GPRC5D chimeric antigen receptors and cells expressing these receptors

Abstract

The present disclosure provides a chimeric antigen receptor (CAR) that specifically targets a G protein-coupled receptor, i.e., G protein-coupled receptor family group C 5 member D (GPRC5D), and an immune response cell comprising such a CAR, which is used to treat cancer.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on January 9, 2020, is named JBI6043WOPCT1_SL.txt, and is 150,002 bytes in size. Technical Field

[0003] The present invention relates to a chimeric antigen receptor (CAR) targeting GPRC5D comprising a GPRC5D single-chain variable fragment and an engineered immune cell targeting GPRC5D expressing the CAR. Also provided are nucleic acids and expression vectors encoding the CAR, recombinant cells comprising the vector, and compositions comprising engineered immune cells expressing CAR targeting GPRC5D. Also provided are methods for preparing the CAR and engineered immune cells, and methods for treating conditions including cancer using engineered immune cells. Background Art

[0004] T cell therapy utilizes isolated T cells that have been genetically modified to enhance their specificity for specific tumor-associated antigens. The genetic modification may involve the expression of a chimeric antigen receptor (CAR) or an exogenous T cell receptor to provide new antigen specificity to the T cell. 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

[0005] Disclosed herein are chimeric antigen receptors (CARs) (e.g., CARs targeting G protein-coupled receptor, G protein-coupled receptor family C group 5 member D (GPRC5D)), cells comprising the CARs, vectors encoding the CARs (e.g., recombinant expression vectors), and nucleic acid molecules encoding the CARs, methods for preparing the CARs, compositions, polypeptides, proteins, nucleic acids, host cells, cell populations, and methods for treating diseases (e.g., cancer) using the disclosed CARs.

[0006] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0007] a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:66, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:67, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:68;

[0008] a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:58, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:59, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:60;

[0009] a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:39, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:40, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:41;

[0010] a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:42, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:43, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:44; or

[0011] a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:45, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:46, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:47;

[0012] The extracellular antigen binding domain binds to the GPRC5D antigen.

[0013] In one embodiment:

[0014] The extracellular antigen-binding domain comprises a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 66, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 67, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 68, and further comprises a light chain CDR1 having an amino acid sequence of SEQ ID NO: 69, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 70, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 71;

[0015] The extracellular antigen-binding domain comprises a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 58, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 59, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 60, and further comprises a light chain CDR1 having an amino acid sequence of SEQ ID NO: 61, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 62, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 63;

[0016] The extracellular antigen-binding domain comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:39, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:40, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:41, and further comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO:48, a light chain CDR2 having the amino acid sequence of SEQ ID NO:49, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:50;

[0017] The extracellular antigen-binding domain comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:42, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:43, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:44, and further comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO:51, a light chain CDR2 having the amino acid sequence of SEQ ID NO:52, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:53; or

[0018] The extracellular antigen-binding domain comprises a heavy chain CDR1 having an amino acid sequence of SEQ ID NO:45, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO:46, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO:47, and also comprises a light chain CDR1 having an amino acid sequence of SEQ ID NO:54, a light chain CDR2 having an amino acid sequence of SEQ ID NO:55, and a light chain CDR3 having an amino acid sequence of SEQ ID NO:56.

[0019] In one embodiment, the extracellular antigen binding domain comprises: a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:65, SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, or a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:72, SEQ ID NO:64, SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, or a combination of a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:65, SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5 and a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO:72, SEQ ID NO:64, SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.

[0020] In one embodiment, the extracellular antigen binding domain comprises:

[0021] a light chain variable region comprising the amino acid sequence of SEQ ID NO: 73 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72;

[0022] a light chain variable region comprising the amino acid sequence of SEQ ID NO:65 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:64;

[0023] 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: 2;

[0024] 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; or

[0025] A light chain variable region comprising the amino acid sequence of SEQ ID NO:5 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6.

[0026] In one embodiment, 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 and the heavy chain variable region.

[0027] In one embodiment, the linker polypeptide comprises the amino acid sequence of SEQ ID NO:7.

[0028] In one embodiment, the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NO:78, SEQ ID NO:77, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.

[0029] In one embodiment, the extracellular antigen binding domain comprises a signal polypeptide. In some embodiments, the signal polypeptide comprises the amino acid sequence of SEQ ID NO:11.

[0030] In one embodiment, the intracellular signaling domain comprises a polypeptide component selected from the group consisting of a TNF receptor superfamily member 9 (CD137) component, a T cell surface glycoprotein CD3 zeta chain (CD3z) component, a cluster of differentiation (CD27) component, a cluster of differentiation superfamily member (such as, CD28 or inducible T cell co-stimulator (ICOS)) component, and combinations thereof.

[0031] In one embodiment, the CD137 component comprises the amino acid sequence of SEQ ID NO:12.

[0032] In one embodiment, the CD3z component comprises the amino acid sequence of SEQ ID NO:13.

[0033] In one embodiment, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:14.

[0034] 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:15.

[0035] In one embodiment, the CAR further comprises 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 comprises the amino acid sequence of SEQ ID NO: 16.

[0036] In one embodiment, the extracellular antigen binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:82, SEQ ID NO:81, SEQ ID NO:80, SEQ ID NO:79, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22.

[0037] In one embodiment, the CAR of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NO:86, SEQ ID NO:85, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32.

[0038] In one aspect, the present disclosure provides isolated lymphocytes expressing CAR as described herein. In some embodiments, the lymphocytes are T lymphocytes. In some embodiments, the T lymphocytes are initial T cells. In some embodiments, the T lymphocytes are memory stem T cells. In some embodiments, the T lymphocytes are central memory T cells. In some embodiments, the T lymphocytes are CD4+. In some embodiments, the T lymphocytes are CD8+. In some embodiments, the T lymphocytes are CD4+ and CD8+.

[0039] In one aspect, the disclosure provides an isolated nucleic acid molecule encoding any CAR described herein. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of the following items: SEQ ID NO: 90, SEQ ID NO: 89, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO:90, SEQ ID NO:89, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38. In one embodiment, the present disclosure provides a vector comprising the nucleic acid molecule. In one embodiment, the present disclosure provides a cell expressing the nucleic acid molecule.

[0040] In one aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, comprising an effective amount of lymphocytes expressing one or more of the described CARs and a pharmaceutically acceptable excipient.

[0041] In one aspect, the present disclosure provides a CAR according to the present disclosure for use in a method of treatment.

[0042] In one aspect, the present disclosure provides lymphocytes according to the present disclosure for use in a method of treatment. In one aspect, the present disclosure provides a composition, such as a pharmaceutical composition, according to the present disclosure for use in a method of treatment.

[0043] In one aspect, the present disclosure provides a CAR according to the present disclosure for use in a method for treating cancer. In one aspect, the present disclosure provides a lymphocyte according to the present disclosure for use in a method for treating cancer. In one aspect, the present disclosure provides a composition according to the present disclosure for use in a method for treating cancer, such as a pharmaceutical composition. In one embodiment, the cancer is selected from the group consisting of bladder cancer, metastatic bladder cancer, esophageal cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, prostate cancer, urothelial carcinoma, small cell lung cancer, endometrial cancer, bile duct cancer, hepatocellular carcinoma, sarcoma, solid tumors of squamous epithelial origin, lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), smoldering multiple myeloma (SMM), multiple myeloma (MM), acute myeloid leukemia (AML), and combinations thereof. In one embodiment, the cancer treated is multiple myeloma.

[0044] In one aspect, the present disclosure provides a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of lymphocytes expressing one or more of the CARs to a subject in need thereof, whereby the lymphocytes induce killing of cancer cells in the subject. In one embodiment, the cancer is selected from the group consisting of bladder cancer, metastatic bladder cancer, esophageal cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, prostate cancer, urothelial carcinoma, small cell lung cancer, endometrial cancer, bile duct cancer, hepatocellular carcinoma, sarcoma, solid tumors of squamous epithelial origin, lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), smoldering multiple myeloma (SMM), multiple myeloma (MM), acute myeloid leukemia (AML), and combinations thereof. In one embodiment, the cancer treated in the subject is multiple myeloma.

[0045] In one aspect, a method for targeted killing of cancer cells is disclosed, the method including contacting cancer cells with lymphocytes expressing one or more of the CAR, whereby the lymphocytes induce killing of cancer cells. In some embodiments, cancer cells are selected from the group consisting of lung cancer cells, gastric cancer cells, colon cancer cells, hepatocellular cancer cells, renal cell cancer cells, bladder urothelial cancer cells, metastatic melanoma cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, head and neck cancer cells, pancreatic cancer cells, glioma cells, glioblastoma cells and non-Hodgkin's lymphoma (NHL) cells, acute lymphocytic leukemia (ALL) cells, chronic lymphocytic leukemia (CLL) cells, chronic myeloid leukemia (CML) cells, smoldering multiple myeloma (SMM) cells, multiple myeloma (MM) cells, acute myeloid leukemia (AML) cells, and combinations thereof. In one embodiment, the cancer cell is a multiple myeloma cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The foregoing will be apparent from the following more particular description of exemplary embodiments, as illustrated in the accompanying drawings.

[0047] Figure 1 Flow cytometry analysis of primary human pan T cells electroporated with the following is shown: no mRNA (simulation) is used, or 10 μg of mRNA expressing α-GPRC5D scFv CAR or isotype control CAR. 24 hours after electroporation, after staining with biotinylated L-protein and streptavidin-conjugated PE, CAR surface expression is measured by flow cytometry. The hollow histogram is simulated, and the filled gray histogram is a CAR-T population.

[0048] Figure 2 Flow cytometry analysis of transiently transfected pan-T cells co-cultured with multiple myeloma cell lines is shown. Twenty-four hours after transient transfection, primary pan-T cells were labeled with Cell Trace Violet (CTV) fluorescent proliferation dye and then co-cultured with multiple myeloma cell line H929. After four days of co-culture, cells were pre-gated for CD8+CD4- populations, and the surface expression of activation markers CD25 and CD71 on CAR-T was compared with T cells cultured alone or T cells cultured in the presence of α-CD3 / CD28 beads.

[0049] Figure 3The cytokine profile analysis of primary pan-T cells co-cultured with various myeloma cell lines and transiently transfected with mRNA expressing CAR is shown.Primary pan-T cells transiently transfected 24 hours before expressing mRNA for CAR and various myeloma cell lines expressing target antigen GPRC5D are co-cultured with a ratio of 1:1 with high (+++) level, medium (++) level, low (+) level or negative (-) level.After co-cultivation for sixteen hours, supernatant is harvested and cytokine profile analysis is performed on the supernatant from co-culture by Meso Scale Discovery (MSD).

[0050] Figure 4 The cytokine profile analysis of the primary pan-T cells co-cultured with autologous peripheral blood mononuclear cells (PBMC) and multiple myeloma cell lines by mRNA transient transfection expressing CAR is shown.24 hours after the mRNA transient transfection expressing CAR, primary pan-T cells are co-cultured with autologous PBMC and multiple myeloma cell line H929 (with 1:1:1 ratio).After co-cultivation for sixteen hours, supernatant is harvested.The cytokine profile analysis carried out by MSD to the supernatant from the co-culture comprising all three groups (CAR-T, H929, autologous PBMC) and the control co-culture lacking CAR-T cells, PBMC or H929, is compared with each cell colony of separation and culture.

[0051] Figure 5 CAR-T cells are shown to express GPRC5D myeloma cells cytotoxic potential flow cytometry analysis.Primary pan T cells (transiently expressing one of the three CARs as described above) are co-cultured with fluorescently labeled myeloma cell lines MM1R, H929 and K562 with specified effectors: target (E: T) ratio for eight hours, and then the co-culture is dyed with viability dye.Kill percentage is the absolute number of live (viability dye negative) target (CTV positive) cells remaining in the co-culture relative to the ratio of the number of live targets not cultured with CAR-T cells.

[0052] Figure 6 CAR-T cells are shown to express GPRC5D myeloma cells cytotoxic potential flow cytometry analysis.Primary multiple myeloma patient T cells (transiently expressing one of the two CARs as described above) are co-cultured with a specified effector: target (E: T) ratio and a fluorescently labeled myeloma cell line MM.1S or a control GPRC5d negative cell line KG-1 for 48 hours, at which point the co-culture is stained with a viability dye.Cytotoxicity percentage is determined as the percentage of fluorescently labeled cells that are positive for viability dye staining (indicating cell death).

[0053] Figure 7The results of flow cytometric analysis of the proliferation capacity of α-GPRC5D-CAR-T cells are shown. Fluorescently labeled pan-T cells (transiently expressing α-GPRC5D CAR or isotype CAR control) were co-cultured with H929 at an E:T ratio of 1 for four days. Proliferation is measured as the absolute number of live (viability dye negative) CAR-T cells with diluted fluorescent labels (CTV). The proliferation in response to H929 and α-CD3 / CD28 beads after four days of stimulation were compared with CAR-T proliferation and CAR-T cultured alone (no stimulation control).

[0054] Figure 8 A bar graph is shown, which depicts the percentage of GPRC5D-CAR expression on healthy donor T cells measured by flow cytometry. The shaded gray column represents background CAR detection in untransduced (simulated) cells. The black column shows GPRC5DGC5B680-N68S-LH in transduced CAR-T cells. The values ​​shown represent the mean ± SD of 6 healthy donors.

[0055] Figure 9A to Fig.9F Depicted are data showing the expression of CD4 markers, CD8 markers, and memory markers on GPRC5D CAR-T cells. Figure 9A to Fig. 9C Figure 9A shows a bar graph in which the data collectively demonstrate that GPRC5D GC5B680-N68S-LH CAR was detected in both CD4 T and CD8 T cells by flow cytometry and that the CD4 / CD8 ratio was not significantly different from the untransduced population. + Cells and CD4 + The cells each contained a percentage of the mock (untransduced) cell population. Fig. 9C Each shows the CD8 + Cells and CD4 + CARs contained in each cell - The percentage of cells (transduced cells that do not express GPRC5D CAR) and the percentage of cells transduced by CD8 + Cells and CD4 + CARs contained in each cell + Figure 9D shows a flow cytometric plot prepared from one donor, which shows the gating strategy for selecting different indicative memory populations in the CAR-T population using surface markers (CD45RA and CD62L). Figure 9E and Fig.9F Depicts bar graphs showing that GPRC5D GC5B680-N68S-LH is mainly in T N / SCM and TCM It has been shown to have increased proliferation capacity, survival rate and therapeutic efficacy. Figure 9A to Fig. 9C and Figure 9E to Fig.9F The values ​​shown represent the mean ± SD of 6 healthy donors.

[0056] Figure 10A to Fig. 10B Scatter plots demonstrating GPRC5D CAR-T toxicity are shown. GPRC5D GC5B680-N68S-LH or untransduced (mock) T cells were added to GPRC5D containing a luciferase transgene at various E:T ratios. + MM.1S cells or GPRC5D – K562 cells. CAR-T cells were made from six healthy donors. Values ​​are expressed as mean ± SD at 6 hours (Figure 10A) or mean ± SD at 24 hours ( Fig. 10B ). The specific lysis percentage was calculated by: (i) measuring the luciferase signal in tumor cells in the presence of CAR-T for 6 hours or 24 hours divided by the luciferase signal in tumor cells alone at the same time point and multiplied by 100, and (ii) subtracting this number from 100. The formula is: 100-[(average of CAR-T+tumor luminescence / tumor luminescence alone)×100]. The dotted line represents a specific lysis percentage equal to zero. Negative lysis indicates cell growth.

[0057] FIG. 11A to FIG. 11F Scatter plots are shown showing the cytotoxicity of GPRC5D CAR-T cells added at the indicated E:T ratios using GPRC5D + H929, MM.1S, MM.1R, MOLP-2, and EJM cells or GPRC5D – K562 cells were evaluated. GPRC5D CAR-T cells were resistant to all GPRC5D + All cell lines showed cytotoxicity.

[0058] Fig.12 A bar graph is shown depicting the surface CD25 expression level (%CD25) by untransduced (mock) cells or GC5B680-N68S-LH cells in the presence of K562 cells, using an E:T ratio of 3:1. CAR-T was made from six healthy donors. Data are expressed as mean ± SD.

[0059] Fig.13Flow cytometry plots are shown, which show that GC5B680-N68S-LH specifically proliferates in the presence of GPRC5D+ target cells (H929 or MM.1S) or positive controls (agonist CD3 / CD28 beads) and in the absence of target cells or in the presence of GPRC5D- cells (K562). Cell proliferation was measured using CELLTRACE Violet (CTV) (X axis). CAR was detected using anti-idiotypic antibodies (Y axis) + event.

[0060] Fig.14 A bar graph is shown which shows that with GPRC5D + The level of cleaved caspase 3 / 7 in CAR-T (GC5B680-N68S-LH) cells incubated with cell line H929 for 24 h remained unchanged compared to controls (staurosporine, GPRC5D–K562 cells, or alone). An E:T ratio of 1:2 was used for H929 cells and K562 cells. - Indicates transduced cells that do not express GPRC5D CAR. + Represents transduced cells expressing GPRC5D CAR.

[0061] Fig.15 A graph showing the effect of GPRC5D-directed CAR-T cells on established MM.1S human MM xenografts in NSG-B2M mice according to Example 15 is shown. GC5B680-N68S-LH, GC5B680-N68S-HL, GC5B83-N24T-N31S-LH and GC5B83-N24T-N31S-HL, GPRC5D-directed CAR-T, mock, untransduced CAR-T, PBS, phosphate-buffered saline, NSG, non-obese diabetic severe combined immunodeficiency gamma, B2M, beta-2 microglobulin, MM, multiple myeloma. The volume of the tumor group is plotted as mean ± SEM. Tumor cells were implanted on day 0, 1×10 6 CAR+T cells were implanted on day 13. The symbol "*" indicates a significant difference relative to the PBS control on day 26 (p≤0.05, n=10 / group).

[0062] Fig.16A graph showing the effect of GC5B680-N68S-LH on the survival of NSG mice bearing disseminated H929 human MM xenografts according to Example 16 is shown. GC5B680-N68S-LH, GC5B680-N68S-HL, GC5B83-N24T-N31S-LH and GC5B83-N24T-N31S-HL, GPRC5D-directed CAR-T, mock, untransduced CAR-T, PBS, phosphate-buffered saline, NSG, non-obese diabetic severe combined immunodeficiency gamma, MM, multiple myeloma. Tumor cells implanted at day 0; 1×10 6 CAR+T cells were engrafted on day 9. The symbol "*" indicates a significant difference relative to the PBS control on day 111. DETAILED DESCRIPTION

[0063] A description of an exemplary embodiment follows:

[0064] The present disclosure provides chimeric antigen receptors (CARs) targeting a G protein-coupled receptor, i.e., G protein-coupled receptor family group C 5 member D (GPRC5D), cells comprising such CARs, and methods of using the CARs described herein to treat cancer (e.g., hematological malignancies and solid tumors).

[0065] The CAR of the present invention has antigen specificity for GPRC5D (e.g., a human GPRC5D polypeptide having an amino acid sequence of SEQ ID NO: 57, or a fragment thereof). As used herein, the phrases "having antigen specificity" and "eliciting an antigen-specific response" mean that CAR can specifically bind to and immunorecognize an antigen, so that the binding of CAR to the GPRC5D antigen induces an immune response. Methods for testing the antigen specificity of CAR and the ability to identify target cells are known in the art.

[0066] The present 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.

[0067] Hereinafter, several aspects of the present invention are described with reference to examples only for illustrative purposes. It should be understood that many specific details, relationships and methods have been described to provide a comprehensive understanding of the present invention. However, it will be readily appreciated by those skilled in the relevant art that the present invention can be implemented without one or more specific details, or can be implemented with other methods, schemes, reagents, cell lines and animals. The present invention is not limited by the displayed order of actions or events, because some actions can occur in different orders and / or occur simultaneously with other actions or events. In addition, not all displayed actions, steps or events are necessary for implementing the method according to the present invention. Many techniques and regulations described herein or quoted are well known to those skilled in the art, and are usually adopted by those skilled in the art using conventional methods.

[0068] Unless otherwise defined, all technical terms, symbols and other scientific terms or technical terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention belongs. In some cases, for the sake of clarity and / or for ease of reference, the terms with commonly understood meanings are defined herein, and these definitions contained herein should not necessarily be interpreted as indicating that there are significant differences with the meanings commonly understood in the art. It should also be understood that terms (such as those defined in commonly used dictionaries) should be interpreted as having the meanings consistent with their meanings in the context of the relevant field and / or as otherwise defined herein.

[0069] It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.As used herein, the indefinite articles "a", "an" and "the" should be understood to include plural references unless the context clearly indicates otherwise.

[0070] Chimeric Antigen Receptor

[0071] The present invention as a whole relates to the use of T cells that are genetically modified to stably express the desired chimeric antigen receptor. Chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide containing an antigen binding domain of an antibody (scFv) connected to a T cell signaling domain. The characteristics of CAR can include that they can redirect T cell specificity and reactivity to a selected target in a non-MHC restricted manner, thereby utilizing the antigen binding properties of monoclonal antibodies. Non-MHC restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thereby bypassing the main mechanism of tumor escape. In addition, when expressed in T cells, CAR advantageously does not dimerize with the alpha and beta chains of endogenous T cell receptors (TCR).

[0072] 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 a functional signaling domain derived from a stimulatory molecule defined as follows. 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. The cell can be genetically modified to stably express an antibody binding domain on its surface, thereby conferring a novel antigen specificity that is independent of MHC.

[0073] 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 FcγRI protein into a single chimeric protein. In one embodiment, the stimulatory molecule is a ζ chain associated with a T cell receptor complex.

[0074] As used herein, the term "intracellular signaling domain" refers to the intracellular part of a molecule. The functional part of the protein acts by transmitting information within the cell to regulate cell activity via a defined signaling pathway by producing a second messenger, or by responding to such a messenger to act as an effector. The intracellular signaling domain produces a signal that promotes the immune effector function of cells (e.g., CAR-T cells) containing CAR. Examples of immune effector functions (e.g., in CAR-T cells) include cytolytic activity and auxiliary activity, including secreted cytokines.

[0075] 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 stimulation or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may include a costimulatory intracellular domain. Example costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, with respect to CAR-T, the primary intracellular signaling domain may include a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain may include a cytoplasmic sequence from a co-receptor or a costimulatory molecule.

[0076] The primary intracellular signaling domain may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences comprising ITAMs include, but are not limited to, those derived from CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP12.

[0077] The primary intracellular signaling domain can be derived from, for example, the signaling domain of CD3-ζ, CD3ε, CD22, CD79a, CD66d, CD39 DAP10, DAP12, Fcε receptor I γ chain (FCER1G), FcRβ, CD3δ, CD3γ, CD5, CD226, or CD79B.

[0078] The term "ζ" or alternatively "ζ chain", "CD3-ζ" or "TCR-ζ" is defined as the protein provided with GenBank Accession No. BAG36664.1, or the equivalent residues from non-human species (e.g., rats, rabbits, primates, mice, rodents, monkeys, apes, etc.), and a "ζ stimulatory domain" or alternatively "CD3-ζ stimulatory domain" or "TCR-ζ stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the ζ chain that are sufficient to functionally transmit the initial signal necessary for T cell activation. In one aspect, the cytoplasmic domain of ζ comprises residues 52 to 164 of GenBank Accession No. BAG36664.1, or the equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.) that are functionally orthologous to them.

[0079] In a preferred embodiment, the intracellular signaling domain comprises a CD3-zeta stimulatory domain. In one aspect, the "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO:13.

[0080] The term "costimulatory molecule" refers to a cognate binding partner on a T cell, which specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of a T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, BTLA, GITR, CD226, HVEM and ZAP70.

[0081] The costimulatory intracellular signal transduction domain can be the intracellular part of a costimulatory molecule.Costimulatory molecules can be represented in the following protein families: TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signal transduction lymphocyte activation molecule (SLAM protein) and activated NK cell receptor. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand specifically bound to CD83, etc.

[0082] The intracellular signaling domain may comprise the entire intracellular portion of the molecule from which it is derived or the entire native intracellular signaling domain, or a functional fragment thereof.

[0083] The term "4-1BB" refers to a member of the TNFR superfamily having an amino acid sequence provided as GenBank Accession No. AAA62478.2, or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.); and "4-1BB costimulatory domain" is defined as amino acid residues 214 to 255 of GenBank Accession No. AAA62478.2, or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.). In one aspect, "4-1BB costimulatory domain" is a sequence provided as SEQ ID NO: 12, or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.).

[0084] In some embodiments, the cytoplasmic signaling domain also includes one or more functional signaling domains derived from at least one costimulatory molecule as defined herein. In one embodiment, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, CD3-ζ and / or CD28. CD28 is a very important T cell marker in T cell costimulation. CD27 is a member of the tumor necrosis factor receptor superfamily and acts as a costimulatory immune checkpoint molecule. 4-1BB delivers potent costimulatory signals to T cells, thereby promoting T lymphocyte differentiation and enhancing the long-term survival of T lymphocytes. CD3-ζ associates with TCR to generate signals, and comprises an activation motif (ITAM) based on immunoreceptor tyrosine.

[0085] In a preferred embodiment, the intracellular signaling domain comprises a co-stimulatory intracellular signaling domain, wherein the co-stimulatory intracellular signaling domain is a 4-1BB co-stimulatory domain. In a preferred embodiment, the 4-1BB co-stimulatory domain is a sequence provided as SEQ ID NO: 12.

[0086] In one embodiment, a transmembrane domain naturally associated with a domain in CAR is used. In another embodiment, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex. In an exemplary embodiment, the transmembrane domain is a CD8 alpha transmembrane domain.

[0087] In one embodiment, the CAR comprises a hinge domain that includes a CD8 alpha hinge domain.

[0088] In one embodiment, the CAR comprises a CD8α hinge domain and a CD8α transmembrane domain.

[0089] In one embodiment, the CAR comprises a hinge domain and an intracellular signaling domain, wherein the hinge domain comprises a CD8α hinge domain and the intracellular signaling domain comprises CD28, 4-1BB, and CD3-ζ.

[0090] 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"), which intracellular signaling domain includes, for example, a functional signaling domain derived from a stimulatory molecule as defined below.

[0091] In one embodiment, CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain includes a functional signaling domain derived from a stimulatory molecule. In one embodiment, CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain includes a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one embodiment, CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain includes at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule.

[0092] The CAR of the present invention can be designed to include a CD28 signaling domain and / or a 4-1BB signaling domain by itself, or in combination with any other desired cytoplasmic domain in the context of the CAR of the present invention. In one embodiment, the cytoplasmic domain of CAR may further include a signaling domain CD3-ζ. For example, the cytoplasmic domain of CAR may include, but is not limited to, CD3-ζ, 4-1BB and CD28 signaling modules, and combinations thereof.

[0093] In a preferred embodiment, the CAR comprises a CD8α hinge domain, a CD8α transmembrane domain, and an intracellular signaling domain, which includes the signaling domain CD3-ζ.

[0094] In a preferred embodiment, the CAR comprises a CD8α hinge domain, a CD8α transmembrane domain, and an intracellular signaling domain, which includes the signaling domain CD3-ζ and a 4-1BB co-stimulatory domain.

[0095] Therefore, the present invention provides CAR T cells and methods for using them in adoptive therapy.

[0096] The disclosure also provides variants of CAR, nucleic acid, polypeptide and protein described herein, such as functional variants. "Variant" refers to a polypeptide or polynucleotide different from a reference polypeptide 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, polypeptide or protein having substantial or significant sequence identity or similarity to a parent CAR, polypeptide or protein, and the functional variant retains the biological activity of the CAR, polypeptide or protein for which it is a variant. Functional variants include those variants of CAR, polypeptide or protein (parent CAR, polypeptide or protein) as described herein, and the variant retains the ability to identify target cells to a degree similar to that of a parent CAR, polypeptide or protein, the same degree or a higher degree. With respect to the parent CAR, polypeptide or protein, the functional variant can, 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 more identity with the amino acid sequence of the parent CAR, polypeptide or protein.

[0097] Herein, the structure of a polypeptide is at a position defined based on the % sequence identity to a reference sequence (with a given SEQ ID NO). In this context, the % sequence identity between two amino acid sequences can be determined by comparing the two sequences aligned in an optimal manner, and wherein the amino acid sequence to be compared may contain additions or deletions relative to the reference sequence for optimal alignment between the two sequences. The percent identity is calculated by determining the number of identical positions at which the amino acid residues are identical 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 percent identity 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 2 sequences-a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250), which is available on the website http: / / www.ncbi.nlm.nih.gov / gorf / bl2.html, can be used, the parameters used are those given by default (especially for the parameters "open gap penalty": 5, and "extend gap penalty": 2; the matrix selected is, for example, the matrix "BLOSUM 62" recommended by the program), and the percent identity between the two sequences to be compared is calculated directly by the program. Determining the sequence identity of a query sequence to a reference sequence is within the capabilities of a technician and can be performed using commercially available analysis software (such as BLAST TM )conduct.

[0098] Functional variants may, for example, include an amino acid sequence of a parent CAR, polypeptide or protein having at least one conservative amino acid substitution. In another embodiment, the functional variant may include an amino acid sequence of a parent CAR, polypeptide or protein having at least one non-conservative amino acid substitution. In this case, the non-conservative amino acid substitution may not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, so that the biological activity of the functional variant is increased compared to the parent CAR, polypeptide or protein.

[0099] The amino acid substitution of the CAR of the present invention may be a conservative amino acid substitution.Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which an amino acid having a specific physical property and / or chemical property is replaced with another amino acid having the same or similar chemical property or physical property.For example, conservative amino acid substitutions may be acidic amino acids replaced by another acidic amino acid (e.g., Asp or Glu), amino acids with non-polar side chains replaced by another amino acid with non-polar side chains (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), basic amino acids replaced by another basic amino acid (Lys, Arg, etc.), amino acids with polar side chains replaced by another amino acid with polar side chains (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), and the like.

[0100] The CAR, polypeptide or protein can consist essentially of one or more specified amino acid sequences described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0101] The CAR, polypeptide and protein (including functional parts and functional variants) of the embodiments of the present disclosure can have any length, that is, can contain any number of amino acids, provided that the CAR, polypeptide or protein (or its functional part or functional variant) maintains its biological activity, such as the ability to specifically bind to an antigen, detect diseased cells (e.g., cancer cells) in a host, or treat or prevent a disease in a host, etc. For example, the length of the polypeptide can be 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 475, about 50 ... 5, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000 or more amino acids. Polypeptides of the present invention also include oligopeptides.

[0102] The CAR, polypeptide and protein of the embodiments of the present invention (including the functional part and functional variant of the present invention) may include 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, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, α-(2-amino-2-norbornane)-carboxylic acid, α, γ-diaminobutyric acid, α, β-diaminopropionic acid, homophenylalanine, 4-chlorophenylalanine, 4-carboxylphenylalanine, 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, dihydroindoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide and α-tert-butylglycine.

[0103] The CAR, polypeptide and protein (including functional parts and functional variants) of the embodiments of the present invention can be subjected to post-translational modification. They can be glycosylated, esterified, N-acylated, amidated, carboxylated, phosphorylated, esterified, cyclized via, for example, disulfide bridges, or converted into acid addition salts. In some embodiments, they are dimerized or polymerized, or conjugated.

[0104] The CAR, polypeptide and / or protein (including functional parts and functional variants thereof) of the embodiments of the present invention can be obtained by methods known in the art. Suitable methods for synthesizing polypeptides and proteins from scratch 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, Reid, R., ed., Marcel Dekker, Inc., 2000; and Epitope Mapping, Westwood et al., ed., Oxford University Press, Oxford, United Kingdom, 2001. In addition, standard recombinant methods can be used to recombinantly produce polypeptides and proteins using nucleic acids described herein. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, 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. In addition, some of the CARs, polypeptides and proteins of the present invention (including functional parts and functional variants thereof) can be separated and / or purified from sources such as plants, bacteria, insects, mammals, etc. Methods of separation and purification are known in the art. Alternatively, the CARs, polypeptides and / or proteins described herein (including functional parts and functional variants thereof) can be commercially synthesized. In this regard, the CARs, polypeptides and proteins can be synthetic, recombinant, separated and / or purified.

[0105] 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-N 6-Isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, braided glycoside, β-D-galactosyl braided glycoside, inosine, N 6 -isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetate methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil and 2,6-diaminopurine.

[0106] The nucleic acid may comprise any isolated or purified nucleotide sequence encoding a CAR, a polypeptide or a protein or any of its functional parts or functional variants. Alternatively, the nucleotide sequence may include a degenerate nucleotide sequence or a combination of degenerate sequences with any of the sequences.

[0107] Some embodiments of the present invention also provide isolated or purified nucleic acids comprising a nucleotide sequence that is complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any nucleic acid described herein.

[0108] Nucleotide sequences hybridized under stringent conditions can hybridize under high stringency conditions.So-called "high stringency conditions" means that the nucleotide sequence specifically hybridizes with a target sequence (nucleotide sequence of any nucleic acid described herein) in an amount that can be detected as stronger than non-specific hybridization.High stringency conditions include conditions for distinguishing polynucleotides with precise complementary sequences, or polynucleotides containing only a small amount of dispersed mismatches from random sequences that just have a small amount of small regions (e.g., 3 to 12 bases) that match the nucleotide sequence.This small complementary region is easier to melt than the full-length complementary sequence of 14 to 17 or more bases, and high stringency hybridization enables them to be easily distinguished.Relatively high stringency conditions will include, for example, low salt conditions and / or high temperature conditions, such as provided by about 0.02M NaCl to 0.1M NaCl or equivalents at a temperature of about 50°C to 70°C.Such high stringency conditions allow very few (if any) mismatches between nucleotide sequences and templates or target chains, 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 incremental formamide.

[0109] In one embodiment, the nucleic acid of the present invention can be incorporated into a recombinant expression vector. The present disclosure provides a recombinant expression vector comprising any nucleic acid of the present invention. As used herein, the term "recombinant expression vector" means the following genetically modified oligonucleotide or polynucleotide construct: when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide or peptide, and the vector contacts a host cell under conditions sufficient to allow the mRNA, protein, polypeptide or peptide to be expressed in the cell, it allows the host cell to express the mRNA, protein, polypeptide or peptide. The vectors described herein are not naturally occurring as a whole; however, portions of these vectors may be naturally occurring. The recombinant expression vector may contain any type of nucleotides, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from a natural source, and it may contain natural, non-natural or altered nucleotides. The recombinant expression vector may contain naturally occurring or non-natural internucleotide bonds, or both types of bonds. Non-naturally occurring or altered nucleotides or internucleotide bonds do not hinder transcription or replication of the vector.

[0110] In one embodiment, the recombinant expression vector of the present invention can be any suitable recombinant expression vector, and can be used for transformation or transfection of any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. Vectors can be selected from the group consisting of the following items: 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 and λNM1149, λZapII (Stratagene) can be used. The example of plant expression vectors includes pBI01, pBI01.2, pBI121, pBI101.3 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). The recombinant expression vector may be a viral vector, such as a retroviral vector, such as a gammaretroviral vector.

[0111] 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. (supra) and Ausubel et al. (supra). Circular or linear expression vector constructs can be prepared to contain a replication system that is functional in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, ColE1, SV40, 2μ plasmid, λ, bovine papilloma virus, etc.

[0112] Recombinant expression vectors may contain regulatory sequences, such as transcription and translation start and stop codons, that are specific for the type of host (e.g., bacteria, plants, fungi, or animals) into which the vector is to be appropriately introduced, and consider whether the vector is DNA-based or RNA-based.

[0113] The recombinant expression vector may comprise one or more marker genes that allow selection of transformed or transfected hosts. Marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.), complementation in auxotrophic hosts to provide prototrophy, etc. Suitable marker genes for the expression vector include, for example, neomycin / G418 resistance genes, histidinol x resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0114] The recombinant expression vector may include a nucleotide sequence operably connected to the CAR, polypeptide or protein (including its functional part and functional variant), or may be operably connected to the natural or standard promoter of the nucleotide sequence complementary or hybridized with the nucleotide sequence encoding the CAR, polypeptide or protein. The selection of promoters (such as strong promoters, weak promoters, tissue-specific promoters, inducible promoters and development-specific promoters) is within the skill range of ordinary technicians. Similarly, combining nucleotide sequence with promoters is also within the skill range of technicians. Promoters may be non-viral promoters or viral promoters, such as cytomegalovirus (CMV) promoters, RSV promoters, SV40 promoters, or promoters found in the long terminal repeats of mouse stem cell viruses.

[0115] The recombinant expression vector can be designed for transient expression, stable expression or both. In addition, the recombinant expression vector can be prepared for constitutive expression or for inducible expression.

[0116] In addition, the recombinant expression vector can be prepared to include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes cell death that expresses the suicide gene. The suicide gene can be a gene that confers sensitivity to an agent (e.g., a drug) on ​​cells expressing the gene, and causes cell death when the cell contacts or is exposed to the agent. Suicide genes are known in the art and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) genes, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0117] Included within the scope of the present invention is a conjugate (e.g., a bioconjugate) comprising any of the following: the CAR, polypeptide or protein (including any of its functional part or functional variant), host cell, nucleic acid, recombinant expression vector, host cell colony, or antibody or its antigen binding portion. In general, conjugates and methods for synthesizing conjugates are known in the art (see, e.g., Hudecz, F., Methods Mol. Biol. 298: 209-223 (2005) and Kirin et al., Inorg Chem. 44 (15): 5405-5415 (2005)).

[0118] 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 a CAR of the present invention.

[0119] The antibody can be any type of immunoglobulin known in the art. Immunoglobulins can be divided into five major classes: IgA, IgD, IgE, IgG and IgM. IgA and IgG are further divided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. The antibody light chain of a vertebrate species can be designated as one of two types, i.e., kappa and lambda, based on the amino acid sequence of its constant domain. Antibodies can be of any type or isotype.

[0120] Antibodies include immunoglobulin molecules, specifically 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 multimeric antibodies, single-chain antibodies, domain antibodies, and any other modified configurations of immunoglobulin molecules containing antigen-binding sites with desired specificity. Antibodies can be naturally occurring antibodies, such as antibodies isolated and / or purified from mammals (e.g., mice, primates, mice, rabbits, goats, horses, chickens, hamsters, humans, etc.). Alternatively, antibodies can be engineered (e.g., genetically engineered) antibodies.

[0121] "Human antibody" refers to an antibody that is optimized to have 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 comprises a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. If the variable region of a human antibody is obtained by using a system of human germline immunoglobulins or rearranged immunoglobulin genes, the human antibody comprises a heavy chain variable region and a light chain variable region "derived from" a sequence of human origin. Such exemplary systems are human immunoglobulin gene libraries displayed on phages, and transgenic non-human animals, such as mice or rats carrying human immunoglobulin loci. Due to the differences between the systems for obtaining human antibodies and human immunoglobulin loci, the introduction of somatic mutations or the intentional introduction of substitutions into the framework or CDR or both, so "human antibodies" generally include amino acid differences compared to immunoglobulins expressed in humans. Typically, the amino acid sequence of a "human antibody" is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence encoded by a human germline immunoglobulin gene or a rearranged immunoglobulin gene. In some cases, a "human antibody" may comprise a consensus framework sequence derived from human framework sequence analysis (e.g., as described in Knappik et al., (2000) J Mol Biol 296:57-86), or a synthetic HCDR3 bound to a human immunoglobulin gene library displayed on a phage (e.g., as described in Shi et al., (2010) J Mol Biol 397:385-96 and International Patent Publication No. WO2009 / 085462). The definition of "human antibody" does not include antibodies in which at least one CDR is derived from a non-human species.

[0122] "Humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. A humanized antibody may comprise substitutions in the framework so that the framework may not be an exact copy of an expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0123] Typically, humanized antibodies have an antigen binding site derived from a non-human species and the variable region framework is derived from human immunoglobulin sequences. Human antibodies have a heavy chain variable region and a light chain variable region, wherein both the framework and the antigen binding site are derived from sequences of human origin.

[0124] In addition, the antibody can have any level of affinity or avidity for the functional portion of the CAR. In some embodiments, the antibody can have a range of affinities (K D) binds to the GPRC5D antigen. In one embodiment according to the invention, and in some embodiments of each of the numbered embodiments listed below, the antibody binds to the GPRC5D antigen with high affinity, e.g., as determined by surface plasmon resonance or Kinexa method, K D 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 therein, as practiced by those skilled in the art. An exemplary affinity is equal to or less than 1×10 -8 M. Another example has an affinity equal to or less than 1×10 -9 M.

[0125] Methods for testing the ability of an antibody to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and competitive inhibition assays.

[0126] Suitable methods for preparing antibodies are known in the art. For example, standard hybridoma methods are 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 ed., 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, e.g., Huse et al., Science, 246, 1275-81 (1989)) are known in the art. Additionally, methods of raising antibodies in non-human animals are described in, for example, U.S. Patent Nos. 5,545,806, 5,569,825, and 5,714,352 and U.S. Patent Application Publication No. 2002 / 0197266A1.

[0127] Phage display can also be used to produce antibodies. In this regard, phage libraries encoding antigen-binding variable (V) domains of antibodies can be produced using standard molecular biology techniques and recombinant DNA techniques (see, for example, Sambrook et al. (ibid.) and Ausubel et al. (ibid.)). Select the phage encoding the variable region with the desired specificity for specific binding with the desired antigen (i.e., GPRC5D), and rebuild the complete or partial antibody comprising the selected variable region. The nucleic acid sequence encoding the reconstructed antibody is introduced into a suitable cell line (such as a myeloma cell for producing a hybridoma) so that the antibody with monoclonal antibody characteristics is secreted by the cell (see, for example, Janeway et al. (ibid.), Huse et al. (ibid.) and U.S. Patent number 6,265,150).

[0128] 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 in, for example, U.S. Patent Nos. 5,545,806 and 5,569,825 and Janeway et al. (supra).

[0129] Methods for producing humanized antibodies are known in the art and are described, for example, in Janeway et al. (supra), U.S. Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No. 0239400B1, and British Patent No. 2188638. Humanized antibodies can also be produced using the antibody resurfacing technique described in U.S. Pat. No. 5,639,641 and Pedersen et al., J. Mol. Biol., 235, 959-973 (1994).

[0130] As used herein, an antibody may be multi-chain or single chain, or an intact immunoglobulin, and may be derived from natural or recombinant sources.An antibody may be a tetramer of immunoglobulin molecules.

[0131] In some embodiments, the antibody is a bispecific antibody. "Bispecific" refers to a molecule (such as an antibody) that specifically binds to two different antigens or two different epitopes within the same antigen. Bispecific molecules may have cross-reactivity to other related antigens, for example, to the same antigen from other species (homologs) (such as humans or monkeys, for example, cynomolgus (Macacafascicularis) (cynomolgus, cyno) or chimpanzees (Pan troglodytes)), or may bind to an epitope shared between two or more different antigens. The VL region and / or VH region of an existing antibody or the VL region and VH region identified de novo as described herein may be engineered into a bispecific full-length antibody. Such bispecific antibodies can be prepared by modulating CH3 interactions in antibody Fc to form bispecific antibodies using techniques such as those described in the following patents: U.S. Patent No. 7,695,936; International Patent Publication No. WO04 / 111233; U.S. Patent Publication No. U52010 / 0015133; U.S. Patent Publication No. US2007 / 0287170; International Patent Publication No. WO2008 / 119353; U.S. Patent Publication No. US2009 / 0182127; U.S. Patent Publication No. US52010 / 0286374; U.S. Patent Publication No. US2011 / 0123532; International Patent Publication No. WO2011 / 131746; International Patent Publication No. WO2011 / 143545; or U.S. Patent Publication No. US2012 / 0149876. For example, the bispecific antibodies of the present invention can be produced in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 region of two monospecific homodimeric antibodies according to the method described in International Patent Publication No. WO2011 / 131746, and forming a bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies under reducing conditions that allow disulfide bond isomerization. In the method, the first monospecific bivalent antibody and the second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; these antibodies are incubated together under reducing conditions sufficient to cause disulfide bond isomerization of cysteine ​​in the hinge region; thereby generating a bispecific antibody by Fab arm exchange. The incubation conditions are ideally restored to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine ​​and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine.For example, the following conditions can be used: incubation for at least 90 minutes at a pH of 5 to 8 (eg, pH 7.0 or pH 7.4) and a temperature of at least 20°C in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol.

[0132] The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof that retains the antigen binding properties of a parent full-length antibody. It refers to, for example, an antigen-binding domain, e.g., an antigen-determining variable region of an intact antibody, which is sufficient to confer recognition and binding, e.g., 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), Camelidae VHH domains, and multispecific antibodies formed from antibody fragments.

[0133] The term "scFv" refers to a protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region. In some embodiments, the light chain variable region and the heavy chain variable region are continuously connected via a short flexible polypeptide linker and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise indicated, as used herein, scFv can have VL variable regions and VH variable regions in either order, for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv can comprise VL-linker-VH or can comprise VH-linker-VL.

[0134] One embodiment of the present invention also provides an antigen binding portion of any of the antibodies described herein. The antigen binding portion can be any portion having at least one antigen binding site, such as Fab, F(ab')2, dsFv, sFv, diabodies and triabodies.

[0135] In some embodiments, the antigen binding fragment is a heavy chain complementary determining region (HCDR) 1, 2 and / or 3, a light chain complementary determining region (LCDR) 1, 2 and / or 3, a heavy chain variable region (VH) or a light chain variable region (VL), Fab, F(ab')2, Fd and Fv fragments, and a domain antibody (dAb) comprising (e.g., consisting of any of the following) one VH domain or one VL domain. The VH domain and the VL domain can be linked together via a linker (e.g., a synthetic linker).

[0136] "Complementarity determining region (CDR)" is an antigen binding site in an antibody. CDRs can be defined using various terms: (i) three in VH (HCDR1, HCDR2, HCDR3) and three in 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 ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) three in VH (H1, H2, H3) and three in VL (L1, L2, L3) "hypervariable region", "HVR" or "HV" refers to the region of the antibody variable domain that is structurally hypervariable, 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 numbering and definitions of 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" used herein include CDRs defined by any of the above methods (Kabat, Chothia or IMGT).

[0137] Additionally, the antibodies or antigen-binding portions thereof can be modified to include detectable labels, such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0138] The present disclosure also provides a nucleic acid comprising a nucleotide sequence encoding any one of the CARs, polypeptides or proteins described herein (including functional portions and functional variants thereof).

[0139] The part of the CAR comprising an antibody or its antibody fragment can exist in various forms, wherein the antigen binding domain is represented as a part of a continuous polypeptide chain, including, for example, a single domain antibody fragment (sdAb), scFv and human chimeric antibodies 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 242: 423-426). In one aspect, the antigen binding domain of the CAR composition of the present invention comprises an antibody fragment. In one aspect, the CAR comprises an antibody fragment with scFv.

[0140] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage or yeast expression system. The term should also be understood to mean an antibody that has been 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 sequence technology available and known in the art.

[0141] The term "antigen" refers to a molecule that triggers an immune response. The immune response may involve antibody production, or the activation of specific immunocompetent cells, or both. It will be appreciated by the skilled person that any macromolecule (including nearly all proteins or peptides) may be used as an antigen. In addition, antigens may be derived from recombinant DNA or genomic DNA. It will be appreciated by the skilled person that any DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that causes an immune response thus encodes the term "antigen" as used herein. In addition, it will be appreciated by those skilled in the art that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. It is apparent that the present disclosure includes, but is not limited to, using partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to encode polypeptides that cause a desired immune response.

[0142] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain binds to a GPRC5D antigen.

[0143] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0144] a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:66, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:67, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:68;

[0145] a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:58, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:59, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:60;

[0146] a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:39, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:40, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:41;

[0147] a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:42, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:43, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:44; or

[0148] a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:45, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:46, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:47;

[0149] The extracellular antigen binding domain binds to the GPRC5D antigen.

[0150] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0151] A heavy chain complementary determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:66, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:67, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:68; wherein the extracellular antigen binding domain binds to the GPRC5D antigen.

[0152] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0153] A heavy chain complementary determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:58, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:59, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:60; wherein the extracellular antigen binding domain binds to the GPRC5D antigen.

[0154] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0155] A heavy chain complementary determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:39, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:40, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:41; wherein the extracellular antigen binding domain binds to the GPRC5D antigen.

[0156] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0157] A heavy chain CDR1 having the amino acid sequence of SEQ ID NO:42, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:43, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:44, wherein the extracellular antigen binding domain binds to the GPRC5D antigen.

[0158] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises: a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 45, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 46, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 47;

[0159] The extracellular antigen binding domain binds to the GPRC5D antigen.

[0160] In one embodiment, the extracellular antigen binding domain comprises

[0161] The extracellular antigen-binding domain comprises a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 66, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 67, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 68, and further comprises a light chain CDR1 having an amino acid sequence of SEQ ID NO: 69, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 70, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 71;

[0162] The extracellular antigen-binding domain comprises a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 58, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 59, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 60, and further comprises a light chain CDR1 having an amino acid sequence of SEQ ID NO: 61, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 62, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 63;

[0163] A heavy chain CDR1 having the amino acid sequence of SEQ ID NO:39, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:40, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:41, and further comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO:48, a light chain CDR2 having the amino acid sequence of SEQ ID NO:49, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:50;

[0164] The extracellular antigen-binding domain comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:42, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:43, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:44, and further comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO:51, a light chain CDR2 having the amino acid sequence of SEQ ID NO:52, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:53; or

[0165] The extracellular antigen-binding domain comprises a heavy chain CDR1 having an amino acid sequence of SEQ ID NO:45, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO:46, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO:47, and also comprises a light chain CDR1 having an amino acid sequence of SEQ ID NO:54, a light chain CDR2 having an amino acid sequence of SEQ ID NO:55, and a light chain CDR3 having an amino acid sequence of SEQ ID NO:56.

[0166] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0167] A heavy chain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:66, SEQ ID NO:58, SEQ ID NO:39, SEQ ID NO:42 and SEQ ID NO:45 and conservative modifications thereof, wherein the extracellular antigen-binding domain binds to the GPRC5D antigen.

[0168] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0169] A heavy chain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:67, SEQ ID NO:59, SEQ ID NO:40, SEQ ID NO:43 and SEQ ID NO:46 and conservative modifications thereof, wherein the extracellular antigen-binding domain binds to the GPRC5D antigen.

[0170] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0171] A heavy chain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:68, SEQ ID NO:60, SEQ ID NO:41, SEQ ID NO:44 and SEQ ID NO:47 and conservative modifications thereof, wherein the extracellular antigen binding domain binds to the GPRC5D antigen.

[0172] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0173] a heavy chain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:66, SEQ ID NO:58, SEQ ID NO:39, SEQ ID NO:42 and SEQ ID NO:45, and conservative modifications thereof;

[0174] a heavy chain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:67, SEQ ID NO:59, SEQ ID NO:40, SEQ ID NO:43 and SEQ ID NO:46, and conservative modifications thereof; and

[0175] a heavy chain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:68, SEQ ID NO:60, SEQ ID NO:41, SEQ ID NO:44 and SEQ ID NO:47, and conservative modifications thereof;

[0176] The extracellular antigen binding domain binds to the GPRC5D antigen.

[0177] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0178] A light chain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:69, SEQ ID NO:54, SEQ ID NO:48, SEQ ID NO:51 and SEQ ID NO:54 and conservative modifications thereof, wherein the extracellular antigen-binding domain binds to the GPRC5D antigen.

[0179] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0180] A light chain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:70, SEQ ID NO:55, SEQ ID NO:49, SEQ ID NO:52 and SEQ ID NO:55 and conservative modifications thereof, wherein the extracellular antigen-binding domain binds to the GPRC5D antigen.

[0181] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0182] A light chain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:56, SEQ ID NO:50, SEQ ID NO:53 and SEQ ID NO:56 and conservative modifications thereof, wherein the extracellular antigen binding domain binds to the GPRC5D antigen.

[0183] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0184] a light chain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:69, SEQ ID NO:54, SEQ ID NO:48, SEQ ID NO:51 and SEQ ID NO:54 and conservative modifications thereof;

[0185] a light chain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:70, SEQ ID NO:55, SEQ ID NO:49, SEQ ID NO:52 and SEQ ID NO:55, and conservative modifications thereof; and

[0186] A light chain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:56, SEQ ID NO:50, SEQ ID NO:53 and SEQ ID NO:56 and conservative modifications thereof; wherein the extracellular antigen binding domain binds to the GPRC5D antigen.

[0187] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0188] a heavy chain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:66, SEQ ID NO:58, SEQ ID NO:39, SEQ ID NO:42 and SEQ ID NO:45, and conservative modifications thereof;

[0189] a heavy chain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:67, SEQ ID NO:59, SEQ ID NO:40, SEQ ID NO:43 and SEQ ID NO:46, and conservative modifications thereof; and

[0190] a heavy chain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:68, SEQ ID NO:60, SEQ ID NO:41, SEQ ID NO:44 and SEQ ID NO:47, and conservative modifications thereof; and

[0191] a light chain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO:69, SEQ ID NO:54, SEQ ID NO:48, SEQ ID NO:51 and SEQ ID NO:54 and conservative modifications thereof;

[0192] a light chain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO:70, SEQ ID NO:55, SEQ ID NO:49, SEQ ID NO:52 and SEQ ID NO:55, and conservative modifications thereof; and

[0193] a light chain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:56, SEQ ID NO:50, SEQ ID NO:53 and SEQ ID NO:56 and conservative modifications thereof;

[0194] The extracellular antigen binding domain binds to the GPRC5D antigen.

[0195] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0196] A heavy chain CDR1 having the amino acid sequence of SEQ ID NO:66, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:67, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:68, and also includes a light chain CDR1 having the amino acid sequence of SEQ ID NO:69, 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.

[0197] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0198] A heavy chain CDR1 having the amino acid sequence of SEQ ID NO:58, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:59, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:60, and also includes a light chain CDR1 having the amino acid sequence of SEQ ID NO:61, a light chain CDR2 having the amino acid sequence of SEQ ID NO:62, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:63.

[0199] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0200] A heavy chain CDR1 having the amino acid sequence of SEQ ID NO:39, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:40, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:41, and also includes a light chain CDR1 having the amino acid sequence of SEQ ID NO:48, a light chain CDR2 having the amino acid sequence of SEQ ID NO:49, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:50.

[0201] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0202] A heavy chain CDR1 having the amino acid sequence of SEQ ID NO:42, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:43, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:44, and also includes a light chain CDR1 having the amino acid sequence of SEQ ID NO:51, a light chain CDR2 having the amino acid sequence of SEQ ID NO:52, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:53.

[0203] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0204] A heavy chain CDR1 having the amino acid sequence of SEQ ID NO:45, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:46, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:47, and also includes a light chain CDR1 having the amino acid sequence of SEQ ID NO:54, a light chain CDR2 having the amino acid sequence of SEQ ID NO:55, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:56.

[0205] In one embodiment, the extracellular antigen binding domain comprises: a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:65, SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, or a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:72, SEQ ID NO:64, SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, or a combination of a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:65, SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5 and a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO:72, SEQ ID NO:64, SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.

[0206] In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising the amino acid sequence of any one of SEQ ID NO: 73, SEQ ID NO: 65, SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 73, SEQ ID NO: 65, SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 73. In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 65. In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1.In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3. In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:5.

[0207] In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising the amino acid sequence of any one of SEQ ID NO: 72, SEQ ID NO: 64, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In some embodiments, the extracellular antigen binding domain comprises a heavy chain variable region (HCVR) comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 72, SEQ ID NO: 64, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the extracellular antigen binding domain comprises a heavy chain variable region (HCVR) comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 72. In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the extracellular antigen binding domain comprises a heavy chain variable region (HCVR) comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:64. In some embodiments, the extracellular antigen binding domain comprises a heavy chain variable region (HCVR) comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:2.In some embodiments, the extracellular antigen binding domain comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the extracellular antigen binding domain comprises a heavy chain variable region (HCVR) comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. In some embodiments, the extracellular antigen binding domain comprises a heavy chain variable region (HCVR) comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:6.

[0208] In one embodiment, the extracellular antigen binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO:73, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:73; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:72, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:73. NO:72 has an amino acid sequence with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0209] In one embodiment, the extracellular antigen binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 65; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 64. NO:64 has an amino acid sequence with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0210] In one embodiment, the extracellular antigen binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 1; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 1. NO:2 has an amino acid sequence with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0211] In one embodiment, the extracellular antigen binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:3; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:4. NO:4 has an amino acid sequence with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0212] In one embodiment, the extracellular antigen binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:5; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:6. NO:6 has an amino acid sequence with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0213] In one embodiment, the extracellular antigen binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO:73 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:72.

[0214] In one embodiment, the extracellular antigen binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO:65 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:64.

[0215] 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:2.

[0216] 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.

[0217] In one embodiment, the extracellular antigen binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO:5 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6.

[0218] Herein, in embodiments where the amino acid sequence of the VH domain exhibits less than 100% sequence identity with a given reference VH sequence, it may still include the same heavy chain CDR as the HCDR1, HCDR2, and HCDR3 of the reference sequence, while exhibiting amino acid sequence variations in the framework region. Similarly, embodiments where the amino acid sequence of the VL domain exhibits less than 100% sequence identity with a given reference sequence may still include the same light chain CDR as the LCDR1, LCDR2, and LCDR3 of the reference sequence, while exhibiting amino acid sequence variations in the framework region. Similarly, embodiments where the scFv or extracellular domain of the CAR disclosed herein exhibits less than 100% sequence identity with a given reference VH sequence may still include the same heavy chain CDR as the HCDR1, HCDR2, and HCDR3 of the reference sequence, and the same light chain CDR as the LCDR1, LCDR2, and LCDR3 of the reference sequence, while exhibiting amino acid sequence variations in the framework region.

[0219] In one embodiment, the extracellular antigen binding domain comprises an scFv. In some embodiments, the scFv comprises a linker polypeptide between a light chain variable region and a heavy chain variable region. In certain embodiments, the extracellular antigen binding domain is an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 78, SEQ ID NO: 77, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, and specifically binds to a GPRC5D polypeptide (e.g., a human GPRC5D polypeptide having an amino acid sequence of SEQ ID NO: 57, or a fragment thereof).

[0220] In some embodiments, a scFv comprises, from N-terminus to C-terminus, a VH, a linker, and a VL (VH-linker-VL).

[0221] In some embodiments, a scFv comprises, from N-terminus to C-terminus, a VL, a linker, and a VH (VL-linker-VH).

[0222] In a recombinant expression system, the linker is a peptide linker and may comprise any naturally occurring amino acid. Exemplary amino acids that may be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His and The. The length of the linker should be sufficient to allow VH and VL to form a correct conformation relative to each other so that they maintain the desired activity (such as binding to GPRC5D) in a manner connected.

[0223] The length of the joint can be about 5 to 50 amino acids. In some embodiments, the length of the joint is about 10 to 40 amino acids. In some embodiments, the length of the joint is about 10 to 35 amino acids. In some embodiments, the length of the joint is about 10 to 30 amino acids. In some embodiments, the length of the joint is about 10 to 25 amino acids. In some embodiments, the length of the joint is about 10 to 20 amino acids. In some embodiments, the length of the joint is about 15 to 20 amino acids. In some embodiments, the length of the joint is 6 amino acids. In some embodiments, the length of the joint is 7 amino acids. In some embodiments, the length of the joint is 8 amino acids. In some embodiments, the length of the joint is 9 amino acids. In some embodiments, the length of the joint is 10 amino acids. In some embodiments, the length of the joint is 11 amino acids. In some embodiments, the length of the joint is 12 amino acids. In some embodiments, the length of the joint is 13 amino acids. In some embodiments, the length of the joint is 14 amino acids. In some embodiments, the length of the joint is 15 amino acids. In some embodiments, the length of the joint is 16 amino acids. In some embodiments, the length of the linker is 17 amino acids. In some embodiments, the length of the linker is 18 amino acids. In some embodiments, the length of the linker is 19 amino acids. In some embodiments, the length of the linker is 20 amino acids. In some embodiments, the length of the linker is 21 amino acids. In some embodiments, the length of the linker is 22 amino acids. In some embodiments, the length of the linker is 23 amino acids. In some embodiments, the length of the linker is 24 amino acids. In some embodiments, the length of the linker is 25 amino acids. In some embodiments, the length of the linker is 26 amino acids. In some embodiments, the length of the linker is 27 amino acids. In some embodiments, the length of the linker is 28 amino acids. In some embodiments, the length of the linker is 29 amino acids. In some embodiments, the length of the linker is 30 amino acids. In some embodiments, the length of the linker is 31 amino acids. In some embodiments, the length of the linker is 32 amino acids. In some embodiments, the length of the linker is 33 amino acids. In some embodiments, the length of the linker is 34 amino acids. In some embodiments, the length of the linker is 35 amino acids. In some embodiments, the length of the linker is 36 amino acids. In some embodiments, the length of the linker is 37 amino acids. In some embodiments, the length of the linker is 38 amino acids. In some embodiments, the length of the linker is 39 amino acids. In some embodiments, the length of the linker is 40 amino acids.Exemplary linkers that can be used are Gly-rich linkers, linkers containing Gly and Ser, linkers containing Gly and Ala, linkers containing Ala and Ser, and other flexible linkers.

[0224] In one embodiment, the linker polypeptide comprises the amino acid sequence of SEQ ID NO:7.

[0225] Other linker sequences may include portions of immunoglobulin hinge regions, CL or CH1 derived from any immunoglobulin heavy or light chain isotype. Exemplary linkers that may be used are shown in Table 1. Additional linkers are described, for example, in International Patent Publication No. WO2019 / 060695, which is incorporated herein by reference in its entirety.

[0226] In one embodiment, the linker polypeptide comprises the amino acid sequence of any one of SEQ ID NO:91 to SEQ ID NO:123.

[0227] Table 1.

[0228] Connector Name Amino acid sequence SEQ ID NO: Connector 1 GGSEGKSSGSGSESKSTGGS 91 Connector 2 GGGSGGGS 92 Connector 3 GGGSGGGSGGGS 93 Connector 4 GGGSGGGSGGGSGGGS 94 Connector 5 GGGSGGGSGGGSGGGSGGGS 95 Connector 6 GGGGSGGGGSGGGGS 96 Connector 7 GGGGSGGGGSGGGGSGGGGS 97 Connector 8 GGGGSGGGGSGGGGSGGGGSGGGGS 98 Connector 9 GSTSGSGKPGSGEGSTKG 99 Connector 10 IRPRAIGGSKPRVA 100 Connector 11 GKGGSGKGGSGKGGS 101 Connector 12 GGKGSGGKGSGGKGS 102 Connector 13 GGGKSGGGKSGGGKS 103 Connector 14 GKGKSGKGKSGKGKS 104 Connector 15 GGGKSGGKGSGKGGS 105 Connector 16 GKPGSGKPGSGKPGS 106 Connector 17 GKPGSGKPGSGKPGSGKPGS 107 Connector 18 GKGKSGKGKSGKGKSGKGKS 108 Connector 19 STAGDTHLGGEDFD 109 Connector 20 GEGGSGEGGSGEGGS 110 Connector 21 GGEGSGGEGSGGEGS 111 Connector 22 GEGESGEGESGEGES 112 Connector 23 GGGESGGEGSGEGGS 113 Connector 24 GEGESGEGESGEGESGEGES 114 Connector 25 GSTSGSGKPGSGEGSTKG 115 Connector 26 PRGASKSGSASQTGSAPGS 116 Connector 27 GTAAAGAGAAGGAAAGAAG 117 Connector 28 GTSGSSGSGSGGSGSGGGG 118 Connector 29 GKPGSGKPGSGKPGSGKPGS 119 Connector 30 GSGS 120 Connector 31 APAPAPAPAP 121 Connector 32 APAPAPAPAPAPAPAPAPAP 122 Connector 33 AEAAAKEAAAKEAAAAKEAAAAKEAAAAKAAA 123

[0229] In one embodiment, the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NO:78, SEQ ID NO:77, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26. In some embodiments, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 78, SEQ ID NO: 77, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 78. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 77. In some embodiments, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 77. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 75. In some embodiments, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 75.In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 76. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 25.In some embodiments, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 25. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the scFv comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26.

[0230] In one embodiment, the extracellular antigen binding domain comprises a signal polypeptide. In some embodiments, the signal polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, 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 SEQ ID NO: 11.

[0231] In one aspect, the present disclosure provides a CAR comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 82, SEQ ID NO: 81, SEQ ID NO: 80, SEQ ID NO: 79, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, 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 thereto. Another feature of the CAR having an extracellular antigen binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:82, SEQ ID NO:81, SEQ ID NO:80, SEQ ID NO:79, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22 is that the extracellular antigen binding domain binds to the GPRC5D antigen.

[0232] In one embodiment, the intracellular signaling domain comprises a polypeptide component selected from the group consisting of a TNF receptor superfamily member 9 (CD137) component, a T cell surface glycoprotein CD3 zeta chain (CD3z) component, a cluster of differentiation (CD27) component, a cluster of differentiation superfamily member (such as, CD28 or inducible T cell co-stimulator (ICOS)) component, and combinations thereof.

[0233] In one embodiment, the CD137 component comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, 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 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12.

[0234] In one embodiment, the CD3z component comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, 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 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13.

[0235] In some embodiments, the intracellular signaling domain comprises a CD137 component and a CD3z component.

[0236] In one embodiment, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the intracellular signaling domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14.

[0237] The transmembrane domain of the CAR can be derived from the transmembrane domain of the following items: CD8, α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2Rβ, IL2Rγ, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI Id, ITGAE, CD103, ITGAL, CDI la, LFA-1, ITGAM, CDI lb, ITGAX, CDI lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD15 0, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C.

[0238] 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: 15. 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 95%, at least 98% or at least 99% sequence identity with SEQ ID NO: 15.

[0239] In one embodiment, the transmembrane domain comprises at least the transmembrane region of the following items: α, β or ζ chain of 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 transmembrane domain of the following items: ζ, η or FcεR1γ and -β, MB1 (Igα.), B29 or CD3-γ, ζ or η. In another embodiment, the transmembrane domain is synthetic, for example, mainly comprising hydrophobic residues, such as leucine and valine, phenylalanine triplet or tryptophan.

[0240] In one embodiment, the CAR further comprises 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 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the CD8a hinge region comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with SEQ ID NO: 16. In some embodiments, the hinge region comprises the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 124), 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 95%, at least 98%, or at least 99% sequence identity to EPKSCDKTHTCPPCP (SEQ ID NO: 124). In some embodiments, the hinge region comprises the sequence ERKCCVECPPCP (SEQ ID NO: 125), 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 95%, at least 98%, or at least 99% sequence identity to ERKCCVECPPCP (SEQ ID NO: 125). In some embodiments, the hinge region comprises the sequence ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3SEQ ID NO:126), or comprises an amino acid sequence that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3SEQ ID NO:126). In some embodiments, the hinge region comprises the sequence ESKYGPPCPSCP (SEQ ID NO: 127), 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 95%, at least 98%, or at least 99% sequence identity to ESKYGPPCPSCP (SEQ ID NO: 127).

[0241] In one embodiment, CAR comprises an extracellular antigen binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain. In one such embodiment, the hinge region is a CD8α hinge region, the transmembrane domain is a CD8α-TM domain, and the intracellular signaling domain includes a CD3-ζ domain and a 4-1BB / CD137 domain. In one such embodiment, the hinge region is a CD8α hinge region comprising an amino acid sequence of SEQ ID NO: 16, the transmembrane domain is a CD8α-TM domain comprising an amino acid sequence of SEQ ID NO: 15, and the intracellular signaling domain includes a CD3-ζ domain and a 4-1BB / CD137 domain, wherein the intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 14.

[0242] In one embodiment, the extracellular antigen binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:82, SEQ ID NO:81, SEQ ID NO:80, SEQ ID NO:79, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22. In some embodiments, the extracellular antigen binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NO:82, SEQ ID NO:81, SEQ ID NO:80, SEQ ID NO:79, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.

[0243] In some embodiments, the extracellular antigen binding domain comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the extracellular antigen binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 82.

[0244] In some embodiments, the extracellular antigen binding domain comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the extracellular antigen binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 81.

[0245] In some embodiments, the extracellular antigen binding domain comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, the extracellular antigen binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 80.

[0246] In some embodiments, the extracellular antigen binding domain comprises the amino acid sequence of SEQ ID NO: 79. In some embodiments, the extracellular antigen binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 79.

[0247] In some embodiments, the extracellular antigen binding domain comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the extracellular antigen binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17.

[0248] In some embodiments, the extracellular antigen binding domain comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the extracellular antigen binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18.

[0249] In some embodiments, the extracellular antigen binding domain comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the extracellular antigen binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19.

[0250] In some embodiments, the extracellular antigen binding domain comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the extracellular antigen binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20.

[0251] In some embodiments, the extracellular antigen binding domain comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the extracellular antigen binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21.

[0252] In some embodiments, the extracellular antigen binding domain comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the extracellular antigen binding domain comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 22.

[0253] In one embodiment, the CAR of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NO:86, SEQ ID NO:85, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32. In some embodiments, the CAR of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO:86, SEQ ID NO:85, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32.

[0254] In some embodiments, the CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, the CAR of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 86.

[0255] In some embodiments, the CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the CAR of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 85.

[0256] In some embodiments, the CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the CAR of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 83.

[0257] In some embodiments, the CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the CAR of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 84.

[0258] In some embodiments, the CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CAR of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 27.

[0259] In some embodiments, the CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the CAR of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 28.

[0260] In some embodiments, the CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the CAR of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 29.

[0261] In some embodiments, the CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the CAR of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 30.

[0262] In some embodiments, the CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the CAR of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 31.

[0263] In some embodiments, the CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CAR of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 32.

[0264] In certain embodiments, the CAR may include a signal peptide. In certain embodiments, the signal peptide is located at the N-terminus of the CAR sequence. In certain embodiments, the signal peptide is located at the C-terminus of the CAR sequence. In certain embodiments, the signal peptide includes or consists of the amino acid sequence of SEQ ID NO: 11.

[0265] CAR constructs and CAR-expressing immune response cells

[0266] In one aspect, the present disclosure provides an isolated nucleic acid molecule encoding a CAR as described herein. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 90, SEQ ID NO: 89, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of SEQ ID NO:90, SEQ ID NO:89, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38.

[0267] In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 90. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 90. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 89. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 89. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 87. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 87. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 88. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 88. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 34.In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 35. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 36. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 36. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 37. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 37. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 38. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 38.

[0268] The term "coding" refers to the intrinsic properties of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA or mRNA) for serving as a template for synthesizing other polymers and macromolecules in a biological process, and the polymer and macromolecule have a limited nucleotide sequence (e.g., rRNA, tRNA and mRNA) or a limited amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene, cDNA or RNA encodes the protein. The coding strand (its nucleotide sequence is identical to the mRNA sequence) and the non-coding strand (which is used as a template for gene or cDNA transcription) can all be referred to as encoding the protein, or other products of the gene or cDNA.

[0269] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence" encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may contain introns in some versions.

[0270] The term "isolated" refers to a homogenous population of molecules (such as synthetic polynucleotides or polypeptides) that have been substantially separated from and / or purified from other components in a system (such as a recombinant cell) in which the molecules were produced, as well as proteins that have been subjected to at least one purification or separation step. "Isolated" refers to molecules that are substantially free of other cellular materials and / or chemicals, and encompasses molecules isolated to higher purities, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0271] In some embodiments, the present disclosure provides an expression vector comprising the nucleic acid molecule (e.g., SEQ ID NO:90, SEQ ID NO:89, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38). In some embodiments, the expression vector comprises a nucleic acid molecule comprising a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:90, SEQ ID NO:89, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38.

[0272] In some embodiments, the expression vector comprises a nucleic acid molecule of SEQ ID NO: 90. In some embodiments, the expression vector comprises a nucleic acid molecule comprising a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 90. In some embodiments, the expression vector comprises a nucleic acid molecule of SEQ ID NO: 89. In some embodiments, the expression vector comprises a nucleic acid molecule comprising a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 89. In some embodiments, the expression vector comprises a nucleic acid molecule of SEQ ID NO: 87. In some embodiments, the expression vector comprises a nucleic acid molecule comprising a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 87. In some embodiments, the expression vector comprises a nucleic acid molecule of SEQ ID NO: 88. In some embodiments, the expression vector comprises a nucleic acid molecule comprising a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 88. In some embodiments, the expression vector comprises a nucleic acid molecule of SEQ ID NO: 33. In some embodiments, the expression vector comprises a nucleic acid molecule comprising a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the expression vector comprises a nucleic acid molecule of SEQ ID NO: 34.In some embodiments, the expression vector comprises a nucleic acid molecule comprising a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the expression vector comprises a nucleic acid molecule of SEQ ID NO: 35. In some embodiments, the expression vector comprises a nucleic acid molecule comprising a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the expression vector comprises a nucleic acid molecule of SEQ ID NO: 36. In some embodiments, the expression vector comprises a nucleic acid molecule comprising a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 36. In some embodiments, the expression vector comprises a nucleic acid molecule of SEQ ID NO: 37. In some embodiments, the expression vector comprises a nucleic acid molecule comprising a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO: 37. In some embodiments, the expression vector comprises a nucleic acid molecule of SEQ ID NO: 38. In some embodiments, the expression vector comprises a nucleic acid molecule comprising a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of SEQ ID NO:38.

[0273] The nucleic acid molecule encodes an amino acid having a sequence selected from the group consisting of SEQ ID NO:86, SEQ ID NO:85, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, or a variant thereof. In some embodiments, the expression vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:86. In some embodiments, the expression vector comprises a nucleic acid molecule encoding an amino acid sequence having 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 to the amino acid sequence of SEQ ID NO:86. In some embodiments, the expression vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:85. In some embodiments, the expression vector comprises a nucleic acid molecule encoding an amino acid sequence having 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 to the amino acid sequence of SEQ ID NO: 85. In some embodiments, the expression vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 83. In some embodiments, the expression vector comprises a nucleic acid molecule encoding an amino acid sequence having 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 to the amino acid sequence of SEQ ID NO: 83. In some embodiments, the expression vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 84. In some embodiments, the expression vector comprises a nucleic acid molecule encoding an amino acid sequence having 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 to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the expression vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 27.In some embodiments, the expression vector comprises a nucleic acid molecule encoding an amino acid sequence having 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 to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the expression vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 28. In some embodiments, the expression vector comprises a nucleic acid molecule encoding an amino acid sequence having 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 to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the expression vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 29. In some embodiments, the expression vector comprises a nucleic acid molecule encoding an amino acid sequence having 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 to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the expression vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 30. In some embodiments, the expression vector comprises a nucleic acid molecule encoding an amino acid sequence having 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 to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the expression vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 31. In some embodiments, the expression vector comprises a nucleic acid molecule encoding an amino acid sequence having 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 to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the expression vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:32.In some embodiments, the expression vector comprises a nucleic acid molecule encoding an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:32.

[0274] Herein, the structure of a nucleic acid molecule is at a position defined based on the % sequence identity with the reference sequence (with a given SEQ ID NO). In this context, the % sequence identity of a nucleic acid molecule refers to the similarity between at least two different nucleic acid sequences. When the position in both of the two comparison sequences is occupied by the same base, for example, if the position in each of the two DNA molecules is occupied by adenine, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of matching positions or homologous positions shared by the two sequences divided by the number of comparison positions and multiplied by 100. For example, if 6 of the 10 positions in the two sequences are matched or homologous, then the two sequences are 60% identical (or homologous). For example, the DNA sequences ATTGCC and TATGGC share 50% identity (or homology). Generally speaking, comparison is made when the two sequences are aligned to give maximum homology. The corresponding percent identity can be determined by a standard alignment algorithm, for example, the basic local alignment search tool (BLAST) described by Altshul et al. ((1990) J. MoI. Biol. 215:403-10); the algorithm of Needleman et al. ((1970) J. MoI. Biol. 48:444-53); or the algorithm of Meyers et al. ((1988) Comput. Appl. Biosci. 4:11-17). One set of parameters can be the Blosum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller ((1989) CABIOS 4:11-17) (which has been incorporated into the ALIGN program (version 2.0)) using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Percent identity is typically calculated by comparing sequences of similar length.

[0275] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked plasmids or plasmids contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) to which the recombinant polynucleotide is incorporated.

[0276] In one embodiment, the disclosure provides cells expressing nucleic acid molecules described herein. In one embodiment, the disclosure provides cells expressing CAR of the disclosure. In one embodiment, the CAR of the disclosure comprises an amino acid sequence selected from the group consisting of the following items: SEQ ID NO: 86, SEQ ID NO: 85, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32. In some embodiments, the CAR of the present disclosure comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO:86, SEQ ID NO:85, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32.

[0277] In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 86. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 85. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 83. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 84. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 28. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 31. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 32.

[0278] In some embodiments, the CAR comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 86. In some embodiments, the CAR comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 85. In some embodiments, the CAR comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 83. In some embodiments, the CAR comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 84. In some embodiments, the CAR comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 27. In some embodiments, the CAR comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 28.In some embodiments, the CAR comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 29. In some embodiments, the CAR comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 30. In some embodiments, the CAR comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 31. In some embodiments, the CAR comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 32.

[0279] In one aspect, the present disclosure provides an immune response cell comprising a separation of CAR described herein. In some embodiments, the separated immune response cell is transduced with CAR, for example, CAR is constitutively expressed on the surface of the immune response cell. In certain embodiments, the separated immune response cell is further transduced with at least one costimulatory ligand so that the immune response cell expresses the at least one costimulatory ligand. In certain embodiments, the at least one costimulatory ligand is selected from the group consisting of the following items: 4-1BBL, CD48, CD70, CD80, CD86, OX40L, TNFRSF14, and combinations thereof. In certain embodiments, the separated immune response cell is further transduced with at least one cytokine so that the immune response cell secretes the at least one cytokine. In certain embodiments, the at least one cytokine is selected from the group consisting of the following items: IL-2, IL-3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17, IL-21, and combinations thereof. In some embodiments, the isolated immune response cell is selected from the group consisting of: T lymphocytes (T cells), natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, human embryonic stem cells, lymphoid progenitor cells, T cell precursor cells, and pluripotent stem cells from which lymphoid cells can be differentiated.

[0280] The nucleic acid sequence encoding CAR of the present invention can be introduced into cells by "transfection", "conversion" or "transduction". As used herein, "transfection", "conversion" or "transduction" refers to the introduction of one or more exogenous polynucleotides into host cells by using physical methods or chemical methods. Many transfection techniques are known in the art, and include, for example, calcium phosphate DNA coprecipitation (see, for example, Murray EJ (editor), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; microparticle bombardment promoted by tungsten particles (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). After the infection particles grow in suitable packaging cells, phage vectors or viral vectors can be introduced into host cells, many of which are commercially available.

[0281] In one embodiment, the CAR T cells of the present disclosure can be produced by introducing a lentiviral vector comprising a desired CAR (e.g., a CAR comprising anti-GPRC5D, CD8α hinge and transmembrane domains, and human 4-1BB and CD3ζ signaling domains) into a cell. The CAR T cells of the present invention are capable of replicating in vivo, resulting in long-term persistence that can cause sustained tumor control.

[0282] Embodiments of the present invention also provide host cells comprising any recombinant expression vector described herein. As used herein, the term "host cell" refers to any type of cell that can contain a recombinant expression vector. The host cell can be a eukaryotic cell (e.g., a plant, an animal or algae, a fungus), or can be a prokaryotic cell (e.g., a bacterium or a protozoan). The host cell can be a cultured cell or a primary cell (i.e., directly isolated from an organism (e.g., a human). The host cell can be an adherent cell or a suspension cell, i.e., a cell grown in a suspension. Suitable host cells are known in the art, and include, for example, DH5α Escherichia coli (E.coli) cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. In order to amplify or replicate a recombinant expression vector, the host cell can be a prokaryotic cell, such as a DH5α cell. In order to produce a recombinant CAR, a polypeptide or a protein, the host cell can be a mammalian cell. The host cell can be a human cell. Although the host cell can be any cell type, can be derived from any type of tissue, and can be at any stage of development, the host cell can be a peripheral blood lymphocyte (PBL). The host cell may be a T cell.

[0283] For the purposes of this article, T cells can be any T cells, such as cultured T cells (e.g., primary T cells), or T cells from cultured T cell lines (e.g., Jurkat, SupT1, etc.), or T cells obtained from mammals. If obtained from mammals, T cells can be obtained from a variety of sources, including but not limited to bone marrow, blood, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. T cells can be human T cells. T cells can be T cells isolated from humans. T cells can be any type of T cell and can be at any developmental stage, including but not limited to CD4 + / CD8 + Double positive T cells, CD8 + T cells (e.g., cytotoxic T cells), CD4 + Helper T cells (e.g., Th1 cells and Th2 cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating cells, memory T cells, naive T cells, etc. T cells can be CD8 + T cells or CD4+ T cells.

[0284] T cells can also include "NKT cells", which refer to the expression of semi-invariant αβT cell receptors, but also express a specialized T cell population of multiple molecular markers commonly associated with NK cells (such as NK1.1). NKT cells include NK1.1+ cells and NK1.1- cells, as well as CD4+ cells, CD4- cells, CD8+ cells and CD8- cells. The uniqueness of the TCR on NKT cells is that it recognizes glycolipid antigens presented by MHC I-like molecules CD Id. NKT cells can have protective or deleterious effects due to their ability to produce cytokines that promote inflammation or immune tolerance. Also included is "γ-δT cells (γδT cells)", which refer to specialized populations, i.e., a small subset of T cells with unique TCR on their surface, and different from most T cells in which TCR is composed of two glycoprotein chains named as α-TCR chains and β-TCR chains, the TCR in γδT cells is composed of γ-chains 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 refer to T cells that inhibit abnormal or excessive immune responses and play a role in immune tolerance. Treg cells are usually transcription factor Foxp3-positive CD4+T cells, and may also include transcription factor Foxp3-negative regulatory T cells for CD4+T cells that produce IL-10.

[0285] T cell lines are available from, for example, the American Type Culture Collection (ATCC, Manassas, VA) and the German Collection of Microorganisms and Cell Cultures (DSMZ), and include, for example, Jurkat cells (ATCC TIB-152), Sup-T1 cells (ATCC CRL-1942), RPMI8402 cells (DSMZ ACC-290), Karpas 45 cells (DSMZ ACC-545), and derivatives thereof.

[0286] In another embodiment, the host cell is a natural killer (NK) cell. NK cells are a class of cytotoxic lymphocytes that work in the innate immune system. NK cells are defined as large granular lymphocytes and constitute the third type of cells differentiated from common lymphocyte progenitor cells, which also produce B lymphocytes and T lymphocytes (see, for example, Immunobiology, the 5th edition, Janeway et al., edited, Garland Publishing, New York, NY (2001)). NK cells differentiate and mature in bone marrow, lymph nodes, spleen, tonsils and thymus. After maturation, NK cells enter the circulation as large lymphocytes with unique cytotoxic granules. NK cells can recognize and kill some abnormal cells, such as some tumor cells and virus-infected cells, and are considered to be important in the innate immune defense against intracellular pathogens. As described above with respect to T cells, NK cells can be any NK cells, such as cultured NK cells (e.g., primary NK cells), or NK cells from cultured NK cell lines, or NK cells obtained from mammals. If obtained from mammals, NK cells can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph nodes, thymus or other tissues or fluids. NK cells can also be enriched or purified. NK cells are preferably human NK cells (e.g., separated from humans). NK cell lines are available from, for example, the American Type Culture Collection (ATCC, Manassas, VA), and include, for example, NK-92 cells (ATCC CRL-2407), NK92MI cells (ATCC CRL-2408), and their derivatives.

[0287] Also provided is a cell colony comprising at least one host cell described herein.Cell colony can be a heterogeneous colony, comprising a host cell with any one of the recombinant expression vector, and in addition comprising at least one other cell, such as a host cell (such as a T cell) without any one of the recombinant expression vector, or a cell except a T cell, such as a B cell, a macrophage, an erythrocyte, a neutrophil, a hepatocyte, an endothelial cell, an epithelial cell, a myocyte, a brain cell, etc. Alternatively, a cell colony can be a substantially homogeneous colony, wherein the colony mainly comprises a host cell (such as, substantially consisting of) with a recombinant expression vector. The colony can also be a clonal colony of a cell, wherein all cells of the colony are clones of a single host cell comprising a recombinant expression vector, so that all cells of the colony comprise a recombinant expression vector. In one embodiment, a cell colony is a clonal colony comprising a host cell with a recombinant expression vector as described herein.

[0288] Pharmaceutical composition / administration

[0289] In embodiments of the present disclosure, cells expressing CAR can be provided in a composition, for example, a suitable pharmaceutical composition comprising cells expressing CAR and a pharmaceutically acceptable carrier. In one aspect, the present disclosure provides a pharmaceutical composition comprising an effective amount of lymphocytes expressing one or more of the CARs and a pharmaceutically acceptable excipient. The pharmaceutical composition of the present disclosure may include a cell expressing CAR (e.g., a plurality of cells expressing CAR as described herein) and a combination of one or more pharmaceutically or physiologically acceptable carriers, excipients or diluents. A pharmaceutically acceptable carrier may be a component other than an active ingredient in a pharmaceutical composition that is nontoxic to a subject.

[0290] Pharmaceutically acceptable carriers may include, but are not limited to, buffers, excipients, stabilizers or preservatives. Examples of pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, such as salts, buffers, antioxidants, sugars, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifiers, or combinations thereof. The amount of the pharmaceutically acceptable carrier in the pharmaceutical composition can be determined experimentally based on carrier activity and desired formulation characteristics (such as stability and / or minimum oxidation).

[0291] Such compositions may include buffers such as acetic acid, citric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, histidine, boric acid, Tris buffer, HEPPSO, HEPES, neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); antibacterial and antifungal agents; and preservatives.

[0292] Compositions disclosed herein can be formulated for parenteral administration or the various modes of non-parenteral administration. In one embodiment, the composition can be formulated for infusion or intravenous administration. Compositions disclosed herein can be provided, for example, as a sterile liquid preparation (e.g., isotonic aqueous solution, emulsion, suspension, dispersion or viscous composition), which can be buffered to the desired pH. Preparations suitable for oral administration can include liquid solutions, capsules, sachets, tablets, lozenges and lozenges, powders, and liquid suspensions in suitable liquids and emulsions.

[0293] The term "pharmaceutically acceptable," as used herein with respect to pharmaceutical compositions, means a material approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and / or humans.

[0294] In one aspect, the present disclosure relates to the use of lymphocyte infusion to administer genetically modified T cells expressing CAR to treat subjects with cancer or at risk of developing cancer. In at least one embodiment, autologous lymphocyte infusion is used in treatment. Autologous PBMCs are collected from subjects in need of treatment, and T cells are activated and amplified using methods described herein and known in the art, and then infused back into the subject.

[0295] In one aspect, the disclosure as a whole relates to treatment of subjects at risk of developing cancer. The present invention also includes treating the following malignancies or autoimmune diseases: wherein chemotherapy and / or immunotherapy of the subject results in significant immunosuppression, thereby increasing the risk of the subject developing cancer. In one aspect, the disclosure provides a method for preventing cancer, the method comprising administering a certain amount of lymphocytes expressing one or more of the CARs to a subject in need.

[0296] In one aspect, the present disclosure provides a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of lymphocytes expressing one or more of the CARs to a subject in need thereof, whereby the lymphocytes induce or regulate killing of cancer cells in the subject.

[0297] On the other hand, the present disclosure provides a method for reducing the tumor burden in a subject with cancer, the method comprising administering a therapeutically effective amount of lymphocytes expressing one or more of the CARs described herein to a subject in need, whereby the lymphocytes induce killing of cancer cells in the subject. On the other hand, the present disclosure provides a method for extending the survival of a subject with cancer, the method comprising administering a therapeutically effective amount of lymphocytes expressing one or more of the CARs to a subject in need, thereby extending the survival of the subject. Generally speaking, lymphocytes expressing the CAR induce killing of cancer cells in the subject, and result in a reduction or eradication of tumor cells / cancer cells in the subject. A non-limiting list of cancers that can be targeted, including metastatic lesions, includes lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), smoldering multiple myeloma (SMM), multiple myeloma (MM), acute myeloid leukemia (AML), and combinations thereof. In one embodiment, the cancer treated in the subject is multiple myeloma.

[0298] In one aspect, the methods described herein are useful for treating non-cancerous conditions with a risk of developing a cancerous condition, such as monoclonal gammopathy of undetermined significance (MGUS), with a risk of developing a blood cancer such as MM or lymphoma, and myelodysplastic syndrome with a risk of developing a blood cancer such as leukemia.

[0299] In one aspect, a method for treating a subject with cancer is provided, comprising administering a therapeutically effective amount of lymphocytes expressing CAR (the CAR having an extracellular antigen binding domain that binds to a GPRC5D antigen) to a subject in need, whereby the lymphocytes induce killing of cancer cells in the subject. In some embodiments, at least one of the CARs comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 86, SEQ ID NO: 85, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.

[0300] In one aspect, a method for targeted killing of cancer cells is disclosed, the method including contacting cancer cells with lymphocytes expressing one or more of the CAR, whereby the lymphocytes induce killing of cancer cells. A non-limiting list of cancer cells (including metastatic cancer cells) that can be targeted includes lung cancer cells, gastric cancer cells, colon cancer cells, hepatocellular cancer cells, renal cell cancer cells, bladder urothelial cancer cells, metastatic melanoma cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, head and neck cancer cells, pancreatic cancer cells, glioma cells, glioblastoma cells and non-Hodgkin's lymphoma (NHL) cells, acute lymphocytic leukemia (ALL) cells, chronic lymphocytic leukemia (CLL) cells, chronic myeloid leukemia (CML) cells, smoldering multiple myeloma (SMM) cells, multiple myeloma (MM) cells, acute myeloid leukemia (AML) cells, and combinations thereof. In one embodiment, the cancer cell is a multiple myeloma cell.

[0301] The pharmaceutical composition of the present disclosure can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the subject's condition and the type and severity of the subject's disease, but the appropriate dosage can be determined by clinical trials.

[0302] The term "treat" or "treatment" refers to therapeutic treatment, wherein the subject of treatment will slow down (mitigate) an undesirable physiological change or disease, or provide a beneficial or desired clinical outcome during treatment. Beneficial or desired clinical outcomes include alleviation of symptoms, weakening of the extent of the disease, a stable (i.e., non-exacerbated) state of the disease, a delay or slowing of disease progression, an improvement or alleviation of the disease state, and / or relief (whether partial relief or complete relief), whether detectable or undetectable. "Treatment" may also mean extending survival compared to the expected survival when the subject is not receiving treatment. Those subjects in need of treatment include those subjects who have already suffered from undesirable physiological changes or diseases, and those subjects who are prone to suffer from physiological changes or diseases.

[0303] "Therapeutically effective amount" or "effective amount" are used interchangeably herein and refer to an amount effective to achieve the desired therapeutic result at the necessary dosage and for the necessary period of time. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. Example indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improvement in the patient's health status, reduction in tumor burden, arrest or slowing of tumor growth, and / or the absence of metastasis of cancer cells to other parts of the body.

[0304] As used herein, the term "subject" refers to an animal. The terms "subject" and "patient" are used interchangeably herein when referring to a subject. Thus, a "subject" includes a human being who is being treated for a disease or prevented from a disease as a patient.

[0305] The methods described herein can be used to treat animal subjects belonging to any classification. Examples of such animals include mammals. Mammals include, but are not limited to, mammals of the order Rodentia (such as mice and hamsters) and mammals of the order Logomorpha (such as rabbits). Mammals can be from the order Carnivora, including felines (cats) and canines (dogs). The mammals can be from the order Artiodactyla, including bovines (cows) and suids (pigs), or the order Perssodactyla, including equines (horses). Mammals can be primates, order Ceboid or order Simoid (monkeys), or order Anthropoid (humans and apes). In one embodiment, the mammal is a human.

[0306] When indicating a therapeutically effective amount, the exact amount of the composition of the present disclosure to be administered can be determined by a physician taking into account individual differences in the subject's age, weight, tumor size, degree and condition of infection or metastasis. In general, it can be said that the pharmaceutical composition comprising the T cells described herein can be administered in an amount of about 10 4 cells / kg body weight to about 10 10 cells / kg body weight, in some cases about 10 5 cells / kg body weight to about 10 6 In some embodiments, the pharmaceutical composition comprising the T cells described herein can be administered at a dose of about 10 cells / kg body weight (including all integer values ​​within those ranges). 6 The T cell compositions may also be administered multiple times at these doses. These cells may be administered using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[0307] Delivery systems that can be used in the context of embodiments of the present invention can include timed release delivery systems, delayed release delivery systems, and sustained release delivery systems, so that the delivery of the T cell composition occurs before the sensitization of the site to be treated, and has sufficient time to cause sensitization of the site to be treated. The composition can be used in combination with other therapeutic agents or therapies. Such systems can avoid repeated administration of the composition, thereby increasing convenience for subjects and physicians, and can be particularly suitable for certain composition embodiments of the present invention.

[0308] Many types of release delivery systems are available and are known to those of ordinary skill in the art. They include polymer-based systems such as poly(lactide-glycolide), copolyoxalates, polyesteramides, polyorthoesters, polycaprolactones, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of drugs containing the aforementioned polymers are described, for example, in U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems, which are lipids, including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as monoglycerides, diglycerides, and triglycerides; sylastic systems; peptide-based systems; hydrogel release systems; wax coatings; tablets using conventional binders and excipients; partially molten implants; and the like. Specific examples include, but are not limited to: (a) erosion systems, in which the active composition is contained in a matrix in a certain form, such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034, and 5,239,660; and (b) diffusion systems, in which the active component permeates from the polymer at a controlled rate, such as described in U.S. Pat. Nos. 3,854,480 and 3,832,253. In addition, pump-based hardware delivery systems can be used, some of which are suitable for implantation.

[0309] In some aspects, it may be advantageous to administer activated T cells to a subject, then redraw blood (or perform apheresis), activate T cells according to the disclosure, and re-infuse these activated and amplified T cells to the subject. This process can be performed multiple times every few weeks. In some aspects, T cells in a blood draw of 10cc to 400cc can be activated. In some aspects, T cells in a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc can be activated.

[0310] Administration of CAR-T cells and compositions can be carried out in any manner, for example, by parenteral or non-parenteral administration, including by aerosol inhalation, injection, infusion, ingestion, transfusion, implantation or transplantation. For example, CAR-T cells and compositions described herein can be administered to patients via arteries, intradermal, subcutaneous, intratumoral, intramedullary, intranodal, intramuscular, by intravenous (iv) injection, or intraperitoneal administration. In one aspect, the compositions of the present disclosure are administered by intravenous injection. In one aspect, the compositions of the present disclosure are administered to subjects by intradermal or subcutaneous injection. The compositions of T cells can be injected directly into, for example, tumors, lymph nodes, tissues, organs, or infection sites.

[0311] Administration can be autologous administration or non-autologous administration.For example, immune response cells expressing G protein coupled receptor (e.g., GPRC5D) specific CAR can be obtained from a subject and applied to the same subject or different compatible subjects. Peripheral blood derived T cells of the present disclosure, or amplified T cells (e.g., in vivo, ex vivo or in vitro derived) can be administered via, for example, intravenous injection, local injection, systemic injection, catheter administration or parenteral administration.

[0312] In certain embodiments, the subject can be subjected to leukapheresis, wherein leukocytes are collected, enriched or removed in vitro to select and / or separate cells of interest (eg, T cells). These T cell isolates can be amplified by methods known in the art, and processed so that one or more CAR constructs of the present disclosure can be introduced to form CAR-T cells. Subsequently, subjects in need can be subjected to standard treatment with high-dose chemotherapy, followed by peripheral blood stem cell transplantation. In some aspects, after transplantation or simultaneously with transplantation, the subject receives an infusion of amplified CAR-T cells. In one aspect, amplified cells are administered before or after surgery.

[0313] The dose administered to a patient suffering from a malignancy is sufficient to alleviate or at least partially arrest the disease being treated (a "therapeutically effective amount"). The dose of the above treatment to be administered to a subject will vary with the exact nature of the condition being treated and the recipient of the treatment. Scaling of doses for human administration can be performed according to generally accepted practices in the art.

[0314] The CART T cells of the present invention can be expanded by T cells in the recipient and can establish GPRC5D-specific memory cells that remain at high levels in the blood and bone marrow for an extended period of time. In some cases, the CAR T cells of the present invention infused into the subject can eliminate cancer cells, such as leukemia cells, in the subject with advanced chemotherapy-resistant cancer.

[0315] In one embodiment, CAR of the present disclosure is introduced into T cells, for example, using in vitro transcription, and the subject (e.g., people) receives the initial administration of CAR-T cells disclosed herein, and one or more subsequent administrations of CAR-T cells, wherein the one or more subsequent administrations are less than 15 days (e.g., 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days or 2 days) after the previous administration. In one embodiment, more than one administration of CAR-T cells is applied to the subject (e.g., people) weekly, for example, 2, 3 or 4 administrations of CAR-T cells are applied weekly. In one embodiment, the subject receives more than one CAR-T cell application (e.g., 2, 3 or 4 administrations per week) (also referred to herein as cycle), and then does not receive CAR-T cell administration for one week, and then applies one or more CAR-T cells to the subject for additional administration (e.g., more than one CAR-T cell application per week). In another embodiment, the subject receives more than one CAR-T cell cycle, and the time between each cycle is less than 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days or 3 days. In one embodiment, the CAR-T cells are administered every other day and 3 times a week. In one embodiment, the CAR-T cells are administered for at least two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks or more weeks.

[0316] In one embodiment, administration can be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or more. The course of treatment can also be repeated, as in chronic administration. Repeated administration can be the same dose or different doses.

[0317] CAR-T cells can be administered in the methods of the invention via maintenance therapy, such as once a week for a period of 6 months or longer.

[0318] In one embodiment, a lentiviral vector (such as a lentivirus) is used to generate CAR-T cells. CAR-T cells generated with such viral vectors will generally have stable CAR expression.

[0319] In one embodiment, CAR-T cells transiently express CAR vectors 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days after transduction. The transient expression of CAR can be affected by RNA CAR vector delivery. In one embodiment, CAR RNA is transduced into T cells by electroporation.

[0320] If the patient is at high risk for developing anti-CAR antibody responses (such as those produced by RNA transduction) during the course of transient CAR therapy, the CAR-T infusion interval should not last more than ten to fourteen days.

[0321] The cells expressing CAR described herein can be used in combination with other known agents and therapies. As used herein, "combination" administration means that two (or more) different treatments are delivered to the subject during the treatment course of the subject, for example, two or more treatments are delivered after the subject has been diagnosed with cancer and before the cancer has been cured or eliminated or before the treatment has been stopped for other reasons. In some embodiments, the delivery of one treatment is still ongoing at the beginning of the delivery of the second treatment, so that there is overlap in the administration. This is sometimes referred to as "simultaneous" or "simultaneous delivery" in this article. In other embodiments, the delivery of one treatment ends before the delivery of another treatment begins. In some embodiments of any case, due to combined administration, the treatment is more effective. For example, compared with the similar situation observed when the second treatment is applied in the absence of the first treatment or when the first treatment is used, the second treatment is more effective, for example, when using less second treatment, an equivalent effect is observed, or the second treatment alleviates symptoms to a greater extent. In some embodiments, delivery causes symptom relief or other parameters related to the disease to be reduced to a greater extent than the degree of reduction or reduction observed when delivering one treatment without the presence of another treatment. The effects of the two treatments may be partially additive, fully additive, or greater than additive. The delivery may be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

[0322] In one embodiment, other therapeutic agents (such as factors) can be administered before, after, or simultaneously with (concurrently with) the CAR-T cells, including but not limited to interleukins (e.g., IL-2, IL-3, IL 6, IL-7, IL-11, IL-12, IL-15, IL-21, and other interleukins), colony stimulating factors (such as G-CSF, M-CSF, and GM-CSF), and interferons (e.g., gamma-interferon).

[0323] The cells expressing CAR described herein and at least one additional therapeutic agent can be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the cells expressing CAR described herein can be administered first, and the additional agent can be administered secondly, or the order of administration can be reversed.

[0324] In additional embodiments, the CAR-expressing cells described herein can be used in combination with surgery, radiation, chemotherapy, immunosuppressants (such as methotrexate, cyclosporine, azathioprine, mycophenolic acid and FK506, antibodies or other immunosuppressants (such as anti-CD3 antibodies) or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines) and irradiation in a treatment regimen.

[0325] In one embodiment, the cells expressing CAR as described herein can be used in combination with chemotherapeutic agents.Example chemotherapeutic agents include but are not limited to anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzumab, gemtuzumab, rituximab, tositumomab), antimetabolites (including, for example, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-induced TNFR-related proteins (GITR) agonists, proteasome inhibitors (e.g., aclarubicin A, gliotoxin or bortezomib), immunomodulators such as thalidomide or thalidomide derivatives (e.g., lenalidomide).

[0326] A non-exhaustive list of chemotherapeutic agents considered for use in combination therapy includes anastrozole Bicalutamide Bleomycin sulfate Busulfan Calcium folinate, melphalan 6-Mercaptopurine Methotrexate Mitoxantrone Mylotarg, paclitaxel phoenix (Yttrium90 / MX-DTPA), pentostatin, polyphenylpropanone 20 with carmustine implant Dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride Daunorubicin citrate liposome injection Dexamethasone, docetaxel Doxorubicin Hydrochloride Etoposide Busulfan Injection Capecitabine N4-pentyloxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin Carmustine Chlorambucil Cisplatin Cladribine Cyclophosphamide ( or ), cytosine arabinoside, cytosine arabinoside Cytarabine liposome injection Dacarbazine (DTIC- ), Fludarabine phosphate 5-Fluorouracil Flutamide Tezacitabine, gemcitabine (difluorodeoxycytidine), hydroxyurea Idabi Ifosfamide Irinotecan L-asparaginase Tamoxifen citrate Teniposide 6-thioguanine, thiotepa, tirapazamine Topotecan Hydrochloride for Injection Vinblastine Vincristine Vinorelbine

[0327] Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes: Aminouracil mustard Uracil Nitrogen ), Nitrogen mustard Cyclophosphamide ( Revimmune TM ), ifosfamide Melphalan Chlorambucil Pipobrom Triethylenemelamine Triethylenethiophosphamide, temozolomide Thiotepa Busulfan Carmustine Lomustine Streptozotocin and dacarbazine Additional exemplary alkylating agents include, but are not limited to, oxaliplatin Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, ); Hexamethylmelamine (also known as hexamethylmelamine (HMM), ); Carmustine Bendamustine Busulfan and ); Carboplatin Temozolomide ( and ); Dactinomycin (also known as actinomycin D, ); lomustine (also known as CCNU, ); cisplatin (also known as CDDP, and AQ); Chlorambucil Cyclophosphamide ( and ); Dacarbazine (also known as DTIC, DIC, and imidazole carboxamide, DTIC- ); Hexamethylmelamine (also known as hexamethylmelamine (HMM), ); ifosfamide Prednumustine; Procarbazine Mechlorethamine (also known as nitrogen mustard, mustard, and mechlorethamine hydrochloride, ); Streptozotocin Thiotepa (also known as thiophosphoramide, TESPA and TSPA, ); Cyclophosphamide Bendamustine hydrochloride

[0328] Examples of immunomodulators that can be used herein include, but are not limited to, ofatumumab (available from ); Pegfilgrastim Lenalidomide (CC-5013, ); thalidomide actimid (CC4047); and IRX-2 (a mixture of human cytokines (including interleukin 1, interleukin 2 and interferon γ), CAS951209-71-5, purchased from IRX Therapeutics).

[0329] In one embodiment, an agent that enhances the activity of cells expressing CAR can be administered to a subject. For example, in one embodiment, the agent can be an agent that inhibits inhibitory molecules. In some embodiments, inhibitory molecules (e.g., programmed death 1 (PD1)) can reduce the ability of cells expressing CAR to produce immune effector responses. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFRβ.

[0330] A description of an exemplary embodiment follows:

[0331] Embodiment 1. A chimeric antigen receptor (CAR), comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises:

[0332] a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:66, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:67, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:68;

[0333] a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:58, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:59, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:60;

[0334] a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:39, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:40, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:41;

[0335] a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:42, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:43, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:44; or

[0336] a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:45, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:46, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:47;

[0337] The extracellular antigen binding domain binds to the anti-G protein receptor family C group 5 member D (GPRC5D) antigen.

[0338] Embodiment 2. The CAR according to embodiment 1, wherein

[0339] The extracellular antigen-binding domain comprises a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 66, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 67, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 68, and further comprises a light chain CDR1 having an amino acid sequence of SEQ ID NO: 69, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 70, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 71;

[0340] The extracellular antigen-binding domain comprises a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 58, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 59, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 60, and further comprises a light chain CDR1 having an amino acid sequence of SEQ ID NO: 61, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 62, and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 63;

[0341] The extracellular antigen-binding domain comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:39, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:40, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:41, and further comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO:48, a light chain CDR2 having the amino acid sequence of SEQ ID NO:49, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:50;

[0342] The extracellular antigen-binding domain comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:42, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:43, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:44, and further comprises a light chain CDR1 having the amino acid sequence of SEQ ID NO:51, a light chain CDR2 having the amino acid sequence of SEQ ID NO:52, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:53; or

[0343] The extracellular antigen-binding domain comprises a heavy chain CDR1 having an amino acid sequence of SEQ ID NO:45, a heavy chain CDR2 having an amino acid sequence of SEQ ID NO:46, and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO:47, and also comprises a light chain CDR1 having an amino acid sequence of SEQ ID NO:54, a light chain CDR2 having an amino acid sequence of SEQ ID NO:55, and a light chain CDR3 having an amino acid sequence of SEQ ID NO:56.

[0344] Embodiment 3. A CAR according to embodiment 1 or 2, wherein the extracellular antigen binding domain comprises: a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 73, SEQ ID NO: 65, SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, or a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 72, SEQ ID NO: 64, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, or a combination of a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 73, SEQ ID NO: 65, SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 and a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 72, SEQ ID NO: 64, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.

[0345] Embodiment 4. The CAR according to embodiments 1 to 3, wherein the extracellular antigen binding domain comprises:

[0346] a light chain variable region comprising the amino acid sequence of SEQ ID NO: 73 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72;

[0347] a light chain variable region comprising the amino acid sequence of SEQ ID NO:65 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:64;

[0348] 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: 2;

[0349] 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; or

[0350] A light chain variable region comprising the amino acid sequence of SEQ ID NO:5 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6.

[0351] Embodiment 5. A CAR according to any one of Embodiments 1 to 4, wherein the extracellular antigen binding domain comprises a single-chain variable fragment (scFv).

[0352] Embodiment 6. A CAR according to embodiment 5, wherein the scFv comprises a linker polypeptide located between the light chain variable region and the heavy chain variable region.

[0353] Embodiment 7. The CAR according to embodiment 6, wherein the linker polypeptide comprises the amino acid sequence of SEQ ID NO:7.

[0354] Embodiment 8. A CAR according to any one of embodiments 5 to 7, wherein the scFv comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:78, SEQ ID NO:77, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.

[0355] Embodiment 9. A CAR according to any one of embodiments 1 to 8, wherein the extracellular antigen binding domain comprises a signal polypeptide.

[0356] Embodiment 10. The CAR according to embodiment 9, wherein the signal polypeptide comprises the amino acid sequence of SEQ ID NO:11.

[0357] Embodiment 11. A CAR according to any one of embodiments 1 to 10, wherein the intracellular signaling domain comprises a polypeptide component selected from the group consisting of: a TNF receptor superfamily member 9 (CD137) component, a T cell surface glycoprotein CD3 ζ chain (CD3z) component, a cluster of differentiation (CD27) component, a cluster of differentiation superfamily member component, and combinations thereof.

[0358] Embodiment 12. A CAR according to embodiment 11, wherein the CD137 component comprises the amino acid sequence of SEQ ID NO:12.

[0359] Embodiment 13. A CAR according to embodiment 11, wherein the CD3z component comprises the amino acid sequence of SEQ ID NO:13.

[0360] Embodiment 14. A CAR according to embodiment 11, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 14.

[0361] Embodiment 15. A CAR according to any one of Embodiments 1 to 14, wherein the transmembrane domain comprises a CD8a transmembrane region (CD8a-TM) polypeptide.

[0362] Embodiment 16. A CAR according to embodiment 15, wherein the CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO:15.

[0363] Embodiment 17. According to any one of Embodiments 1 to 16, the CAR further comprises a hinge region connecting the transmembrane domain to the extracellular antigen binding domain.

[0364] Embodiment 18. A CAR according to Embodiment 17, wherein the hinge region is a CD8a hinge region.

[0365] Embodiment 19. A CAR according to Embodiment 18, wherein the CD8a hinge region comprises the amino acid sequence of SEQ ID NO:16.

[0366] Embodiment 20. A CAR according to any one of embodiments 1 to 19, wherein the extracellular antigen binding domain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:82, SEQ ID NO:81, SEQ ID NO:80, SEQ ID NO:79, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22.

[0367] Embodiment 21. A CAR according to any one of embodiments 1 to 20, wherein the CAR comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:86, SEQ ID NO:85, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32.

[0368] Embodiment 22. An isolated lymphocyte expressing the CAR according to any one of Embodiments 1 to 21.

[0369] Embodiment 23. The isolated lymphocyte according to embodiment 22, wherein the lymphocyte is a T lymphocyte.

[0370] Embodiment 24. An isolated nucleic acid molecule encoding the CAR according to any one of embodiments 1 to 21.

[0371] Embodiment 25. An isolated nucleic acid molecule according to embodiment 24, wherein the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO:90, SEQ ID NO:89, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38.

[0372] Embodiment 26. The isolated nucleic acid molecule of embodiment 24, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO:90, SEQ ID NO:89, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38.

[0373] Embodiment 27. A vector comprising the nucleic acid molecule according to any one of embodiments 24 to 26.

[0374] Embodiment 28. A cell expressing a nucleic acid molecule according to any one of embodiments 24 to 26.

[0375] Embodiment 29. A pharmaceutical composition comprising an effective amount of lymphocytes according to any one of embodiments 22 to 23.

[0376] Embodiment 30. A pharmaceutical composition comprising an effective amount of lymphocytes according to any one of embodiments 22 to 23 and a pharmaceutically acceptable excipient.

[0377] Embodiment 31. A CAR according to any one of Embodiments 1 to 21 or a pharmaceutical composition according to Embodiment 29 or 30 for use in therapy.

[0378] Embodiment 32. A CAR according to any one of Embodiments 1 to 21 or a pharmaceutical composition according to Embodiment 29 or 30, for use in a method for treating a subject having cancer.

[0379] Embodiment 33. A method of treating a subject having cancer, the method comprising:

[0380] A therapeutically effective amount of the lymphocytes according to any one of Embodiments 22 to 23 is administered to a subject in need thereof, whereby the lymphocytes induce killing of cancer cells in the subject.

[0381] Embodiment 34. The method of embodiment 33 or the CAR or pharmaceutical composition for use in the method of embodiment 32, wherein the cancer is selected from the group consisting of lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), smoldering multiple myeloma (SMM), multiple myeloma (MM), acute myeloid leukemia (AML), and combinations thereof.

[0382] Embodiment 35. The method of Embodiment 33 or 34 or the CAR or pharmaceutical composition for use in the method of Embodiment 32 or 34, wherein the cancer is multiple myeloma.

[0383] Embodiment 36. A method for targeted killing of cancer cells, the method comprising:

[0384] A cancer cell is contacted with a lymphocyte according to any one of embodiments 22 to 23, whereby the lymphocyte induces killing of the cancer cell.

[0385] Embodiment 37. The method according to embodiment 36, wherein the cancer cells are selected from the group consisting of lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), smoldering multiple myeloma (SMM), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors, and combinations thereof.

[0386] Embodiment 38. The method of embodiment 36 or 37, wherein the cancer cells are multiple myeloma cells.

[0387] Embodiment 39. A method of detecting the presence of cancer in a subject, comprising:

[0388] (a) contacting a cell sample obtained from a subject with the CAR according to claim 1, thereby forming a CAR cell complex, and

[0389] (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the subject. Preferably, this embodiment is performed in vitro.

[0390] Example

[0391] The following examples are provided to further describe some of the embodiments disclosed herein. These examples are intended to illustrate, but not to limit, the embodiments disclosed herein.

[0392] Example 1 - Expression of α-GPRC5DscFv CAR

[0393] Primary human pan T cells were electroporated with either no mRNA (mock), or 10 μg of mRNA expressing α-GPRC5D scFv CAR or isotype control CAR. CAR surface expression was measured by flow cytometry 24 hours after electroporation after staining with biotinylated L-protein and streptavidin-conjugated PE. Figure 1 ). The percentage of CAR expression was determined by staining with protein L. Expression was determined to be 95%, 88%, 70%, and 67% positive for CAR A, CAR B, CAR C, and isotype CAR, respectively. Only mock T cells were electroporated without adding mRNA to these cells. As expected, the mock T cells did not express any CAR structure. These data show that T cells highly express CAR structures. The hollow histograms are mock and the filled gray histograms are CAR-T populations.

[0394] The amino acid sequences of the components of the CAR construct targeting GPRC5D are shown in Table 2.

[0395] Table 2

[0396]

[0397]

[0398]

[0399] Example 2 - Induction of activation markers in antigen-stimulated CAR-T cells

[0400] Twenty-four hours after transient transfection, primary pan-T cells were labeled with Cell Trace Violet (CTV) fluorescent proliferation dye and then co-cultured with the multiple myeloma cell line H929. After four days of co-culture, cells were pre-gated for the CD8+CD4- population, and the surface expression of activation markers CD25 and CD71 on CAR-T was compared with T cells cultured alone or in the presence of α-CD3 / CD28 beads. ( Figure 2 ). Analysis from Figure 1 The same CAR T cells of the T cell activation response to H929 cells expressing GPRC5D antigen. Four days after electroporation, CAR A and CAR B show an increase in T cell activation response, as shown in the increase in CD25 and CD71 expression in response to culture with H929 cells. In contrast, simulated cells and cells expressing CAR C show that T cell activation in response to H929 cells is reduced. As a negative control, the same cells grow in the absence of any GPRC5D or T cell antigens, and the minimum increase of CD25 or CD71 is shown. As a positive control, simulated cells, CAR A cells, CAR B cells and CAR C cells are co-cultured with CD3 / CD28 beads. Each of these four cell populations shows a similar increase in CD25 expression and CD71 expression. Overall, these data show that CAR A cells and CAR B cells show the maximum T cell activation response to H929 cells expressing GPRC5D.

[0401] Example 3 - Cytokine profile analysis of antigen-stimulated CAR-T cells

[0402] Primary pan-T cells transiently transfected 24 hours before using mRNA expressing CAR were co-cultured with various myeloma cell lines expressing the target antigen GPRC5D at a ratio of 1:1 at high (+++) levels, medium (++) levels, low (+) levels or negative (-) levels. After sixteen hours of co-culture, the supernatant was harvested and the supernatant from the co-culture was analyzed for cytokine profiles by Meso Scale Discovery (MSD). ( Figure 3 ). The supernatant from GPRC5D CAR-T cells showed an increase in proinflammatory cytokines INF-γ, TNF-α, or IL-2. The increase in each of the proinflammatory cytokines is an increase in GPRC5D expression in the cell lines co-cultured with GPRC5D CAR-T cells, respectively. Co-culture with MM.1S resulted in the highest INF-γ and TNF-α expression, while co-culture with KMS11 cells resulted in the highest IL-2 levels. The K562 cell line was used as a negative control to show the basal background cytokine levels of the cell lines that do not express GPRC5D.

[0403] 24 hours after transient transfection with mRNA expressing CAR, primary pan T cells were co-cultured with autologous peripheral blood mononuclear cells (PBMC) and multiple myeloma cell line H929 (at a ratio of 1: 1: 1). After co-culture for sixteen hours, the supernatant was harvested. The cytokine profile analysis of the supernatant from the co-culture containing all three groups (CAR-T, H929, autologous PBMC) by MSD and the control co-culture lacking CAR-T cells, PBMC or H929 were compared with each cell population isolated and cultured. ( Figure 4 ). Similar to Figure 3 , mock cells, CAR-TA cells, CAR-T B cells, CAR-T C cells, or isotype CAR-T cells were co-cultured with H929 and / or autologous PBMCs. When H929 cells were co-cultured with PBMCs, increases in TNF-α and IL-2 were observed with CAR-T A cells and CAR-T B cells. When CAR-T C cells were co-cultured with H929 and PBMCs, limited to undetectable levels of INF-γ release were observed. In summary, CAR-T C cells showed reduced cytokine release, which is consistent with Figure 2 CAR-T cells, H929 cells, and PBMC cells were cultured alone as negative controls, and background cytokine levels were shown.

[0404] Example 4 - Tumor cell lines expressing GPRC5D: Targeting cells of target cell lines expressing high / low / no GPRC5D toxicity

[0405] The cytotoxic potential of CAR-T cells against myeloma cells expressing GPRC5D was assessed by flow cytometry. Primary pan-T cells (transiently expressing one of the three CARs described above) were co-cultured with fluorescently labeled myeloma cell lines MM1R, H929, and K562 at a specified effector: target (E:T) ratio for eight hours, at which time the co-cultures were stained with viability dyes. The killing percentage is the ratio of the absolute number of live (viability dye-negative) target (CTV-positive) cells remaining in the co-culture to the number of live targets not cultured with CAR-T cells. ( Figure 5). CAR A cells, CAR B cells, CAR C cells, isotype CAR cells and simulated T cells were co-cultured with GPRC5D+ cell lines MM1R and H929 or GPRC5D-K562 cells for eight hours. Various effectors: target ratios were analyzed, showing that CAR A cells, CAR B cells and CAR C cells were able to induce cytotoxicity of MM1R cells. The increase in cytotoxicity was associated with a higher effector: target ratio. Isotype controls and simulated controls were included as controls and showed approximately 20% to 30% cytotoxicity. Similarly, CAR A cells and CAR B cells induced cytotoxicity of H929 cells. CAR A cells and CAR B cells exhibited cytotoxic activity similar to isotype cells and simulated cells. These data show the specificity of CAR A cells and CAR B cells to MM1R cells and H929 cells rather than to K562 cells (GPRC5D).

[0406] Example 5 - Tumor cell lines expressing GPRC5D: Targeting cells for multiple myeloma patient-derived T cells toxicity

[0407] The cytotoxic potential of CAR-T cells against myeloma cells expressing GPRC5D was assessed by flow cytometry. Primary multiple myeloma patient T cells (transiently expressing one of the two CARs described previously) were co-cultured with the fluorescently labeled myeloma cell line MM.1S or the control GPRC5D-negative cell line KG-1 at the specified effector: target (E:T) ratio for 48 hours, at which time the co-cultures were stained with a viability dye. The percentage of cytotoxicity was determined as the percentage of fluorescently labeled cells that stained positive for the viability dye (indicating cell death). ( Figure 6 ). Compared with simulated T cells, primary multiple myeloma patient T cells transduced with GC5B81_LH or GC5B483_LH induced cytotoxicity of GPRC5D+MM.1S cells and increased effector cells. When co-cultured with the GPRC5D-KG-1 cell line, there was no observable cytotoxicity in both GC5B81_LH cells and GC5B483_LH GPRC5D CAR-T cells. These data show that multiple myeloma T cells transduced with GPRC5D CAR can induce killing of GPRC5D+ cells, but not GPRC5D cells.

[0408] Example 6 - α-GPRC5D Proliferative response of CAR-T cells to antigen stimulation

[0409] The proliferation capacity of α-GPRC5D CAR-T cells was evaluated by flow cytometry. Fluorescently labeled pan T cells (transiently expressing α-GPRC5D CAR or isotype CAR control) were co-cultured with H929 at a 1:1 effector:target ratio for four days. Proliferation was measured as the absolute number of live (viability dye negative) CAR-T cells with diluted fluorescent label (CTV). Proliferation in response to H929 was compared with CAR-T proliferation after four days of α-CD3 / CD28 bead stimulation and CAR-T cultured alone (no stimulation control). ( Figure 7 ). Simulated cells, isotype CAR cells, CAR-T A cells, CAR-T B cells and CAR-T C cells were co-cultured with GPRC5D+H929 cells, CD3 / CD28 beads or co-cultured without stimulation. Four days later, the total live counts of CD4 and CD8 populations were analyzed, reflecting the proliferation capacity of cells in response to GPRC5D antigens. In the CD4 population, CD3 / CD28 beads were used as positive controls, and all cell populations showed an increase in CD4 events compared to no stimulation. Compared with isotypes and simulations, only CAR-T A cells and CAR-TB cells showed a robust increase in counts in response to H929 cells. Similar results were observed for CD8 populations; however, CAR-T C cells did not show proliferation in response to H929 cells. In summary, these data show that CAR-T A cells and CAR-T B cells proliferate in response to GPRC5D+H929 cells.

[0410] Example 7 - Expression of GPRC5D-CAR on healthy donor T cells

[0411] Six primary human pan T cells were activated and expanded using the Miltenyi Biotec T cell TRANSACT system. 24 hours after activation, T cells were transduced with a lentivirus containing a nucleotide sequence encoding the GPRC5D GC5B680-N68S-LH CAR (SEQ ID NO: 90). The cells were expanded for 12 days, and surface CAR expression was measured by flow cytometry on days 6 and 10 using a commercially available rabbit anti-human H+L detection antibody. Results were Figure 8 , where data represent mean ± SD from 6 healthy donors. The shaded gray column (simulation) immediately to the left of the black column (GCB6800-N68S-LH) represents background CAR detection in untransduced (simulation) cells using commercial antibodies. The black column represents GPRC5D GC5B680-N68S-LH transduced CAR-T cells.

[0412] Example 8—GPRC5D Expression of CD4 markers, CD8 markers, and memory markers on CAR-T cells

[0413] Pan T cells from six healthy donors were transduced with lentivirus containing a nucleotide sequence encoding the GPRC5D GC5B680-N68S-LH CAR (GPRC5D CAR; SEQ ID NO: 90). Mock (untransduced) cells and transduced cells ("T cell pools", including cells with surface CAR (CAR-expressing cells or CAR + cells) and cells without surface CAR (cells not expressing CAR or CAR - Cells)] Evaluation of cells with surface CD4 (CD4 + ) and the proportion of cells with surface CD8 (CD8 + ) ratio. The data is Figure 9A to Fig. 9C Shown in. Fig. 9C The data in Figure 2 show that, as expected, GPRC5DCAR is expressed in CD4 + T cells and CD8 + T cells were detected in both. Figure 9A to Fig. 9C Also shown are the differences in all cell populations evaluated (including CAR + Cells, CAR - The CD4:CD8 ratios were similar in WT and Mock cells.

[0414] Two surface markers, CD45RA and CD62L, were used to determine T cell subtypes within the CAR-T population to distinguish four memory cell populations: effector memory RA + T cells (T EM RA+ ), effector memory cells T EM , naive T cells and memory stem cells T N / SCM , and central memory T cells (T CM ). The data is in Figure 9D to Fig.9F FIG9D is a flow cytometric plot of one donor sample, highlighting the gating strategy using two surface markers to distinguish four different memory populations. FIG9E and Fig.9F GPRC5D GC5B680-N68S-LH is shown mainly in T N / SCM Cells and T CM These cells are known to have high proliferation capacity, survival rate and therapeutic efficacy.

[0415] Example 9 - Determination of GPRC5D CAR-T cell toxicity at 6 hours and 24 hours

[0416] Untransduced (mock) T cells or GPRC5DGC5B680-N68S-LH-transduced T cells from one of six healthy donors were added at various E:T ratios to: (i) GPRC5D containing a luciferase transgene. + Cultures of MM.1S tumor cells, or (ii) GPRC5D containing a luciferase transgene – Cultures of K562 tumor cells. After incubation for 6 hours or 24 hours, cytotoxicity at each E:T ratio was evaluated. The data at the 6 hour time point are shown in FIG10A , and Fig. 10B Data at the 24-hour time point are shown in . The specific lysis percentage is calculated by (i) measuring the luciferase signal in tumor cells incubated in the presence of CAR-T for 6 hours or 24 hours, divided by the luciferase signal in tumor cells incubated alone at the same time point and multiplied by 100, and (ii) subtracting this number from 100. The formula is: 100-[(CAR-T+tumor luminescence) / (average value of tumor luminescence alone)]×100. The dotted line highlights zero lysis. Lysis is negative indicating cell growth.

[0417] Example 10 - Determination of GPRC5D CAR-T cell toxicity at 48 hours

[0418] T cells transduced with GPRC5D GC5B680-N68S-LH and untransduced (mock) T cells were prepared from healthy donors. (i) T cells transduced with GPRC5D GC5B680-N68S-LH or (ii) untransduced (mock) T cells were added to carboxyfluorescein succinimidyl ester (CFSE)-labeled GPRC5D + H929 cells, GPRC5D + MM.1S cells, GPRC5D + MM.1R cells, GPRC5D + MOLP-2 cells, GPRC5D + EJM cells and GPRC5D – K562 cells. After 48 hours of incubation, cells were stained with live / dead dye and dead cells and CFSE were evaluated. + The percentage of cells (% cytotoxicity) was plotted using a 4-parameter nonlinear regression curve fit against the log-transformed x-axis (E:T ratio). FIG. 11A to FIG. 11F Shown are mean ± SD. Mock negative control T cells were expressed in all GPRC5D +A certain degree of cytotoxicity was indeed induced in the cell line, but the degree of induction was lower than that of GPRC5DGC5B680-N68S-LH cells, especially at higher E:T ratios. Without wishing to be bound by theory, the reduction in cytotoxicity may be caused by allogeneic responses. In addition, the cytotoxicity of GPRC5DCAR cells varies depending on the cell line.

[0419] Example 11 - Determination of GPRC5D at 48 hours CAR-T activation

[0420] T cells transduced with GPRC5D GC5B680-N68S-LH and untransduced (mock) T cells were prepared from healthy donors. T cells transduced with GPRC5D GC5B680-N68S-LH or untransduced (mock) T cells were added to carboxyfluorescein succinimidyl ester (CFSE)-labeled GPRC5D + H929 cells, GPRC5D + MM.1S cells, GPRC5D + MOLP-2 cells, GPRC5D + EJM cells and GPRC5D – K562 cells. After 48 hours of incubation, cells were stained with live / dead dye, anti-CD25, anti-CD3 and anti-CAR visual markers. Cells were then evaluated for surface CD25 expression. Data are available at Fig.12 The data are expressed as mean ± SD. Fig.12 As shown, less than 13% background surface CD25 expression was observed in mock cells (i.e., not activated). In contrast, GC5B680-N68S-LH CAR-T cells in GPRC5D + In both (i) mock cells and (ii) GC5B680-N68S-LH CAR-T cells alone, low levels of surface CD25 expression were observed when incubated with K562 cells or when incubated alone.

[0421] Example 12 - Determination of GPRC5D CAR-T multifunctionality

[0422] GC5B680-N68S-LH transduced (CAR-T) cells were incubated for 18 h alone or with: PMA / ionomycin (as a positive control), GPRC5D + Cell lines H929 (H929), GPRC5D –K562 (K562) cells (CFSE labeled). When incubated in the presence of H929 cells or K562 cells, these cells are incubated with an E:T ratio of 1:2. Then the cells are first surface stained with live / dead dyes, anti-CD3 and anti-idiotype, fixed, permeabilized, and intracellular staining with anti-IFN-γ, anti-IL-2 and anti-TNF-α. Then the cells stained by flow cytometry analysis. Using Boolean (grading) gating, the percentage of each cytokine level is calculated after analyzing the flow cytometry graph, wherein the percentage is represented in Table 3 to Table 6 below. Also in Table 3 to Table 6, the multifunctional cytokine release level of each group is highlighted in gray, indicating the sum of mean value.

[0423] Table 3. Cytokine expression in CAR-T cells incubated in the presence of H929

[0424]

[0425] Table 4. Cytokine expression in CAR-T cells incubated in the presence of K562

[0426]

[0427] Table 5. Cytokine expression in CAR-T cells cultured alone

[0428]

[0429] Table 6 Cytokine expression in CAR-T cells incubated in the presence of PMA / ionomycin

[0430]

[0431] Example 13 - Determination of GPRC5D CAR-T proliferation

[0432] GC5B680-N68S-LH cells were prepared using cells from one donor. (i) GC5B680-N68S-LH transduced cells and (ii) mock cells were transduced in the presence or absence of GPRC5D + Mock (untransduced) cells were used as negative controls. After 6 days of incubation, the extent of cell proliferation was measured by determining the dilution of the CELLTRACE Violet (CTV) dye concentration in the cells, where this amount is determined by Fig.13 The X-axis of the graph represents the CAR using anti-idiotypic antibodies. + Cell staining, where the degree of staining is determined by Fig.13 The Y-axis of the graph represents.

[0433] like Fig.13 shown, either individually or in GPRC5D –Mock T cells and CTV-labeled CAR T cells in the presence of K562 cells showed little or no cell proliferation, as seen from low to zero dilutions of the CTV dye. As predicted, agonist CD3 / CD28 beads (used as a positive control) caused proliferation of all T cell populations evaluated. When CAR-T cells were co-expressed with GPRC5D + When incubated with target cells (H929 or MM.1S), proliferation was mainly enriched in cells expressing GC5B680-N68S-LH (CAR + ) in transduced cells (e.g. Fig.13 This result indicates that GC5B680-N68S-LH cell proliferation is regulated by GPRC5D + The presence of target cells is enhanced. Fig.13 The data shown are representative of similar observations made using cells prepared from other donors.

[0434] Example 14 - Determination of GPRC5D CAR-T proliferation

[0435] GC5B680-N68S-LH (CAR-T) cells were mixed with positive control staurosporine, GPRC5D + H929 cells, GPRC5D – K562 cells were incubated together or alone for 24 hours. GC5B680-N68S-LH (CAR-T) cells were incubated with H929 cells or K562 cells using an E:T ratio of 1:2. The cells were then stained with live / dead dye, anti-CD3, anti-idiotype, and anti-cleaved caspase 3 / 7 dye. The stained cells were analyzed by flow cytometry. Data are available at Fig.14 As shown in the figure. Fig.14 As shown, staurosporine is expressed in CAR + and CAR – Cleaved caspase 3 / 7 staining was induced in both T cell populations. In addition, starting 4 hours after the 24 hour time point, there were 50% to 65% positive cells. + T cell populations and CARs – Neither T cell population showed significant differences in caspase 3 / 7 levels between negative control incubations (K562 and CAR alone) and incubations in the presence of H929.

[0436] Example 15 - Determination of GC5B680-N68S-LH, GC5B680-N68S-HL, GP5B83_N24T_N31S-LH and Efficacy of GP5B83_N24T_N31S-HL against established MM.1S human MM xenografts in NSG-B2M mice

[0437] In female NSG B2m (alternatively referred to as NOD-scid Il2rg null B2m nullThe anti-tumor efficacy of T cells (CAR-T cells) transduced with GC5B680-N68S-LH (SEQ ID NO: 86), GC5B680-N68S-HL (SEQ ID NO: 85), GP5B83_N24T_N31S-LH (SEQ ID NO: 84) and GP5B83_N24T_N31S-HL (SEQ ID NO: 83) was evaluated in an established subcutaneous (sc) MM.1S human multiple myeloma (MM) xenograft model in 1×10 6 CAR + cells or 5×10 6 CAR + Dosage of cells (i) GC5B680-N68S-LH cells, (ii) GC5B680-N68S-HL cells, (iii) GP5B83_N24T_N31S-LH cells or (iv) GP5B83_N24T_N31S-HL CAR-T cells were administered intravenously (iv) for a total of one dose. Three groups of mice bearing MM.1S xenografts were evaluated: (i) mice treated with PBS, (ii) mice that underwent mock transduction, and (iii) mice transduced with GPRC5D. On day 26 after tumor implantation, the percentage Δ tumor growth inhibition (% ΔTGI) of the three groups of mice bearing SC (subcutaneous) MM.1S xenografts was calculated using the following formula: ([(TVc-TVc0)-(TVt-TVt0)] / (TVc-TVc0))×100, where “TVc” is the average tumor burden of a given control group, “TVc0” is the average initial tumor burden of a given control group, “TVt” is the average tumor burden of the treatment group, and “TVt0” is the average initial tumor burden of the treatment group.

[0438] Statistical significance was calculated using linear mixed effects analysis in R, with treatment and time as fixed effects and animal as a random effect. If each longitudinal response trajectory was not linear, a logarithmic transformation (base 10) was performed. Information derived from this statistical model was used for pairwise treatment comparisons with either the PBS control group or the mock transduced group, with p values ​​considered statistically significant at <0.05.

[0439] Compared with PBS control and mock CAR-T control, all four GPRC5D-directed CAR-T constructs showed significant differences in the expression of CAR-T cells at 1 × 10 6 and 5×10 6 CAR + All T cell dose levels induced statistically significant %ΔTGI, as shown in Tables 7 and Fig.15Complete response (complete tumor regression with no visible tumor) was assessed on day 47 after tumor implantation.

[0440] Table 7. Efficacy of GPRC5D-directed CAR-T against established MM.1S human MM xenografts in NSG-B2M mice Summary

[0441]

[0442]

[0443] p<0.05 relative to PBS control and mock CAR-T control, except where noted as not significant (ns).

[0444] CR = Complete Response

[0445] Example 16 GC5B680-N68S-LH, GC5B680-N68S-HL, GP5B83_N24T_N31S-LH and GP5B83_ Efficacy of N24T_N31S-HL against disseminated H929IV human MM xenografts in NSG mice

[0446] The efficacy of T cells transduced with GC5B680-N68S-LH (SEQ ID NO:86; encoded by the nucleotides of SEQ ID NO:90), GC5B680-N68S-HL (SEQ ID NO:85; encoded by the nucleotides of SEQ ID NO:89), GP5B83_N24T_N31S-LH (SEQ ID NO:84; encoded by the nucleotides of SEQ ID NO:88), and GP5B83_N24T_N31S-HL (SEQ ID NO:83; encoded by the nucleotides of SEQ ID NO:87) in female NSG TM (Alternatively referred to as NOD scidγ or NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ) mice in the disseminated (iv) H929 human MM model. In this example, on day 9 after tumor implantation, 1×10 6 CAR + cells or 5×10 6 CAR + A single iv dose of cells expressing GC5B680-N68S-LH, GC5B680-N68S-HL, GP5B83_N24T_N31S-LH or GP5B83_N24T_N31S-HL (CAR + )T cells.

[0447] Survival rate assessment is carried out, wherein the survival rate percentage of each study day is plotted relative to the study day.Other clinical signs of hind limb paralysis or disseminated tumor overload are used as alternative endpoints of death.Kaplan-Meier survival analysis is used to determine the median survival rate.ILS percentage (%ILS) is limited to the difference between the median survival rate of the treatment group and the control group, and is calculated as ILS=([MSt-MSc] / MSc)×100, wherein "MSc" is the median survival rate of a given control group, and "MSt" is the median survival rate of a specific treatment group.Animals that fail to reach alternative endpoints due to adverse clinical signs (such as ulcerative tumors, weight loss, etc.) or death unrelated to treatment are reviewed for survival assessment.Survival rate is graphically represented using Kaplan-Meier curves, and is evaluated by log-rank (Mantel-Cox) test using GraphPad Prism software (version 7.0).

[0448] For PBS-treated control mice, by day 52 after tumor implantation (CAR + The median survival rate was 50% at 43 days after injection. For mice treated with mock CAR-T cells, the median survival rate was 50% at 44 days after tumor implantation (35 days after CAR-T injection). 6 GC5B680-N68S-LH CAR administered at a dose of + Mice treated with 1×10 6 The GC5B680-N68S-HL CAR was administered at a dose of + The median survival rate of mice treated with 1×10 6 GP5B83_N24T_N31S-LH CAR administered at a dose of 10 cells + The median survival rate of mice treated with 1×10 6 GP5B83_N24T_N31S-HL CAR administered at a dose of 10 cells + The cell-treated mice achieved a median survival rate of 50% after day 111 (102 days after CAR-T injection). The percentage of life extension (ILS) was assessed via Kaplan-Meier curves and evaluated by the log-rank Mantel-Cox test.

[0449] Compared with PBS control and mock CAR-T control, all four GPRC5D-directed CAR-T constructs showed significant differences in the expression of CAR-T cells at 1 × 10 6 All dose levels elicited statistically significant %ILS, as discussed below and shown in Tables 8 and Fig.16 Animals surviving until day 111 were considered CR.

[0450] Table 8. Summary of the efficacy of GPRC5D-directed CAR-T against disseminated H929 human MM xenografts in NSG mice

[0451]

[0452] p<0.05 relative to PBS control and mock CAR-T control, except where noted as not significant (ns).

[0453] CR = Complete Response

[0454] Adverse clinical signs associated with progressive tumor disease burden were observed in the PBS-treated control group starting at day 52 after tumor transplantation, while clinical signs of graft-versus-host disease did not develop until day 48 after tumor transplantation in either the mock-transduced or GPRC5D CAR-T-treated groups.

[0455] The teachings of all patents, published patent applications, and references cited herein are incorporated by reference in their entirety for all purposes.

[0456] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments as covered by the following claims.

[0457] Sequence Listing

[0458] Light chain sequence 1 (GC5B483) (SEQ ID NO: 1)

[0459] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIK

[0460] Heavy chain sequence 1 (GC5B483) (SEQ ID NO: 2)

[0461] EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSFGGRHKALFDYWGQGTLVTVSS

[0462] Light chain sequence 2 (GC5B81) (SEQ ID NO: 3)

[0463] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK

[0464] Heavy chain sequence 2 (GC5B81) (SEQ ID NO: 4)

[0465] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESRWRGYKLDYWGQGTLVTVSS

[0466] Light chain sequence 3 (GC5B596) (SEQ ID NO: 5)

[0467] DIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIK

[0468] Heavy chain sequence 3 (GC5B596) (SEQ ID NO: 6)

[0469] QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSS

[0470] Linker polypeptide sequence (SEQ ID NO:7)

[0471] GTEGKSSGSGSESKST

[0472] scFv sequence 1 (GC5B483) (SEQ ID NO: 8)

[0473] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIKGTEGKSSGSGSESKSTEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSFGGRHKALFDYWGQGTLVTVSS

[0474] scFv sequence 2 (GC5B81) (SEQ ID NO: 9)

[0475] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKGTEGKSSGSGSESKSTQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESRWRGYKLDYWGQGTLVTVSS

[0476] scFv sequence 3 (GC5B596) (SEQ ID NO: 10)

[0477] DIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIKGTEGKSSGSGSESKSTQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSS

[0478] Signal sequence (SEQ ID NO: 11)

[0479] MAWVWTLLFLMAAAQSIQA

[0480] CD137 sequence (SEQ ID NO: 12)

[0481] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0482] CD3z sequence (SEQ ID NO: 13)

[0483] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0484] Intracellular signaling domain (SEQ ID NO: 14)

[0485] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0486] CD8a-TM sequence (SEQ ID NO: 15)

[0487] IYIWAPLAGTCGVLLLSLVITLYC

[0488] CD8a hinge sequence (SEQ ID NO: 16)

[0489] TSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0490] Extracellular binding domain (GC5B483) (SEQ ID NO: 17)

[0491] MAWVWTLLFLMAAAQSIQAEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIKGTEGKSSGSGSESKSTEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSFGGRHKALFDYWGQGTLVTVSS

[0492] Extracellular binding domain (GC5B81) (SEQ ID NO: 18)

[0493] MAWVWTLLFLMAAAQSIQADIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKGTEGKSSGSGSESKSTQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESRWRGYKLDYWGQGTLVTVSS

[0494] Extracellular binding domain (GC5B596) (SEQ ID NO: 19)

[0495] MAWVWTLLFLMAAAQSIQADIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIKGTEGKSSGSGSESKSTQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSS

[0496] Extracellular binding domain (GC5B483) (SEQ ID NO: 20)

[0497] MAWVWTLLFLMAAAQSIQAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSFGGRHKALFDYWGQGTLVTVSSGTEGKSSGSGSESKSTEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIK

[0498] Extracellular binding domain (GC5B81) (SEQ ID NO: 21)

[0499] MAWVWTLLFLMAAAQSIQAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESRWRGYKLDYWGQGTLVTVSSGTEGKSSGSGSESKSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK

[0500] Extracellular binding domain (GC5B596) (SEQ ID NO: 22)

[0501] MAWVWTLLFLMAAAQSIQAQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSSGTEGKSSGSGSESKSTDIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIK

[0502] scFv sequence 4 (GC5B483) (SEQ ID NO: 24)

[0503] EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSFGGRHKALFDYWGQGTLVTVSSGTEGKSSGSGSESKSTEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIK

[0504] scFv sequence 5 (GC5B81) (SEQ ID NO: 25)

[0505] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESRWRGYKLDYWGQGTLVTVSSGTEGKSSGSGSESKSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK

[0506] scFv sequence 6 (GC5B596) (SEQ ID NO: 26)

[0507] QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSSGTEGKSSGSGSESKSTDIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIK

[0508] pDR000074490GC5B483-LH-CAR sequence (SEQ ID NO: 27)

[0509] MAWVWTLLFLMAAAQSIQAEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIKGTEGKSSGSGSESKSTEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSFGGRHKALFDYWGQGTLVTVSSTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0510] pDR000074489GC5B81-LH-CAR sequence (SEQ ID NO: 28)

[0511] MAWVWTLLFLMAAAQSIQADIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKGTEGKSSGSGSESKSTQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESRWRGYKLDYWGQGTLVTVSSTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0512] pDR000074488GC5B596-LH-CAR sequence (SEQ ID NO: 29)

[0513] MAWVWTLLFLMAAAQSIQADIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIKGTEGKSSGSGSESKSTQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSSTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0514] pDR000074483GC5B483-HL-CAR sequence (SEQ ID NO: 30)

[0515] MAWVWTLLFLMAAAQSIQAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSFGGRHKALFDYWGQGTLVTVSSGTEGKSSGSGSESKSTEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIKTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO:30

[0516] pDR000074482GC5B81-HL-CAR sequence (SEQ ID NO: 31)

[0517] MAWVWTLLFLMAAAQSIQAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESRWRGYKLDYWGQGTLVTVSSGTEGKSSGSGSESKSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0518] pDR000074481GC5B596-HL-CAR sequence (SEQ ID NO: 32)

[0519] MAWVWTLLFLMAAAQSIQAQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSSGTEGKSSGSGSESKSTDIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIKTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0520] DNA sequence of pDR000074490GC5B483-LH-CDS (SEQ ID NO: 33)

[0521]

[0522] DNA sequence of pDR000074489GC5B81-LH-CDS (SEQ ID NO: 34)

[0523]

[0524] DNA sequence of pDR000074488GC5B596-LH-CDS (SEQ ID NO: 35)

[0525]

[0526] DNA sequence of pDR000074483GC5B483-HL-CDS (SEQ ID NO: 36)

[0527]

[0528] DNA sequence of pDR000074482GC5B81-HL-CDS (SEQ ID NO: 37)

[0529]

[0530] DNA sequence of pDR000074481GC5B596-HL-CDS (SEQ ID NO: 38)

[0531]

[0532] HCDR1 (GC5B81) (SEQ ID NO: 39)

[0533] GGTFSSY

[0534] HCDR2 (GC5B81) (SEQ ID NO:40)

[0535] ESRWRGYKLDY

[0536] HCDR3 (GC5B81) (SEQ ID NO:41)

[0537] IPIFGT

[0538] HCDR1 (GC5B483) (SEQ ID NO:42)

[0539] GYSFTSY

[0540] HCDR2 (GC5B483) (SEQ ID NO: 43)

[0541] YPGKSD

[0542] HCDR3 (GC5B483) (SEQ ID NO: 44)

[0543] VYSFGGRHKALFDY

[0544] HCDR1 (GC5B596) (SEQ ID NO: 45)

[0545] GYSFTGY

[0546] HCDR2 (GC5B596) (SEQ ID NO: 46)

[0547] NPYNSD

[0548] HCDR3 (GC5B596) (SEQ ID NO: 47)

[0549] VALRVALDY

[0550] LCDR1 (GC5B81) (SEQ ID NO:48)

[0551] RASQSISSYLN

[0552] LCDR2 (GC5B81) (SEQ ID NO: 49)

[0553] AASSLQS

[0554] LCDR3 (GC5B81) (SEQ ID NO: 50)

[0555] QQSYSTPLT

[0556] LCDR1 (GC5B483) (SEQ ID NO:51)

[0557] RASQSVSSYLA

[0558] LCDR2 (GC5B483) (SEQ ID NO: 52)

[0559] DASNRAT

[0560] LCDR3 (GC5B483) (SEQ ID NO: 53)

[0561] QQRSNWPLT

[0562] LCDR1 (GC5B596) (SEQ ID NO: 54)

[0563] KASQNVATHVG

[0564] LCDR2 (GC5B596) (SEQ ID NO: 55)

[0565] SASYRYS

[0566] LCDR3 (GC5B596) (SEQ ID NO: 56)

[0567] QQYNRYPYT

[0568] Human GPRC5D polypeptide (SEQ ID NO: 57)

[0569] MYKDCIESTGDYFLLCDAEGPWGIILESLAILGIVVTILLLLAFLFLMRKIQDCSQWNVLPTQLLFLLSVLGLFGLAFAFIIELNQQTAPVRYFLFGVLFALCFSCLLAHASNLVKLVRGCVSFSWTTILCIAIGCSLLQIIIATEYVTLIMTRGMMFVNMTPCQLNVDFVVLLVYVLFLMALTFFVSKATFCGPCENWKQHGRLIFITVLFSIIIWVVWISMLLRGNPQFQRQPQWDDPVVCIALVTNAWVFLLLYIVPELCILYRSCRQECPLQGNACPVTAYQHSFQVENQELSRARDSDGAEEDVALTSYGTPIQPQTVDPTQECFIPQAKLSPQQDAGGV

[0570] HCDR1 (GP5B83) (SEQ ID NO:58)

[0571] GGSLSSSSY

[0572] HCDR2 (GP5B83) (SEQ ID NO:59)

[0573] YYSGN

[0574] HCDR3 (GP5B83) (SEQ ID NO: 60)

[0575] HVGYSYGRRFWYFDL

[0576] LCDR1 (GP5B83) (SEQ ID NO:61)

[0577] RASQSVSSYLA

[0578] LCDR2 (GP5B83) (SEQ ID NO:62)

[0579] DASNRAT

[0580] LCDR3 (GP5B83) (SEQ ID NO: 63)

[0581] QQRSNWPPT

[0582] Heavy chain sequence (GP5B83) (SEQ ID NO: 64)

[0583] QLQLQESGPGLVKPSETLSLTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFWYFDLWGRGTLVTVSS

[0584] Light chain sequence (GP5B83) (SEQ ID NO: 65)

[0585] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK

[0586] HCDR1 (GC5B680) (SEQ ID NO: 66)

[0587] GFSLTNIRM

[0588] HCDR2 (GC5B680) (SEQ ID NO: 67)

[0589] FSNDE

[0590] HCDR3 (GC5B680) (SEQ ID NO: 68)

[0591] MRLPYGMDV

[0592] LCDR1 (GC5B680) (SEQ ID NO: 69)

[0593] RSSQSLVHSDGNTYLS

[0594] LCDR2 (GC5B680) (SEQ ID NO: 70)

[0595] KISNRFF

[0596] LCDR3 (GC5B680) (SEQ ID NO: 71)

[0597] MQATQFPHT

[0598] Heavy chain sequence (GC5B680) (SEQ ID NO: 72)

[0599] QVTLKESGPVLVKPTETLTLTCTVSGFSLTNIRMSVSWIRQPPGKALEWLAHIFSNDEKSYSSSLKSRLTISRDTSKSQVVLTLTNVDPVDTATYYCARMRLPYGMDVWGQGTTVTVSS

[0600] Light chain sequence (GC5B680) (SEQ ID NO: 73)

[0601] DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFFGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPHTFGQGTKLEIK

[0602] Linker polypeptide sequence (SEQ ID NO:74)

[0603] GGSEGKSSGSGSESKSTGGS

[0604] scFv sequence (GP5B83-HL) (SEQ ID NO: 75)

[0605] QLQLQESGPGLVKPSETLSLTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFWYFDLWGRGTLVTVSSGGSEGKSSGSGSESKSTGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK

[0606] scFv sequence (GP5B83-LH) (SEQ ID NO: 76)

[0607] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKGGSEGKSSGSGSESKSTGGSQLQLQESGPGLVKPSETLSLTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFWYFDLWGRGTLVTVSS

[0608] scFv sequence (GC5B680-HL) (SEQ ID NO: 77)

[0609] QVTLKESGPVLVKPTETLTLTCTVSGFSLTNIRMSVSWIRQPPGKALEWLAHIFSNDEKSYSSSLKSRLTISRDTSKSQVVLTLTNVDPVDTATYYCARMRLPYGMDVWGQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFFGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPHTFGQGTKLEIK

[0610] scFv sequence (GC5B680-LH) (SEQ ID NO: 78)

[0611] DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFFGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPHTFGQGTKLEIKGGSEGKSSGSGSESKSTGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTNIRMSVSWIRQPPGKALEWLAHIFSNDEKSYSSSLKSRLTISRDTSKSQVVLTLTNVDPVDTATYYCARMRLPYGMDVWGQGTTVTVSS

[0612] Extracellular binding domain (GP5B83-HL) (SEQ ID NO:79)

[0613] MAWVWTLLFLMAAAQSIQAQLQLQESGPGLVKPSETLSLTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFWYFDLWGRGTLVTVSSGGSEGKSSGSGSESKSTGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK

[0614] Extracellular binding domain (GP5B83-LH) (SEQ ID NO: 80)

[0615] MAWVWTLLFLMAAAQSIQAEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKGGSEGKSSGSGSESKSTGGSQLQLQESGPGLVKPSETLSLTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFWYFDLWGRGTLVTVSS

[0616] Extracellular binding domain (GC5B680-HL) (SEQ ID NO: 81)

[0617] MAWVWTLLFLMAAAQSIQAQVTLKESGPVLVKPTETLTLTCTVSGFSLTNIRMSVSWIRQPPGKALEWLAHIFSNDEKSYSSSLKSRLTISRDTSKSQVVLTLTNVDPVDTATYYCARMRLPYGMDVWGQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFFGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPHTFGQGTKLEIK

[0618] Extracellular binding domain (GC5B680-LH) (SEQ ID NO: 82)

[0619] MAWVWTLLFLMAAAQSIQADIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFFGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPHTFGQGTKLEIKGGSEGKSSGSGSESKSTGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTNIRMSVSWIRQPPGKALEWLAHIFSNDEKSYSSSLKSRLTISRDTSKSQVVLTLTNVDPVDTATYYCARMRLPYGMDVWGQGTTVTVSS

[0620] GP5B83-HL-CAR sequence (SEQ ID NO: 83)

[0621] QLQLQESGPGLVKPSETLSLTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFWYFDLWGRGTLVTVSSGGSEGKSSGSGSESKSTGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0622] GP5B83-LH-CAR sequence (SEQ ID NO: 84)

[0623] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKGGSEGKSSGSGSESKSTGGSQLQLQESGPGLVKPSETLSLTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFWYFDLWGRGTLVTVSSTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0624] GC5B680-HL-CAR sequence (SEQ ID NO: 85)

[0625] QVTLKESGPVLVKPTETLTLTCTVSGFSLTNIRMSVSWIRQPPGKALEWLAHIFSNDEKSYSSSLKSRLTISRDTSKSQVVLTLTNVDPVDTATYYCARMRLPYGMDVWGQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFFGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPHTFGQGTKLEIKTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0626] GC5B680-LH-CAR sequence (SEQ ID NO: 86)

[0627] DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFFGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPHTFGQGTKLEIKGGSEGKSSGSGSESKSTGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTNIRMSVSWIRQPPGKALEWLAHIFSNDEKSYSSSLKSRLTISRDTSKSQVVLTLTNVDPVDTATYYCARMRLPYGMDVWGQGTTVTVSSTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0628] DNA sequence of pDR000084454 GP5B83-HL sequence (SEQ ID NO: 87)

[0629]

[0630] DNA sequence of GP5B83-LH sequence (SEQ ID NO: 88)

[0631]

[0632] DNA sequence of GC5B680-HL sequence (SEQ ID NO: 89)

[0633]

[0634] The DNA sequence of GC5B680-LH sequence (SEQ ID NO:90)

[0635]

[0636] Linker sequence (SEQ ID NO:91)

[0637] GGSEGKSSGSGSESKSTGGS

[0638] Linker sequence (SEQ ID NO:92)

[0639] GGGSGGGS

[0640] Linker sequence (SEQ ID NO:93)

[0641] GGGSGGGSGGGS

[0642] Linker sequence (SEQ ID NO:94)

[0643] GGGSGGGSGGGSGGGS

[0644] Linker sequence (SEQ ID NO:95)

[0645] GGGSGGGSGGGSGGGSGGGS

[0646] Linker sequence (SEQ ID NO:96)

[0647] GGGGSGGGGSGGGGS

[0648] Linker sequence (SEQ ID NO:97)

[0649] GGGGSGGGGSGGGGSGGGGS

[0650] Linker sequence (SEQ ID NO:98)

[0651] GGGGSGGGGSGGGGSGGGGSGGGGS

[0652] Linker sequence (SEQ ID NO:99)

[0653] GSTSGSGKPGSGEGSTKG

[0654] Linker sequence (SEQ ID NO: 100)

[0655] IRPRAIGGSKPRVA

[0656] Linker sequence (SEQ ID NO: 101)

[0657] GKGGSGKGGSGKGGS

[0658] Linker sequence (SEQ ID NO: 102)

[0659] GGKGSGGKGSGGKGS

[0660] Linker sequence (SEQ ID NO: 103)

[0661] GGGKSGGGSGGGSGGGS

[0662] Linker sequence (SEQ ID NO: 104)

[0663] GKGKSGKGKSGKGKS

[0664] Linker sequence (SEQ ID NO: 105)

[0665] GGGKSGGKGSGKGGS

[0666] Linker sequence (SEQ ID NO: 106)

[0667] GKPGSGKPGSGKPGS

[0668] Linker sequence (SEQ ID NO: 107)

[0669] GKPGSGKPGSGKPGS

[0670] Linker sequence (SEQ ID NO: 108)

[0671] GKGKSGKGKSGKGKSGKGKS

[0672] Linker sequence (SEQ ID NO: 109)

[0673] STAGDTHLGGEDFD

[0674] Linker sequence (SEQ ID NO: 110)

[0675] GEGGSGEGGGSGEGGS

[0676] Linker sequence (SEQ ID NO: 111)

[0677] GGGGGGGGGGGGGGG

[0678] Linker sequence (SEQ ID NO: 112)

[0679] TALKING TALKING TALKING

[0680] Linker sequence (SEQ ID NO: 113)

[0681] GGGESGGESGGESGGES

[0682] Linker sequence (SEQ ID NO: 114)

[0683] GESEG

[0684] Linker sequence (SEQ ID NO: 115)

[0685] GSTSGSGKPGSGEGSTKG

[0686] Linker sequence (SEQ ID NO: 116)

[0687] PRGASKSGSASQTGSAPGS

[0688] Linker sequence (SEQ ID NO: 117)

[0689] GTAAAGAGAAGGAAAGAAG

[0690] Linker sequence (SEQ ID NO: 118)

[0691] GTSGSSGSGSGGSGSGGGG

[0692] Linker sequence (SEQ ID NO: 119)

[0693] GKPGSGKPGSGKPGSGKPGS

[0694] Linker sequence (SEQ ID NO: 120)

[0695] GSGS

[0696] Linker sequence (SEQ ID NO: 121)

[0697] APAPAPAPAP

[0698] Linker sequence (SEQ ID NO: 122)

[0699] APAPAPAPAPAPAPAPAPAP

[0700] Linker sequence (SEQ ID NO: 123)

[0701] AEAAAKEAAAKEAAAAKEAAAAKEAAAAKAAA

[0702] Hinge sequence (SEQ ID NO: 124)

[0703] EPKSCDKTHTCPPCP

[0704] Hinge sequence (SEQ ID NO: 125)

[0705] ERKCCVECPPCP

[0706] Hinge sequence (SEQ ID NO: 126)

[0707] ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP

[0708] Hinge sequence (SEQ ID NO: 127)

[0709] ESKYGPPCPSCP

Claims

1. A chimeric antigen receptor (CAR), comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises a heavy chain complementary determining region 1 (CDR1) as shown in the amino acid sequence of SEQ ID NO: 66, a heavy chain CDR2 as shown in the amino acid sequence of SEQ ID NO: 67, and a heavy chain CDR3 as shown in the amino acid sequence of SEQ ID NO: 68, and further comprises a light chain CDR1 as shown in the amino acid sequence of SEQ ID NO: 69, a light chain CDR2 as shown in the amino acid sequence of SEQ ID NO: 70, and a light chain CDR3 as shown in the amino acid sequence of SEQ ID NO: 71; The extracellular antigen binding domain binds to anti-G protein receptor family C group 5 member D (GPRC5D) antigen.

2. The CAR of claim 1, wherein the extracellular antigen binding domain comprises: a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 73, or a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 72, or a combination of a LCVR comprising the amino acid sequence of SEQ ID NO: 73 and a HCVR comprising the amino acid sequence of SEQ ID NO:

72.

3. The CAR according to claim 1 or 2, wherein the extracellular antigen binding domain comprises: A light chain variable region comprising the amino acid sequence of SEQ ID NO:73 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

72.

4. The CAR of claim 1 or 2, wherein the extracellular antigen binding domain comprises a single-chain variable fragment (scFv).

5. The CAR of claim 4, wherein the scFv comprises a linker polypeptide located between the light chain variable region and the heavy chain variable region.

6. The CAR of claim 5, wherein the linker polypeptide comprises the amino acid sequence of SEQ ID NO:

7.

7. The CAR of claim 4, wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 78 and SEQ ID NO:

77.

8. The CAR of claim 1 or 2, wherein the extracellular antigen binding domain comprises a signal polypeptide.

9. The CAR according to claim 8, wherein the signal polypeptide comprises the amino acid sequence of SEQ ID NO:

11.

10. The CAR according to claim 1 or 2, wherein the intracellular signaling domain comprises a polypeptide component, and the polypeptide component is selected from the group consisting of: a TNF receptor superfamily member 9 (CD137) component, a T cell surface glycoprotein CD3 ζ chain (CD3z) component, a CD27 component, a CD28 component, an inducible T cell co-stimulator (ICOS) component, and a combination thereof.

11. The CAR of claim 10, wherein the CD137 component comprises the amino acid sequence of SEQ ID NO:

12.

12. The CAR of claim 10, wherein the CD3z component comprises the amino acid sequence of SEQ ID NO:

13.

13. The CAR of claim 10, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

14.

14. The CAR of claim 1 or 2, wherein the transmembrane domain comprises a CD8a transmembrane region (CD8a-TM) polypeptide.

15. The CAR of claim 14, wherein the CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO:

15.

16. The CAR of claim 1 or 2, further comprising a hinge region connecting the transmembrane domain to the extracellular antigen binding domain.

17. The CAR of claim 16, wherein the hinge region is a CD8a hinge region.

18. The CAR of claim 17, wherein the CD8a hinge region comprises the amino acid sequence of SEQ ID NO:

16.

19. The CAR according to claim 1 or 2, wherein the extracellular antigen binding domain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 82 and SEQ ID NO:

81.

20. The CAR of claim 1 or 2, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 86 and SEQ ID NO:

85.

21. An isolated lymphocyte expressing the CAR according to any one of claims 1 to 20.

22. The isolated lymphocyte according to claim 21, wherein the lymphocyte is a T lymphocyte.

23. The isolated lymphocytes according to claim 22, wherein the T lymphocytes are naive T cells.

24. The isolated lymphocytes according to claim 22, wherein the T lymphocytes are memory stem T cells.

25. The isolated lymphocytes of claim 22, wherein the T lymphocytes are central memory T cells.

26. The isolated lymphocyte according to any one of claims 22 to 25, wherein the T lymphocyte is CD4+.

27. The isolated lymphocytes according to any one of claims 22 to 25, wherein the T lymphocytes are CD8+.

28. The isolated lymphocytes according to any one of claims 22 to 25, wherein the T lymphocytes are CD4+ and CD8+.

29. An isolated nucleic acid molecule encoding the CAR according to any one of claims 1 to 28.

30. The isolated nucleic acid molecule of claim 29, wherein the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO:90 and SEQ ID NO:

89.

31. An isolated nucleic acid molecule according to claim 29, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of SEQ ID NO:90 and SEQ ID NO:

89.

32. A vector comprising the nucleic acid molecule according to any one of claims 29 to 31.

33. A cell expressing the nucleic acid molecule according to any one of claims 29 to 31.

34. A pharmaceutical composition comprising: An effective amount of lymphocytes according to any one of claims 21 to 28.

35. A pharmaceutical composition comprising: An effective amount of lymphocytes according to any one of claims 21 to 28 and a pharmaceutically acceptable excipient.

36. The CAR according to any one of claims 1 to 20 or the pharmaceutical composition according to claim 34 or 35, for use in therapy.

37. A CAR according to any one of claims 1 to 20 or a pharmaceutical composition according to claim 34 or 35 for use in a method of treating a subject suffering from cancer.

38. Use of the lymphocytes according to any one of claims 21 to 28 in the preparation of a medicament for treating a subject suffering from multiple myeloma.

39. Use of the lymphocyte according to any one of claims 21 to 28 in the preparation of a medicament for targeted killing of multiple myeloma cells.

40. Use of the CAR according to claim 1 in the preparation of a medicament for use in a method for detecting the presence of multiple myeloma in a subject, the method comprising: (a) contacting a cell sample obtained from the subject with the CAR according to claim 1, thereby forming a CAR cell complex, and (b) detecting the complex, wherein detection of the complex indicates the presence of multiple myeloma in the subject.

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