amino acid sequence of SEQ ID NO: 127, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:

139. The extracellular antigen-binding domain thereunder binds to the hK2 antigen.

6. The CAR according to claim 4, wherein the extracellular antigen-binding domain further comprises: a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 67, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

71. The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68, the light chain CDR2 having the amino acid sequence of SEQ ID NO:70, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, the light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:95, the light chain CDR2 having the amino acid sequence of SEQ ID NO:96, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, the light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:128, the light chain CDR2 having the amino acid sequence of SEQ ID NO:129, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:130; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:125, the light chain CDR2 having the amino acid sequence of SEQ ID NO:131, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:132; The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:133, the light chain CDR2 having the amino acid sequence of SEQ ID NO:134, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:

135. The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:136, the light chain CDR2 having the amino acid sequence of SEQ ID NO:137, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:138; or The light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:97, the light chain CDR2 having the amino acid sequence of SEQ ID NO:127, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:

139.

7. The CAR of claim 6, wherein the extracellular antigen-binding domain comprises: The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:64, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71; The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:68, the light chain CDR2 having the amino acid sequence of SEQ ID NO:70, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71; The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71; The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71; The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:66, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:67, the light chain CDR2 having the amino acid sequence of SEQ ID NO:126, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:71; The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:314, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:315, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:311, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:312, the light chain CDR2 having the amino acid sequence of SEQ ID NO:313, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:91; The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:103, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:104, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:128, the light chain CDR2 having the amino acid sequence of SEQ ID NO:129, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:130; The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:107, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:108, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:109, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:125, the light chain CDR2 having the amino acid sequence of SEQ ID NO:131, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:132; The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:102, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:112, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:113, the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:133, the light chain CDR2 having the amino acid sequence of SEQ ID NO:134, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:135; The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:115, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:116, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:117, and the light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:136, the light chain CDR2 having the amino acid sequence of SEQ ID NO:137, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:138; or The heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 120, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, the heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:

122. The light chain complementarity-determining region 1 (CDR1) of the amino acid sequence of SEQ ID NO: 127, the light chain CDR2 of the amino acid sequence of SEQ ID NO: 138, and the light chain CDR3 of the amino acid sequence of SEQ ID NO:

138.

8. The CAR according to claim 6, wherein the extracellular antigen-binding domain comprises: a heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:86, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:87, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:88, a light chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:89, a light chain CDR2 having the amino acid sequence of SEQ ID NO:90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

91.

9. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises: A light chain variable domain (VL) comprising an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152 and SEQ ID NO:318; and / or A heavy chain variable domain (VH) comprising an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146 and SEQ ID NO:317; The extracellular antigen-binding domain binds to the human kallikrein-2 (hK2) antigen.

10. The CAR of claim 9, wherein the extracellular antigen-binding domain comprises: The light chain variable region containing the amino acid sequence of SEQ ID NO:1 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:4; The light chain variable region containing the amino acid sequence of SEQ ID NO:2 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:4; The light chain variable region containing the amino acid sequence of SEQ ID NO:3 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:6; The light chain variable region containing the amino acid sequence of SEQ ID NO:3 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:4; The light chain variable region containing the amino acid sequence of SEQ ID NO:2 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:6; The light chain variable region containing the amino acid sequence of SEQ ID NO:3 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:4; The light chain variable region containing the amino acid sequence of SEQ ID NO:1 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:6; The light chain variable region containing the amino acid sequence of SEQ ID NO:3 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:5; The light chain variable region containing the amino acid sequence of SEQ ID NO:1 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:5; The light chain variable region containing the amino acid sequence of SEQ ID NO:2 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:5; The light chain variable region containing the amino acid sequence of SEQ ID NO:3 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:5; The light chain variable region containing the amino acid sequence of SEQ ID NO:74 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:76; The light chain variable region containing the amino acid sequence of SEQ ID NO:75 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:77; The light chain variable region containing the amino acid sequence of SEQ ID NO:147 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:140; The light chain variable region containing the amino acid sequence of SEQ ID NO:75 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:141; The light chain variable region containing the amino acid sequence of SEQ ID NO:75 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:140; The light chain variable region containing the amino acid sequence of SEQ ID NO:148 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:142; The light chain variable region containing the amino acid sequence of SEQ ID NO:149 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:143; The light chain variable region containing the amino acid sequence of SEQ ID NO:150 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:144; The light chain variable region containing the amino acid sequence of SEQ ID NO:151 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:145; The light chain variable region containing the amino acid sequence of SEQ ID NO:152 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:146; The light chain variable region containing the amino acid sequence of SEQ ID NO:318 and the heavy chain variable region containing the amino acid sequence of SEQ ID NO:

317.

11. The CAR according to any one of claims 1 to 10, wherein the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv), the scFv comprising a light chain variable region (VL) and a heavy chain variable region (VH).

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

13. The CAR of claim 12, wherein the adapter polypeptide comprises the amino acid sequence of SEQ ID NO:

7.

14. The CAR according to any one of claims 11 to 13, wherein the scFv comprises an amino acid sequence selected from SEQ ID NO:8-23, SEQ ID NO:169-184 and SEQ ID NO:340-343.

15. The CAR according to any one of claims 1 to 14, wherein the extracellular antigen-binding domain comprises a signaling polypeptide.

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

24.

17. The CAR according to any one of claims 4 to 16, wherein the intracellular signal transduction domain comprises a polypeptide component selected from: TNF receptor superfamily member 9 (CD137) component, T cell surface glycoprotein CD3ζ chain (CD3z) component, differentiation cluster (CD27) component, differentiation cluster superfamily member component, and combinations thereof.

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

27.

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

28.

20. The CAR according to any one of claims 17 to 19, wherein the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:45, or optionally, the intracellular signal transduction domain comprises a co-stimulatory domain and a primary signal transduction domain, wherein the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:27, and the primary signal transduction domain comprises the amino acid sequence of SEQ ID NO:

28.

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

22. The CAR of claim 21, wherein the CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO:

26.

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

24. The CAR according to claim 23, wherein the hinge region is a CD8a hinge region.

25. The CAR of claim 24, wherein the CD8a hinge region comprises the amino acid sequence of SEQ ID NO:25, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271 or SEQ ID NO:

272.

26. The CAR according to any one of claims 1 to 25, wherein the extracellular antigen-binding domain comprises an amino acid sequence selected from SEQ ID NO:29-44, SEQ ID NO:78-81, SEQ ID NO:221-236 and SEQ ID NO:273-308.

27. The CAR according to any one of claims 1 to 26, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO:46-61, SEQ ID NO:82-85 and SEQ ID NO:205-220.

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

29. The isolated lymphocytes according to claim 28, wherein the lymphocytes are T lymphocytes.

30. The isolated lymphocytes according to claim 28, wherein the lymphocytes are natural killer (NK) cells.

31. An isolated nucleic acid molecule, said isolated nucleic acid molecule encoding a CAR according to any one of claims 1 to 27.

32. A vector comprising the nucleic acid molecule according to claim 31.

33. A cell that expresses the nucleic acid molecule according to claim 31.

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

35. A pharmaceutical composition comprising an effective amount of lymphocytes according to any one of claims 28 to 30 and a pharmaceutically acceptable excipient.

36. The CAR according to any one of claims 1 to 27 or the pharmaceutical composition according to claim 34 or 35, for use in a therapy.

37. The CAR according to any one of claims 1 to 27 or the pharmaceutical composition according to claim 34 or 35, for use in a method of treating a subject suffering from cancer.

38. A method for treating a subject suffering from cancer, the method comprising: A therapeutically effective amount of lymphocytes according to any one of claims 28 to 30 is administered to a subject in need, thereby inducing the killing of cancer cells in the subject.

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

40. A method for targeted killing of cancer cells, the method comprising: The cancer cells are brought into contact with lymphocytes according to any one of claims 28 to 30, thereby inducing the lymphocytes to kill the cancer cells.

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

42. A method for detecting the presence of cancer in a subject, the method comprising: A cell sample obtained from the subject is contacted with a CAR according to any one of claims 1 to 27 to form a CAR-cell complex, and The complex is detected, and the detection of the complex indicates the presence of cancer in the subject.

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