GPRC5D chimeric antigen receptor and cells expressing the same

Abstract

To provide chimeric antigen receptors (CARs) that specifically target a G-protein coupled receptor, a G-protein coupled receptor family C group 5 member D (GPRC5D), and immunoresponsive cells comprising such CARs, for treatment of cancer.SOLUTION: A chimeric antigen receptor (CAR) comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain consists of a specific sequence and targets a G-protein coupled receptor family C group 5 member D (GPRC5D).SELECTED DRAWING: None

Description

[Technical Field]

[0001] (Sequence Listing) This application has been filed electronically in ASCII format and is incorporated by reference in its entirety. Including the Sequence Listing incorporated herein. An ASCII copy of this document was obtained as of January 2020. It was created on the 9th, named JBI6043WOPCT1_SL.txt, and has a filename of 150,0 It is 02 bytes in size.

[0002] FIELD OF THE INVENTION The present invention provides a GPRC5D-targeting chimeric antigen receptor comprising a GPRC5D single-chain variable fragment. Chimeric antigen receptors (CARs), and genetically engineered cells expressing CARs GPRC5D-targeted immune cells. Also, nucleic acids and expression vectors encoding CARs. , recombinant cells containing the vector, and a gene expressing a GPRC5D-targeting CAR. Compositions comprising the engineered immune cells are also provided. Methods for making and using genetically engineered immune cells to treat conditions, including cancer A method for doing so is also provided. [Background technology]

[0003] T-cell therapy involves the use of single cells genetically modified to enhance their specificity for specific tumor-associated antigens. Genetic modification can be performed using chimeric antigen receptors (CARs) or exogenous T cells. The chimeric antigen receptor (CAR) can be used to express a chimeric antigen receptor (CAR) and provide new antigen specificity to T cells. T cells expressing CAR-T cells can induce tumor immune responses. There is a need for better T cell-based cancer therapies. Summary of the Invention [Means for solving the problem]

[0004] Disclosed herein are chimeric antigen receptors (CARs), e.g., G protein-coupled type receptor, G protein-coupled receptor family C group 5 member D (GPRC5D ), cells containing a CAR, a vector encoding a CAR (e.g., a recombinant expression vector) CARs, methods for producing CARs, and compositions that target nucleic acid molecules encoding CARs , polypeptides, proteins, nucleic acids, host cells, cell populations, and methods for using the disclosed CARs and methods for treating disorders (e.g., cancer) using the compounds of formula (I).

[0005] In one aspect, the present disclosure provides a method for detecting a cellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a transduction domain, wherein the extracellular antigen-binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 66, SEQ ID NO: 6 7, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 68. CDR3, Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 5 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 9, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 60 CDR3, Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 4 0, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 41. CDR3, heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 42, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 43 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 44, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 45; heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 45, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 46 and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 47, The extracellular antigen-binding domain binds to the GPRC5D antigen.

[0006] In one embodiment, The extracellular antigen-binding domain comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 66, a CDR2 having the sequence 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: 69, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 7 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 71. further comprising CDR3, The extracellular antigen-binding domain comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 58, a CDR2 having the sequence 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: 61; a light chain CDR1 having the amino acid sequence of SEQ ID NO: 6 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 63 further comprising CDR3, The extracellular antigen-binding domain comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 39, a CDR2 having the sequence 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: 48; a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 9, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 50; further comprising CDR3, The extracellular antigen-binding domain comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 42, a CDR2 having the sequence 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: 51; a light chain CDR1 having the amino acid sequence of SEQ ID NO: 5 a light chain CDR2 having the amino acid sequence of SEQ ID NO:2, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:53 further comprising a CDR3; or The extracellular antigen-binding domain comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 45, a CDR2 having the sequence 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: 54, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 5 a light chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:56 It further comprises CDR3.

[0007] In one embodiment, the extracellular antigen binding domain is selected from SEQ ID NOs: 73, 65, 1, 3, and 5. A light chain variable region comprising an amino acid sequence selected from the group consisting of: n, LCVR), or an amino acid selected from the group consisting of SEQ ID NOs: 72, 64, 2, 4, and 6 heavy chain variable region (HCVR) containing the amino acid sequence, or sequence LCVR comprising an amino acid sequence selected from the group consisting of numbers 73, 65, 1, 3, and 5. and an amino acid sequence selected from the group consisting of SEQ ID NOs: 72, 64, 2, 4, and 6. Including combination with HCVR.

[0008] 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 light chain variable region comprising the amino acid sequence of SEQ ID NO: 72; a heavy chain variable region comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 65 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64; a heavy chain variable region comprising 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 chain variable region, A light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 chain variable region, or 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 It contains the chain variable region.

[0009] In one embodiment, the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv comprises a linker between the light chain variable region and the heavy chain variable region. - polypeptides.

[0010] In one embodiment, the linker polypeptide comprises the amino acid sequence of SEQ ID NO:7.

[0011] In one embodiment, the scFv is selected from the group consisting of SEQ ID NOs: 78, 77, 75, 76, 8, 9, 10, 24 , 25, and 26.

[0012] In one embodiment, the extracellular antigen-binding domain comprises a signal polypeptide. In one embodiment, the signal polypeptide comprises the amino acid sequence of SEQ ID NO:11.

[0013] In one embodiment, the intracellular signaling domain is a TNF receptor superfamily member. member 9 (CD137) component, T cell surface glycoprotein CD3 zeta chain (CD3z) component , cluster of differentiation (CD27) component, cluster of differentiation superfamily member (e.g., CD28 or inducible T-cell co-stimulator (ICOS) The polypeptide component comprises a polypeptide component selected from the group consisting of:

[0014] In one embodiment, the CD137 component comprises the amino acid sequence of SEQ ID NO:12.

[0015] In one embodiment, the CD3z component comprises the amino acid sequence of SEQ ID NO:13.

[0016] In one embodiment, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 14. .

[0017] In one embodiment, the transmembrane domain is a CD8a transmembrane region (CD8A-TM) polypeptide. In some embodiments, the CD8a-TM polypeptide comprises the sequence of SEQ ID NO: 15. Contains the amino acid sequence.

[0018] In one embodiment, the CAR comprises a cysteine-binding domain linking a transmembrane domain to an extracellular antigen-binding domain. In some embodiments, the hinge region is a CD8a hinge region. In some embodiments, the CD8a hinge region comprises the amino acid sequence of SEQ ID NO: 16. .

[0019] In one embodiment, the extracellular antigen binding domain is selected from the group consisting of SEQ ID NOs: 82, 81, 80, 79, 17 , 18, 19, 20, 21, and 22.

[0020] In one embodiment, the CAR of the present disclosure is selected from the group consisting of SEQ ID NOs: 86, 85, 83, 84, 27, 28, The amino acid sequence is selected from the group consisting of 29, 30, 31 and 32.

[0021] In one aspect, the present disclosure provides an isolated lymphocyte expressing a CAR described herein. In some embodiments, the lymphocytes are T lymphocytes. In some embodiments, the T lymphocytes are naive 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 CD4+ and CD8+. Age 8+.

[0022] In one aspect, the present disclosure provides an isolated nucleic acid encoding any of the CARs described herein. In some embodiments, the nucleic acid molecule is selected from the group consisting of SEQ ID NOs: 90, 89, 87, 88, 33, 34, 35, 36, 37, and 38. In some embodiments, the isolated nucleic acid molecule is selected from the group consisting of SEQ ID NOs: 90, 89, 87, 88, 90, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, , 33, 34, 35, 36, 37 or 38 and at least about 80%, 8 1%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 9 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical In one embodiment, the disclosure provides a vector comprising the nucleic acid molecule. In one embodiment, the disclosure provides a cell that expresses the nucleic acid molecule.

[0023] In one aspect, the present disclosure provides a method for administering an effective amount of a phospholipase C1 receptor agonist (MPA) expressing one or more of the described CARs. Compositions, eg, pharmaceutical compositions, are provided that include the cells and a pharmaceutically acceptable excipient.

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

[0025] In one aspect, the present disclosure provides a lymphocyte according to the present disclosure for use in a method of treatment. In one aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, according to the present disclosure, for use in a method of treatment. The product is provided.

[0026] In one aspect, the present disclosure provides a CAR according to the present disclosure for use in a method of treating cancer. In one aspect, the present disclosure provides a method for treating cancer, comprising administering to a subject a lymphocyte according to the present disclosure. In one aspect, the present disclosure provides a composition according to the present disclosure for use in a method for treating cancer. In one embodiment, the cancer is 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, cholangiocarcinoma, hepatocellular carcinoma, sarcoma, solid tumors of squamous cell 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 Cancer, pancreatic cancer, glioma, glioblastoma, non-Hodgkin's lymphoma, NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (chronic lymphocytic leukemia, CLL), chronic myeloid leukemia (chronic myeloid leukemia) CML), smoldering multiple myeloma, SMM), multiple myeloma (MM), acute myeloid leukemia (MM), multiple myeloma (MM), acute myeloid leukemia leukemia, AML), and combinations thereof. In the method, the cancer to be treated is multiple myeloma.

[0027] In one aspect, the disclosure provides a method of treating a subject having cancer, the method comprising administering to a subject a therapeutically effective amount of a compound selected from the group consisting of benzodiazepines, ... and administering to a subject in need thereof a therapeutically effective amount of lymphocytes expressing one or more CARs. whereby the lymphocytes induce the death of cancer cells in the subject. In one embodiment, the cancer is bladder cancer, metastatic bladder cancer, esophageal cancer, non-small cell lung adenocarcinoma, non-small cell lung adenocarcinoma, non-small cell lung adenocarcinoma, or squamous cell carcinoma, prostate cancer, urothelial carcinoma, small cell lung cancer, endometrial cancer, cholangiocarcinoma, hepatocellular carcinoma, sarcoma, Solid tumors of squamous 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 lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) LL), chronic myeloid leukemia (CML), smoldering multiple myeloma (SMM), multiple myeloma selected from the group consisting of myeloid leukemia (MM), acute myeloid leukemia (AML), and combinations thereof In one embodiment, the cancer being treated in the subject is multiple myeloma.

[0028] In one aspect, a method for targeted killing of cancer cells is disclosed. The method comprises treating the cancer cells with a method as described above. with lymphocytes expressing one or more CARs, thereby The lymphocytes induce the death of cancer cells. In some embodiments, the cancer cells include lung cancer cells, stomach cancer cells, and the like. Cancer cells, colon cancer cells, hepatocellular carcinoma cells, renal cell carcinoma cells, bladder urothelial carcinoma 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 lymphoblastic leukemia (AL) cells L) cells, chronic lymphocytic leukemia (CLL) cells, chronic myeloid leukemia (CML) cells, SMM cells, MM cells, acute myeloid leukemia (AML) cells ML) cells, and combinations thereof. The cells are multiple myeloma cells. [Brief explanation of the drawings]

[0029] The foregoing will be apparent from the following more particular description of exemplary embodiments, as illustrated in the accompanying drawings. It will be. [Figure 1-1] Figures 1-1 and 1-2 show flow cytometry analysis of primary human pan-T cells electroporated with no mRNA (mock) or 10 μg of mRNA expressing either the α-GPRC5D scFv CAR or an isotype control CAR. 24 hours after electroporation, surface expression of CAR was measured by flow cytometry after staining with biotinylated L-protein and streptavidin-conjugated PE. The open histogram represents the mock, and the solid gray histogram represents the CAR-T population. [Figure 1-2] (As mentioned above.) [Figure 2-1] Figures 2-1 and 2-2 show flow cytometry analysis of transiently transfected pan T cells co-cultured with multiple myeloma cell lines. 24 hours after transient transfection, primary pan T cells were labeled with a fluorescent proliferation dye (Cell Trace Violet, CTV) and then co-cultured with the multiple myeloma cell line H929. After 4 days of co-culture, cells were pre-gated on the CD8+CD4 population, and surface expression of activation markers CD25 and CD71 on CAR-T cells was compared with T cells cultured alone or in the presence of α-CD3 / CD28 beads. [Figure 2-2] (As mentioned above.) [Figure 3]Cytokine profile analysis of primary pan T cells transiently transfected with CAR-expressing mRNA and co-cultured with various myeloma cell lines. Primary pan T cells transiently transfected with CAR-expressing mRNA 24 hours prior were co-cultured with various myeloma cell lines expressing the target antigen GPRC5D at a 1:1 ratio at high (+++), medium (++), low (+), or negative (-) levels. After 16 hours of co-culture, supernatants were collected and cytokine profile analysis of the supernatants from the co-cultures was performed using Meso Scale Discovery (MSD). [Figure 4-1] Figures 4-1 and 4-2 show cytokine profile analysis of primary pan T cells transiently transfected with CAR-expressing mRNA and cocultured with autologous peripheral blood mononuclear cells (PBMCs) and multiple myeloma cell lines. Twenty-four hours after transient transfection with CAR-expressing mRNA, primary pan T cells were cocultured with autologous PBMCs and the multiple myeloma cell line H929 (at a 1:1:1 ratio). After 16 hours of coculture, supernatants were collected. Cytokine profile analysis by MSD of supernatants from cocultures containing all three populations (CAR-T, H929, and autologous PBMCs) was compared with control cocultures without CAR-T cells, PBMCs, or H929, and with each population cultured alone. [Figure 4-2] (As mentioned above.) [Figure 5-1] Figures 5-1, 5-2, and 5-3 show flow cytometry analysis of the cytotoxic potential of CAR-T cells against GPRC5D-expressing myeloma cells. Primary pan-T cells (transiently expressing one of the three CARs described above) were cocultured with fluorescently labeled myeloma cell lines, MM1R, H929, and K562, at the indicated effector:target (E:T) ratios for 8 hours. At this time, the cocultures were stained with a viability dye. The mortality rate is the ratio of the absolute number of viable (viability dye-negative) target (CTV-positive) cells remaining in the coculture to the number of viable targets cultured without CAR-T cells. [Figure 5-2] (As mentioned above.) [Figure 5-3] (As mentioned above.) [Figure 6] Flow cytometry analysis of the cytotoxic potential of CAR-T cells against GPRC5D-expressing myeloma cells. Primary multiple myeloma patient T cells (transiently expressing one of the two CARs described above) were cocultured with fluorescently labeled myeloma cell line MM.1S or the control GPRC5d-negative cell line KG-1 at the indicated effector:target (E:T) ratio for 48 hours. At this time, the cocultures were stained with a viability dye. The cytotoxicity rate (indicating cell death) was determined as the percentage of fluorescently labeled cells that stained positive for the viability dye. [Figure 7-1] Figures 7-1, 7-2, and 7-3 show the results of flow cytometry analysis of the proliferative potential of α-GPRC5D-CAR-T cells. Fluorescently labeled pan-T cells (transiently expressing α-GPRC5D CAR or isotype CAR control) were cocultured with H929 at an E:T ratio of 1 for 4 days. Proliferation was measured as the absolute number of viable (viable dye-negative) CAR-T cells bearing the diluted fluorescent label (CTV). Proliferation in response to H929 was compared to CAR-T proliferation 4 days after α-CD3 / CD28 bead stimulation and to CAR-T cells cultured alone (no stimulation control). [Figure 7-2] (As mentioned above.) [Figure 7-3] (As mentioned above.) [Figure 8] 1 shows a bar graph depicting the percentage of GPRC5D-CAR expression in healthy donor T cells as measured by flow cytometry. Shaded gray bars represent background CAR detection in non-transduced (mock) cells. Black bars represent GPRC5D GC5B680-N68S-LH in transduced CAR-T cells. Values ​​shown represent the mean ± SD of six healthy donors. [Figure 9A-C]Figure 9 shows data demonstrating the expression of CD4, CD8, and memory markers on GPRC5D CAR-T cells. Bar graphs are shown, and the data collectively demonstrate that the GPRC5D GC5B680-N68S-LH CAR was detected by flow cytometry in both CD4 and CD8 T cells, and that the CD4 / CD8 ratios were not significantly different from the untransduced population. Figure 9A shows the percentage of the Mock (untransduced) cell population composed of CD8+ and CD4+ cells, respectively. Figures 9B-9C show the percentage of CAR-cells (transduced cells not expressing the GPRC5D CAR) composed of CD8+ and CD4+ cells, respectively, and the percentage of CAR+ cells (transduced cells expressing the GPRC5D CAR) composed of CD8+ and CD4+ cells, respectively. Values ​​shown in Figures 9A-9C represent the mean ± SD of six healthy donors. Values ​​shown in Figure 9B represent the mean ± SD of six healthy donors. [Figure 9D-F] Figure 9 shows data demonstrating the expression of CD4, CD8, and memory markers on GPRC5D CAR-T cells. Figure 9D shows a flow cytogram prepared from one donor, illustrating the gating strategy for the selection of the indicated distinct memory populations in the CAR-T population using surface markers (CD45RA and CD62L). Figures 9E and 9F show bar graphs demonstrating that GPRC5D GC5B680-N68S-LH was primarily expressed on TN / SCM and TCM, demonstrating increased proliferative capacity, survival, and therapeutic efficacy. Values ​​shown in Figures 9E-9F represent the mean ± SD of six healthy donors. [Figure 10A]Scatter plots showing GPRC5D CAR-T toxicity are shown. GPRC5D GC5B680-N68S-LH or non-transduced (Mock) T cells were added to GPRC5D+MM.1S or GPRC5D-K562 cells containing a luciferase transgene at various E:T ratios. CAR-Ts were generated from six healthy donors. Values ​​are expressed as the mean ± SD over 6 hours. Percent specific lysis was calculated by (i) dividing the luciferase signal in tumor cells in the presence of CAR-T for 6 or 24 hours by the luciferase signal in tumor cells at the same time point, multiplying by 100, and (ii) subtracting that number from 100. The formula is 100 - [(CAR-T+tumor luminescence / mean tumor-only luminescence) × 100]. The dotted line represents a specific lysis rate equal to zero. Negative lysis indicates cell growth. [Figure 10B] Scatter plots showing GPRC5D CAR-T toxicity are shown. GPRC5D GC5B680-N68S-LH or non-transduced (Mock) T cells were added to GPRC5D+MM.1S or GPRC5D-K562 cells containing a luciferase transgene at various E:T ratios. CAR-Ts were generated from six healthy donors. Values ​​are expressed as the 24-hour mean ± SD. Percent specific lysis was calculated by (i) dividing the luciferase signal in tumor cells in the presence of CAR-T for 6 or 24 hours by the luciferase signal in tumor cells at the same time point, multiplying by 100, and (ii) subtracting that number from 100. The formula is 100 - [(CAR-T+tumor luminescence / mean tumor-only luminescence) × 100]. The dotted line represents a specific lysis rate equal to zero. Negative lysis indicates cell growth. [Figure 11A] Scatter plots showing that the cytotoxicity of GPRC5D CAR-T cells added at the indicated E:T ratios was assessed using GPRC5D+H929, MM.1S, MM.1R, MOLP-2, and EJM cells or GPRC5D-K562 cells. GPRC5D CAR-T cells exhibited cytotoxicity against all GPRC5D+ cell lines. [Figure 11B]Scatter plots showing that the cytotoxicity of GPRC5D CAR-T cells added at the indicated E:T ratios was assessed using GPRC5D+H929, MM.1S, MM.1R, MOLP-2, and EJM cells or GPRC5D-K562 cells. GPRC5D CAR-T cells exhibited cytotoxicity against all GPRC5D+ cell lines. [Figure 11C] Scatter plots showing that the cytotoxicity of GPRC5D CAR-T cells added at the indicated E:T ratios was assessed using GPRC5D+H929, MM.1S, MM.1R, MOLP-2, and EJM cells or GPRC5D-K562 cells. GPRC5D CAR-T cells exhibited cytotoxicity against all GPRC5D+ cell lines. [Figure 11D] Scatter plots showing that the cytotoxicity of GPRC5D CAR-T cells added at the indicated E:T ratios was assessed using GPRC5D+H929, MM.1S, MM.1R, MOLP-2, and EJM cells or GPRC5D-K562 cells. GPRC5D CAR-T cells exhibited cytotoxicity against all GPRC5D+ cell lines. [Figure 11E] Scatter plots showing that the cytotoxicity of GPRC5D CAR-T cells added at the indicated E:T ratios was assessed using GPRC5D+H929, MM.1S, MM.1R, MOLP-2, and EJM cells or GPRC5D-K562 cells. GPRC5D CAR-T cells exhibited cytotoxicity against all GPRC5D+ cell lines. [Figure 11F] Scatter plots showing that the cytotoxicity of GPRC5D CAR-T cells added at the indicated E:T ratios was assessed using GPRC5D+H929, MM.1S, MM.1R, MOLP-2, and EJM cells or GPRC5D-K562 cells. GPRC5D CAR-T cells exhibited cytotoxicity against all GPRC5D+ cell lines. [Figure 12]

[0023] Figure 1 shows a bar graph depicting surface CD25 expression levels (%CD25) by non-transduced (mock) or GC5B680-N68S-LH cells in the presence of K562 cells at an E:T ratio of 3:1. CAR-Ts were generated from six healthy donors. Data are presented as mean ± SD. [Figure 13-1] Figures 13-1, 13-2, and 13-3 show flow cytograms demonstrating that GC5B680-N68S-LH specifically proliferates in the presence of GPRC5D+ target cells (H929 or MM.1S) or a positive control (agonist CD3 / CD28 beads), but not 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+ events were detected using an anti-idiotypic antibody (Y-axis). [Figure 13-2] (As mentioned above.) [Figure 13-3] (As mentioned above.) [Figure 14] 1 is a bar graph showing no change in cleaved caspase 3 / 7 levels in CAR-T (GC5B680-N68S-LH) cells incubated with the GPRC5D+ cell line H929 for 24 hours compared to controls (staurosporine, GPRC5D-K562 cells, or alone). An E:T ratio of 1:2 was used for H929 and K562 cells. CAR- represents transduced cells that do not express the GPRC5D CAR. CAR+ represents transduced cells that express the GPRC5D CAR. [Figure 15]1 shows a graph demonstrating the effect of GPRC5D-directed CAR-T cells on MM.1S human MM xenografts established in NSG-B2M mice, according to Example 15. GC5B680-N68S-LH, GC5B680-N68S-HL, GC5B83-N24T-N31S-LH, and GC5B83-N24T-N31S-HL, GPRC5D-directed CAR-T; Mock, non-transduced CAR-T; PBS, phosphate-buffered saline; NSG, non-obese diabetic severe combined immunodeficiency gamma; B2M, beta-2 microglobulin; MM, multiple myeloma. Group tumor volumes are graphed as mean ± SEM. Tumor cells were implanted on day 0, and 1 × 10 CAR+T cells were implanted on day 13. The symbol "*" indicates a significant difference from the PBS control on day 26 (p≦0.05, n=10 / group). [Figure 16] 1 shows a graph demonstrating the effect of GC5B680-N68S-LH on survival of NSG mice bearing disseminated H929 human MM xenografts, according to Example 16. GC5B680-N68S-LH, GC5B680-N68S-HL, GC5B83-N24T-N31S-LH, and GC5B83-N24T-N31S-HL, GPRC5D-directed CAR-T; Mock; non-transduced CAR-T; PBS; phosphate-buffered saline; NSG; non-obese diabetic severe combined immunodeficiency gamma; MM; multiple myeloma. Tumor cells were implanted on day 0. 1×10 CAR+ T cells were implanted on day 9. The symbol "*" indicates a significant difference from the PBS control on day 111. DETAILED DESCRIPTION OF THE INVENTION

[0030] A description of exemplary embodiments follows.

[0031] The present disclosure relates to a G protein-coupled receptor, G protein-coupled receptor family C group Chimeric antigen receptors (CARs) targeting 5 member D (GPRC5D), such CARs and cancers (e.g., hematological malignancies and solid tumors) using the CARs described herein. The present invention provides a method for treating cancer (tumor-like tumors).

[0032] The CAR of the present invention is a human CAR having the amino acid sequence of GPRC5D (e.g., SEQ ID NO: 57). GPRC5D polypeptide or a fragment thereof). As used herein, the phrases "having antigen specificity" and "eliciting an antigen-specific response" The CAR specifically binds to the antigen and can immunologically recognize the antigen, resulting in This means that the binding of CAR to the GPRC5D antigen induces an immune response. Methods for testing CARs for their activity and ability to recognize target cells are known in the art. There are.

[0033] The disclosure also relates to related nucleic acids, recombinant expression vectors, host cells, cell populations, antibodies, or also provide antigen-binding portions thereof, and pharmaceutical compositions relating to the CARs of the invention.

[0034] Some aspects of the present invention are described below with reference to examples, which are for illustrative purposes only. Numerous specific details, relationships and methods are set forth in order to provide a thorough understanding of the present invention. However, it will be understood by those skilled in the art that the present invention may be practiced without the aid of specific details. It may be possible to carry out the method without one or more of these, or to use other methods, protocols, or It will be readily apparent that the present invention can be practiced with any number of reagents, cell lines, and animals. Some actions may occur in different orders and / or simultaneously with other actions or events. The present invention is not limited by the illustrated acts or order of events. Not all illustrated acts, steps, or events are required to implement a methodology. Many of the techniques and procedures described or referenced herein are within the skill of the art. It is therefore well understood and commonly used using conventional techniques.

[0035] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein or technical terms have the meanings commonly understood by those skilled in the art to which the present invention pertains. In some cases, terms with their commonly understood meanings have been omitted for the sake of clarity and and / or are defined herein for ready reference and include such definitions herein. The inclusion of a Terms as defined in commonly used dictionaries are not to be construed as meaningless or unrelated terms. shall have a meaning consistent with their meaning in the context of the art and / or this specification. It is further understood that the terms and conditions of the present application must be interpreted as being otherwise defined in the present application. There will be.

[0036] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, unless the context clearly indicates otherwise, The articles "a," "an," and "the" should be understood to include plural referents.

[0037] Chimeric Antigen Receptor The present invention generally relates to a method for producing a chimeric antigen receptor (CAGR) comprising: The present invention relates to the use of T cells containing chimeric antigen receptors (CARs) that are capable of expressing T cell signaling domains. An artificially constructed hybrid containing the antigen-binding domain of an antibody (scFv) bound to CARs are lid proteins or polypeptides. The properties of CARs include selection in a non-MHC restricted manner. Redirecting T cell specificity and reactivity towards targeted targets and This may include the ability to utilize antigen-binding properties to express CARs through non-MHC-restricted antigen recognition. T cells that recognize antigens independently of antigen processing can be used to treat tumors. Furthermore, when expressed in T cells, CARs bypass the primary mechanism of immune evasion. Favourably dimerises with the T cell receptor (TCR) alpha and beta chains do not.

[0038] The CARs described herein comprise at least an extracellular antigen-binding domain, a transmembrane domain, and , an intracellular signaling molecule comprising a functional signaling domain derived from a stimulatory molecule as defined below. and a cytoplasmic signaling domain (also referred to herein as a "cytoplasmic signaling domain"). A recombinant polypeptide construct comprising a CAR is provided herein. A T cell expressing a CAR is defined herein as comprising: These are referred to as CAR T cells, CAR-T cells, or CAR-modified T cells, and these terms are used interchangeably in the present specification. The terms are used interchangeably herein. Cells stably display antibody binding domains on their surfaces. can be genetically engineered to express novel antigen specificities that are MHC-independent .

[0039] In some cases, the antigen recognition domain of a specific antibody is linked to the CD3-zeta chain or FcγRI chain. The CAR is then combined with the intracellular domain of the target protein to form a single chimeric protein. In one embodiment, the T cells are genetically modified to express a stimulatory molecule. The zeta chain is associated with the cytoplasmic receptor complex.

[0040] "Intracellular signaling domain," as the term is used herein, refers to a molecule This refers to the intracellular part of a protein. This is the functional part of the protein that transmits second messengers. to regulate cellular activities through defined signaling pathways by generating It acts by transmitting information within the The intracellular signaling domain acts by functioning as a receptor effector. Produces signals that promote immune effector functions of AR-containing cells (e.g., CAR-T cells) For example, the immune effector functions of CAR-T cells include cytolytic activity. and helper activity (including cytokine secretion).

[0041] In one embodiment, the intracellular signaling domain comprises a primary intracellular signaling domain. Examples of primary intracellular signaling domains include primary stimulation or antigen-dependent signaling domains. In one embodiment, the signaling pathway is derived from a molecule involved in intracellular signaling. The core transduction domain may comprise a costimulatory intracellular domain. Examples of genes include those derived from molecules involved in costimulatory signals or antigen-independent stimulation. For example, in the case of CAR-T, the primary intracellular signaling domain is a T cell The co-stimulatory intracellular signaling domain may comprise the cytoplasmic sequence of a co-receptor or co-stimulatory domain. It may contain cytoplasmic sequences from the cytoplasmic to the cytoplasmic.

[0042] The primary intracellular signaling domain is an immunoreceptor tyrosine-based activation motif or It may contain signaling motifs known as ITAMs. Primary cytoplasmic signaling sequences Examples of ITAMs containing α-aminomethyl-2-methyl-1 ... 3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79 b, and CD66d derived from DAP10 and DAP12, Not limited to:

[0043] Primary intracellular signaling domains include, for example, CD3-zeta, CD3 epsilon, C D22, CD79a, CD66d, CD39, DAP10, DAP12, Fc epsilon Receptor I gamma chain (FCER1G), FcR beta, CD3 delta, CD3 gamma, CD 5, CD226, or CD79B signaling domains.

[0044] "Zeta" or alternatively "Zeta chain", "CD3-zeta" or "TCR-zeta" The term is provided as GenBank accession number BAG36664.1 Proteins, or non-human species, e.g., murine, rabbit, primate, mouse, rodent, monkey , and the equivalent residues from apes, etc., are defined as the "zeta stimulatory domain" or alternatively " The "CD3-zeta stimulatory domain" or "TCR-zeta stimulatory domain" is involved in T cell activation. The access from the cytoplasmic domain of the zeta chain is sufficient to functionally transmit the necessary initial signal. In one embodiment, the cytoplasmic domain of Zeta is defined as a nucleotide sequence of amino acid residues. Residues 52 to 164 of accession number BAG36664.1, or a functional ortholog thereof equivalent residues from non-human species, e.g., mice, rodents, monkeys, apes, etc. Contains a group.

[0045] In a preferred embodiment, the intracellular signaling domain comprises a CD3 zeta stimulatory domain. In one embodiment, the "zeta stimulatory domain" or "CD3 zeta stimulatory domain" comprises the sequence The sequence is provided as number 13.

[0046] The term "costimulatory molecule" refers to the cognate binding partner on a T cell, a costimulatory ligand by specifically binding to, and thereby inducing a costimulatory response by T cells (for example, but not limited to, Costimulatory molecules mediate the proliferation of antigen receptors or their receptors, which are necessary for an efficient immune response. These are cell surface molecules other than ligands. Costimulatory molecules include MHC class 1 molecules and BTL A and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, and CDS , ICAM-1, LFA-1(CD11a / CD18), 4-1BB(CD137), C D30, CD40, CD54(ICAM), CD83, CD134(OX40), CD1 50 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD27 0(HVEM), CD278(ICOS), DAP10, LAT, NKD2C SLP7 6, TRIM, BTLA, GITR, CD226, HVEM, and ZAP70. However, it is not limited to these.

[0047] The costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. The children are involved in the following protein families: TNF receptor proteins, immunoglobulins, Cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Examples include CD27, CD28, 4-1BB (CD137), OX40, GITR, and C D30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LF A-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, B7-H3, and ligands that specifically bind to CD83.

[0048] An intracellular signaling domain may refer to the entire intracellular portion of the molecule from which it is derived, or to the portion of the molecule from which it is derived. It may comprise the entire intracellular signaling domain, or a functional fragment thereof.

[0049] The term "4-1BB" is derived from GenBank accession number AAA62478. a member of the TNFR superfamily having the amino acid sequence provided as in 2, or Refers to equivalent residues in non-human species, e.g., mice, rodents, monkeys, apes, etc., and is referred to as "4-1 The BB costimulatory domain is identified by the GenBank accession number AAA62478.2. amino acid residues 214-255, or non-human species, e.g., mouse, rodent, monkey, and subhuman It is defined as the equivalent residue derived from monkeys etc.

[0050] In one embodiment, the "4-1BB costimulatory domain" comprises the sequence provided as SEQ ID NO: 12: or equivalent residues from non-human species, such as mice, rodents, monkeys, and apes. is.

[0051] In some embodiments, the cytoplasmic signaling domain is at least one of the nucleotides defined herein. and (iii) adding one or more functional signaling domains derived from at least one costimulatory molecule. In one embodiment, the costimulatory molecule includes 4-1BB (i.e., CD137), CD2 7, CD3-zeta and / or CD28. CD28 is involved in T cell costimulation. CD27 is an important T cell marker for tumor necrosis factor receptor superfamily. 4-1BB is a member of the T Delivering a potent costimulatory signal to cells, promoting differentiation and enhancing long-term survival of T lymphocytes CD3-zeta associates with TCR to generate signals and activates the immune receptor tyrosine immunoreceptor tyrosine-based activation motifs (ITAM) ) contains.

[0052] In a preferred embodiment, the intracellular signaling domain is a costimulatory intracellular signaling domain. The co-stimulatory intracellular signaling domain is the 4-1BB co-stimulatory domain. In a preferred embodiment, the 4-1BB costimulatory domain is provided as SEQ ID NO: 12. It is an array.

[0053] In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used. In another embodiment, the transmembrane domain is selected or modified by amino acid substitution to avoiding binding of such domains to transmembrane domains of the same or different surface membrane proteins, Interactions with other members of the receptor complex can be minimized. In this embodiment, the transmembrane domain is a CD8α transmembrane domain.

[0054] In one embodiment, the CAR comprises a hinge domain comprising a CD8α hinge domain.

[0055] In one embodiment, the CAR comprises a CD8α hinge domain and a CD8α transmembrane domain. nothing.

[0056] In one embodiment, the CAR comprises a hinge domain comprising a CD8α hinge domain and a CD2 8, 4-1BB, and CD3-zeta.

[0057] The CARs described herein comprise at least an extracellular antigen-binding domain, a transmembrane domain, and , e.g., an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule as defined below. a signaling domain (also referred to herein as a "cytoplasmic signaling domain"); and a recombinant polypeptide construct comprising:

[0058] In one embodiment, a CAR comprises an extracellular antigen recognition domain, a transmembrane domain, and a stimulatory molecule an intracellular signaling domain comprising a functional signaling domain derived from In one embodiment, the CAR comprises an extracellular antigen recognition domain and a membrane fusion protein. Transmembrane domain and functional signaling domain derived from costimulatory molecules and stimulatory molecules and a chimeric fusion protein comprising an intracellular signaling domain containing a functional signaling domain that In one embodiment, the CAR comprises an extracellular antigen recognition domain and a transmembrane domain. and at least two functional signal transduction pathways derived from one or more costimulatory molecules. Intracellular signal transduction domains containing functional signal transduction domains derived from stimulatory molecules. and a chimeric fusion protein comprising a target domain.

[0059] The CAR of the present invention may contain CD28 and / or 4-1BB signaling domains. or any other desired cytoplasmic domain useful in the context of the CAR of the present invention. In one embodiment, the cytoplasmic domain of the CAR can be designed to be combined with The CAR may further comprise a signaling domain of CD3-zeta. The signal transduction domains include CD3-zeta, 4-1BB, and CD28 signaling modules. These may include, but are not limited to, tetracycline ...

[0060] In a preferred embodiment, the CAR comprises a CD8α hinge domain and a CD8α transmembrane domain. and an intracellular signaling domain containing the signaling domain of CD3-zeta. include.

[0061] In a preferred embodiment, the CAR comprises a CD8α hinge domain and a CD8α transmembrane domain. and cells containing the signaling domain of CD3-zeta and the 4-1BB costimulatory domain. and an inner signaling domain.

[0062] Thus, the present invention provides CAR T cells and methods for their use for adoptive therapy. Provide.

[0063] The present disclosure provides variants of the CARs, nucleic acids, polypeptides, and proteins described herein ( "Variants" include, for example, substitutions, insertions, or deletions. A reference polypeptide or polynucleotide may differ from a reference polypeptide or polynucleotide by one or more modifications. As used herein, "functional mutation" refers to a polypeptide or polynucleotide that is The term "antibody" refers to a polypeptide that has substantial or significant sequence identity with the parent CAR, polypeptide, or protein. It refers to a CAR, polypeptide, or protein that has the same or similar properties as the CAR, and their functional The variants retain the biological activity of the CAR, polypeptide, or protein from which they are variant. Functional variants include, for example, variants of a CAR, polypeptide, or protein (parent CAR, polypeptide, or protein), and includes variants of the parent CAR, polypeptide, or retains the ability to recognize target cells to about the same extent, the same extent, or a greater extent than the protein. With respect to the parent CAR, polypeptide, or protein, a functional variant may be, for example, At least about 30% amino acid sequence identity to the parent CAR, polypeptide, or protein , 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 may be identical.

[0064] As used herein, the structure of a polypeptide is represented by a recited reference sequence (having a given SEQ ID NO: ) at a location defined based on the % sequence identity with the The % sequence identity between sequences is calculated by comparing these two sequences aligned in an optimal way. The amino acid sequences to be compared can be determined by comparing these two sequences The sequence may contain additions or deletions relative to the reference sequence for optimal alignment between the two sequences. The percentage of identity determines the number of identical positions where the amino acid residues between the two sequences are identical. Divide the number of identical positions by the total number of positions in the comparison window and multiply the result by 1. 00 to obtain the percent identity between the two sequences. Typically, the comparison window corresponds to the entire length of the sequences being compared. ,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) This is possible at http: / / www.ncbi.nlm.nih.gov / go rf / bl2.html and the parameters used are the defaults (specifically Specifically, the parameters "open gap penalty": 5 and "extension gap": "Top penalty": 2; the selected matrix is, for example, the one proposed by the program are parameters specified in the matrix "BLOSUM 62" that is used to compare The percentage of identity between the two sequences is calculated directly by the program. Determining the sequence identity of a reference sequence is within the capabilities of one skilled in the art and can be performed using the BLAS This can be performed using commercially available analysis software such as TM.

[0065] Functional variants include, for example, a parent CAR, poly(A) having at least one conservative amino acid substitution. In another embodiment, the amino acid sequence of a functional variant may be a polypeptide or a protein. The subject is a parent CAR, polypeptide, or target polypeptide having at least one non-conservative amino acid substitution. In this case, non-conservative amino acid substitutions may be considered functional variants. Non-conservative amino acid substitutions may not reduce or inhibit the biological activity of the The biological activity of functional variants can be improved by increasing the biological activity of the parent CAR. , polypeptide, or protein.

[0066] The amino acid substitutions in the CAR of the present invention can be conservative amino acid substitutions. Substitutes are known in the art and are compounds that have particular physical and / or chemical properties. Replacing one amino acid with another amino acid that has the same or similar chemical or physical properties For example, a conservative amino acid substitution would be one in which an amino acid is replaced with another acidic amino acid. a polar amino acid (e.g., Asp or Glu), substituted with another amino acid having a nonpolar side chain Amino acids with nonpolar side chains (e.g., Ala, Gly, Val, Ile, Leu, M et, Phe, Pro, Trp, Val, etc.), basic amino acids replaced with other basic amino acids Amino acids (e.g., Lys, Arg), polar side chains replaced with another amino acid with a polar side chain and amino acids having the formula (Asn, Cys, Gln, Ser, Thr, Tyr, etc.). do.

[0067] A CAR, polypeptide, or protein may be a specific amino acid sequence or a polypeptide as described herein. The amino acid sequence may consist essentially of the amino acid sequence, so that other components, e.g., other amino acids, may not function. The biological activity of the target mutant is not substantially altered.

[0068] CARs, polypeptides, and proteins (including functional portions and functional modifications) of the disclosed embodiments The amino acid sequence (including variants) may be of any length, i.e., may contain any number of amino acids, except and the condition is that the CAR, polypeptide, or protein (or functional portion thereof) functional variants) retain their biological activity, e.g., the ability to specifically bind to an antigen, The ability to detect diseased cells (e.g., cancer cells) or to treat or prevent disease in a host For example, a polypeptide may have a length of about 50 to about 5,000 amino acids. acid length, for example, about 50, about 70, about 75, about 100, about 125, about 150, about 175, Approximately 200, approximately 225, approximately 250, approximately 275, approximately 300, approximately 325, approximately 350, approximately 375, Approximately 400, approximately 425, approximately 450, approximately 475, approximately 500, approximately 525, approximately 550, approximately 575, Approximately 600, approximately 625, approximately 650, approximately 675, approximately 700, approximately 725, approximately 750, approximately 775, Approximately 800, approximately 825, approximately 850, approximately 875, approximately 900, approximately 925, approximately 950, approximately 975, The polypeptides of the invention can also be about 1000 or more amino acids in length. Also includes oligopeptides.

[0069] CARs, polypeptides, and proteins of the present invention (including functional moieties and A nucleotide sequence (including functional variants) is a sequence that substitutes a synthetic amino acid for one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and may include: Examples include aminocyclohexanecarboxylic acid, norleucine, and α-amino n-decanoic acid. , homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans- 4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β- Diaminopropionic acid, homophenylalanine, 4-chlorophenylalanine, 4-cal β-phenylserine β-hydroxyphenylalanine, phenyl Cyclohexylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine , N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxy- Ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclopentane Xanthanecarboxylic acid, α-aminocycloheptanecarboxylic acid, indoline-2-carboxylic acid , 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminoaldonic acid, aminoaldonic acid monoamide, and α-tert-butylglycine.

[0070] CARs, polypeptides, and proteins (functional portions and functional modifications) of the present embodiments Proteins (including variants) can undergo post-translational modifications. These include glycosylation, esterification, and N-acylation, amidation, carboxylation, phosphorylation, esterification, e.g., disulfide It can be cyclized via a hydroxyl bridge or converted into an acid addition salt. In form, they are dimerized or polymerized, or conjugated.

[0071] CARs, polypeptides, and / or proteins (including functional portions thereof) of the present invention (including functional variants of) can be obtained by methods known in the art. Suitable methods for de novo synthesis of polypeptides and proteins can be found in the literature, e.g., Cha n et al., Fmoc Solid Phase Peptide Synthesis sis,Oxford University Press,Oxford,Unite d Kingdom,2000;Peptide and Protein Drug Analysis,ed.Reid,R.,Marcel Dekker,Inc.,2 000;and Epitope Mapping,ed.Westwood et a l.,Oxford University Press,Oxford,United Kingdom, 2001. Polypeptides and proteins are also described in Recombinantly produced using the nucleic acids described herein using standard recombinant methods. See, e.g., Sambrook et al., Molecular Cloning ng:A Laboratory Manual,3rd ed.,Cold Spri ng Harbor Press, Cold Spring Harbor, NY2 001; and Ausubel et al.,Current Protocols i n Molecular Biology, Greene Publishing As sociates and John Wiley & Sons, NY, 1994. Further, the CARs, polypeptides, and proteins of the present invention (including functional portions and Some of these proteins (including functional variants thereof) have been isolated from sources such as plants, bacteria, insects, and mammals. and / or purified. Methods for isolation and purification are known in the art. Alternatively, the CARs, polypeptides, and / or proteins (functional In this regard, the target compounds (including target moieties and functional variants thereof) can be commercially synthesized. Therefore, CARs, polypeptides, and proteins may be synthesized, recombinant, isolated, and / or purified. It is possible.

[0072] To generate the recombinant nucleic acids utilized to generate the polypeptides described herein, Examples of modified nucleotides that can be used for this purpose include 5-fluorouracil, 5- Bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, carboxy Methylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, Dihydrouracil, N 6 -substituted adenine, 7-methylguanine, 5-methylaminomethyl Uracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylketone uracil, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthiouracil O-N 6 -Isopentenyladenine, uracil-5-oxyacetic acid (v), wibutoxoxine wybutoxosine, pseudouracil, queosine, beta-D-galactosyl queosine Inosine, N 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methyl Cytosine, 5-methylcytosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2 -Thiouracil, 4-Thiouracil, 5-Methyluracil, Uracil-5-oxyacetate ethyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2 ,6-diaminopurine.

[0073] The nucleic acid may be a CAR, a polypeptide or protein, or a functional portion or function thereof. The present invention may include any isolated or purified nucleotide sequence encoding any of the target variants. Alternatively, the nucleotide sequence may be a nucleotide sequence degenerate to any of the sequences, or It may include a combination of degenerate sequences.

[0074] Some embodiments of the present invention also provide isolated or purified nucleic acids, the nucleic acids comprising the A nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein. or any of the nucleotide sequences of the nucleic acids described herein under stringent conditions. The term "nucleotide sequence" includes a nucleotide sequence that hybridizes under certain conditions.

[0075] Nucleotide sequences that hybridize under stringent conditions are called highly stringent. "High stringency conditions" refer to conditions under which the nucleotide sequence can hybridize. The sequence is capable of detecting the target sequence (as defined herein) in a detectable amount superior to non-specific hybridization. specifically hybridize to any of the nucleotide sequences of the nucleic acids described in High stringency conditions are defined as conditions where a polynucleotide having an exactly complementary sequence is used. A polynucleotide containing only a few scattered mismatches is called a nucleobase. Random sequences that happen to have a few small regions (e.g., 3-12 bases) that match the base sequence. The complementary subregions may be identified by the sequence. It melts more easily than the full-length complement of 1 base, and is more suitable for high stringency hybridization. These conditions are relatively high in stringency. For example, low salt and / or high temperature conditions (about 0.02 to 0.1 M at a temperature of about 50 to 70°C) Such high stringency may be achieved by providing a solution containing 100% NaCl or equivalent. The conditions of operation are rather the relationship between the nucleotide sequence and the template strand or the target strand. mismatches are tolerated to a small extent and are useful for detecting expression of any of the CARs described herein. Conditions can be made more stringent by the addition of increasing amounts of formamide. It is generally understood that this can be a gent.

[0076] In one embodiment, the nucleic acids of the invention can be incorporated into a recombinant expression vector. The disclosure provides recombinant expression vectors comprising any of the nucleic acids of the invention. When used, the term "recombinant expression vector" refers to a vector that expresses a recombinant gene, such as a recombinant human or animal, in a manner similar to that described above. Genetically modified oligonucleotides that allow expression of proteins, polypeptides, or peptides. The term "protein" refers to a nucleic acid or polynucleotide construct, in which the construct may be an mRNA, protein, or a nucleotide sequence encoding a protein, polypeptide, or peptide, sufficient to express mRNA, protein, polypeptide, or peptide in a cell The vectors described herein are not naturally occurring in their entirety. However, some of the vectors may be naturally occurring. Current vectors include DNA and RNA (which can be single-stranded or double-stranded, synthetic or partially may be derived from natural sources and may contain natural, non-natural or modified nucleotides It may contain any type of nucleotide, including but not limited to: A vector may contain naturally occurring internucleotide linkages or non-naturally occurring nucleotide linkages. The non-naturally occurring or modified nucleotides may contain inter-nucleotide bonds, inter-nucleotide bonds, or both types of bonds. The nucleotide or internucleotide bond does not inhibit transcription or replication of the vector.

[0077] In one embodiment, the recombinant expression vector of the present invention is any suitable recombinant expression vector. and can be used to transform or transfect any suitable host. Suitable vectors include plasmids and viruses, which are suitable for propagation and multiplication. Vectors include vectors designed for transcription or expression, or both. UC Series (Fermentas Life Sciences, Glen Burn i.e., Md.), pBluescript series (Stratagene, LaJol la, Calif.), pET series (Novagen, Madison, Wis.) , pGEX series (Pharmacia Biotech, Uppsala, Sweden) en), and pEX series (Clontech, Palo Alto, Calif.) λGT10, λGT11, λEMBL4, and λNM11 49, using bacteriophage vectors such as λZapII (Stratagene). Examples of plant expression vectors include pBI01, pBI01.2, pB Examples include I121, pBI101.3, and pBIN19 (Clontech). Examples of expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clo Recombinant expression vectors include viral vectors, e.g., retroviral vectors. The vector may be a retroviral vector, for example a gammaretroviral vector.

[0078] In one embodiment, the recombinant expression vector of the invention is a vector of the type described, for example, in Sambrook, supra. et al. (supra) and Ausubel et al. (supra). The expression vector constructs, which may be circular or linear, are prepared using recombinant DNA techniques. It can be prepared to contain a replication system that functions in a prokaryotic or eukaryotic host cell. The vectors are derived from, for example, ColEl, SV40, 2μ plasmid, λ, bovine papilloma virus, etc. It can come.

[0079] The recombinant expression vector may be DNA-based or RNA-based, as appropriate. The type of host into which the vector will be introduced (e.g., bacterial, plant, fungal, or animal) will be considered. The gene may include regulatory sequences, such as transcription and translation initiation and termination codons, specific for the target gene (e.g., a target gene).

[0080] Recombinant expression vectors allow for the selection of transformed or transfected hosts. The marker gene may contain one or more marker genes that confer biocide resistance. (e.g., resistance to antibiotics, heavy metals, etc.), providing prototrophy in auxotrophic hosts Suitable marker genes for the described expression vectors include: For example, neomycin / G418 resistance gene, histidinol x resistance gene, histidinol Examples of genes that can be used include the steroid resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene. do.

[0081] The recombinant expression vector can express a CAR, polypeptide, or protein (including functional portions and or to a nucleotide sequence encoding a CAR, polypeptide (including functional variants thereof) or a nucleotide sequence encoding a gene or protein A native or standard promoter operably linked to the hybridizing nucleotide sequence. The promoter selection (e.g., strong, weak, tissue-specific, inducible, and The designation of a nucleotide sequence as a promoter (e.g., a gene encoding a gene for ... It is within the skill of one in the art to combine a promoter with a non-viral promoter. Motor or viral promoters, e.g., cytomegalovirus , CMV) promoter, RSV promoter, SV40 promoter, or murine stem cell The promoter may be one found in the long terminal repeats of yeast viruses.

[0082] Recombinant expression vectors can be used for transient expression, stable expression, or both. Recombinant expression vectors can also be designed for constitutive or inducible expression. can be made.

[0083] Additionally, recombinant expression vectors can be engineered to contain a suicide gene. When used, the term "suicide gene" refers to a gene that causes the death of a cell that expresses the suicide gene. A suicide gene is a gene that makes cells that express the gene resistant to an agent (e.g., a drug). These drugs confer susceptibility to certain diseases, leading to cell death when the cells come into contact with or are exposed to the drug. Suicide genes are known in the art and can be, for example, Herpes Simplex Virus (HSV) thymidine kinase kinase (TK) genes, cytosine deaminase, purine nucleoside phosphorylase, and Nitroreductase is an example.

[0084] Within the scope of the present invention, CARs, polypeptides, or proteins (including functional parts thereof) are host cells, nucleic acids, recombinant expression vectors, host cell populations, or is a conjugate (e.g., Conjugates and methods for synthesizing conjugates are also included. Methods are generally known in the art (e.g., Hudecz, F., Meth. ods Mol.Biol.298:209-223(2005)and Kirin et al., Inorg Chem.44(15):5405-5415(2005) (See

[0085] One embodiment of the present invention is a CAR that binds, e.g., specifically binds, to an epitope of the CAR of the present invention. Further provided is an antibody or antigen-binding portion thereof that

[0086] The antibody can be any type of immunoglobulin known in the art. Globulins are divided into five major classes: IgA, IgD, IgE, IgG, and IgM. IgA and IgG can be assigned isotypes IgA1, IgA2, IgG1, Ig The antibody light chains of vertebrate species are also classified as IgG1, IgG2, IgG3, and IgG4. Based on the amino acid sequences of their constant domains, two types of antibodies, kappa (κ) and lambda (λ), were identified. Antibodies can be of any class or isotype. .

[0087] The antibodies may be monoclonal antibodies, including murine, human, humanized, and chimeric monoclonal antibodies. antibodies, polyclonal, antigen-binding fragments, bispecific or multispecific antibodies, monomers, Dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies, as well as the specificity required immunoglobulin molecules of any other modified configuration containing the antigen binding site of Antibodies include naturally occurring antibodies, such as antibodies from mammals (e.g., mice). isolated from various animals (e.g., primates, mice, rabbits, goats, horses, chickens, hamsters, humans, etc.) and / or purified antibodies. Alternatively, the antibodies may be genetically engineered (e.g., The antibody may be a genetically engineered antibody.

[0088] A "human antibody" is an antibody that is optimized to evoke a minimal immune response when administered to a human subject. Human antibodies refer to antibodies that have been engineered to be human. The variable regions of a human antibody are derived from human immunoglobulin sequences. When the human antibody contains a constant region or a part of a constant region, the constant region is also a human immunoglobulin sequence. A human antibody is one in which the variable regions of the human antibody are derived from human germline immunoglobulins or When obtained from a system that uses rearranged immunoglobulin genes, sequences of human origin are marked with " Such an exemplary system includes heavy and light chain variable regions derived from phage. Splayed human immunoglobulin gene libraries and human immunoglobulin gene loci "Human antibodies" include transgenic non-human animals, such as mice or rats, carrying the " refers to the system used to obtain human antibodies and human immunoglobulin loci. differences, introduction of somatic mutations or intentional introduction of substitutions into the framework or CDRs or both, resulting in a lower amino acid sequence when compared to human-expressed immunoglobulins. Typically, a "human antibody" is a human antibody that contains human germline immunoglobulins or recombinant human antibodies. The amino acid sequence encoded by the assembled immunoglobulin genes is The amino acid sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107 7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 7%, 98% or 99% identical. In some cases, a "human antibody" is a human antibody, e.g., Described in pik et al., (2000) J Mol Biol 296:57-86 a consensus framework sequence obtained from an analysis of human framework sequences, For example, Shi et al., (2010) J Mol Biol 397:385-9 6 and WO2009 / 085462, It may contain a synthetic HCDR3 engineered into a human immunoglobulin gene library. Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of "human antibody."

[0089] A "humanized antibody" is an antibody in which at least one CDR is derived from a non-human species and at least one FDR is derived from a non-human species. A humanized antibody refers to an antibody whose framework is derived from human immunoglobulin sequences. The framework can contain substitutions so that the framework is compatible with expressed human immunoglobulins. may not be an exact copy of the human immunoglobulin germline gene sequence .

[0090] Typically, a humanized antibody has an antigen-binding site derived from a non-human species and a variable region framework. Human antibodies have framework and antigen-binding sequences derived from human immunoglobulin sequences. Both of the binding sites have heavy and light chain variable regions derived from sequences of human origin.

[0091] The antibody may also have any level of affinity or avidity for a functional portion of a CAR. In some embodiments, antibodies are obtained with a range of affinities (K D ) and GPRC5D antigen In one embodiment according to the present invention, the numbered In each of the embodiments and in some of the embodiments, By surface plasmon resonance or equilibrium exclusion (Kinexa) methods performed by the manufacturer As determined, the antibody has high affinity, e.g., about 10 -7 K below M D So, for example, , including but not limited to, 1 to 9.9 (or 1, 2, 3, 4, 5, 6, 7, 8, or 9, etc. (any range or value within this range) x 10 -8 M, 10 -9 M, 10 -10 M, 1 0 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15 M or within this Any range or value of K D , which binds to the GPRC5D antigen. One example of affinity is , 1×10 -8 Another example of affinity is 1×10 -9 It is below M.

[0092] Methods for testing antibodies for their ability to bind to any functional portion of a CAR are known in the art. Any antibody-antigen binding assay known in the art, e.g., radioimmunoassay (radioimmunoassay, RIA), Western blot, enzyme-linked immunosorbent assay (enzyme me-linked immunosorbent assay (ELISA), immunoprecipitation, and competitive inhibition assays Examples include:

[0093] Suitable methods for producing antibodies are known in the art. For example, standard human immunodeficiency virus (HIV) antibodies can be used. The hybridoma method is described, for example, in Kohler and Milstein, Eur. JI mmunol.,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, e.g. For example, the EBV-hybridoma method (Haskard and Archer, J. Immunol. unol.Methods,74(2),361-67(1984),and Rode r et al.,Methods Enzymol.,121,140-67(198 6)), and bacteriophage vector expression systems (e.g., Huse et al., S Science, 246, 1275-81 (1989) are examples of the technology Additionally, methods for producing antibodies in non-human animals are known in the art. Patent Nos. 5,545,806, 5,569,825 and 5,714,352 and in US Patent Application Publication No. 2002 / 0197266(A1).

[0094] Phage display can also be used to generate antibodies. Phage libraries encoding antigen-binding variable (V) domains of can be produced using chemical and recombinant DNA techniques (e.g., Sambrook et al. (supra), and Ausubel et al. (supra). The phage encoding the variable region with the desired specificity are then bound to the desired antigen (i.e., GP RC5D), and a complete or partial vector containing the selected variable domain. The nucleic acid sequence encoding the rearranged antibody is then purified by hybridoma technology. The antibody is then introduced into a suitable cell line, such as a myeloma cell, to be used for production and characterization of the monoclonal antibody. Antibodies having the formula (e.g., Janeway et al., supra) are secreted by the cells. ), Huse et al. (supra), and U.S. Pat. No. 6,265,150. sea ​​bream).

[0095] Antibodies are transgenic antibodies that are transgenic for specific heavy and light chain immunoglobulin genes. Such methods can be used to produce the gene encoding the nucleotide sequence of interest in a transgenic mouse. See, for example, the above-mentioned U.S. Patent Nos. 5,545,806 and 5,569,825; and Janeway et al. (supra).

[0096] Methods for producing humanized antibodies are known in the art, see, e.g., Janew ay et al. (supra), U.S. Patent Nos. 5,225,539 and 5,585,088 9 and 5,693,761, European Patent No. 0239400(B1), and UK Patent No. Humanized antibodies are also described in U.S. Patent No. 5,639, No. 641 and Pedersen et al., J. Mol. Biol., 235, 95 These antibodies can also be generated using the antibody resurfacing technology described in [Publication ID No. 9-973 (1994)]. Cut.

[0097] As used herein, an antibody can be a multi-chain or single-chain, or an intact immunoglobulin. Antibodies may be derived from natural or recombinant sources. It can be a body.

[0098] In some embodiments, the antibody is a bispecific antibody. A molecule, such as an antibody, that specifically binds to two different antigens or two different epitopes within a molecule. Bispecific molecules refer to molecules that bind to other related antigens, e.g., human or monkey, e.g., M acaca cynomolgus (cynomolgus monkey, cyno) or Pan trogl may have cross-reactivity to the same antigen in other species (homologues), such as B. odytes; Alternatively, it may bind to an epitope shared between two or more different antigens. The VL and / or VH regions of an antibody, or newly identified as described herein The resulting VL and VH regions may be genetically engineered into bispecific full-length antibodies. Such bispecific antibodies can be prepared using techniques such as those described below. This can be produced by adjusting CH3 interactions in the antibody Fc to form: 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 / 0182 127, U.S. Patent Publication No. US52010 / 0286374, U.S. Patent Publication No. US20 11 / 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 antibody of the present invention can be prepared by the method described in International Publication No. WO2011 / 131746. According to the method described in the paper, two monospecific homodimers were synthesized in a cell-free environment in vitro. Asymmetric mutations are introduced into the CH3 region of the antibody, followed by reduction to isomerize disulfide bonds. Under these conditions, two parent monospecific homodimeric antibodies are converted into a bispecific heterodimeric antibody. In this method, a first monospecific bivalent antibody may be produced by forming and a second monospecific bivalent antibody in the CH3 domain that promotes heterodimer stability. These antibodies are engineered to have specific substitutions in the hinge region. The cysteines in the covalent bond are reacted under reducing conditions sufficient to isomerize the disulfide bond. The mixture is incubated with 100 μL of Fab-Ag-2000, which generates bispecific antibodies by Fab-arm exchange. Incubation conditions can be optimally returned to non-reducing conditions. Examples of reducing agents are 2-mercaptoethylamine (2-MEA), dithiothreitol (DT T), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl) ) phosphine (TCEP), L-cysteine, and beta-mercaptoethanol. , preferably 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxylate). The reducing agent is selected from the group consisting of (carboxyethyl)phosphine. At a temperature of 20°C, in the presence of at least 25 mM 2-MEA or at least 0. In the presence of 5 mM dithiothreitol, pH 5 to 8, for example, pH 7.0 or pH 7. In 4, an incubation of at least 90 minutes may be used.

[0099] The term "antibody fragment" refers to an intact antibody that retains the antigen-binding properties of the parent full-length antibody. It refers to at least one portion of an antibody or a recombinant variant thereof, e.g., a molecule capable of recognizing and binding ( sufficient to confer specific binding of an antibody fragment to a target such as an antigen "Antigen-binding domain" refers to the antigen-binding domain (e.g., the antigen-determining variable region of an intact antibody) that is An "antibody fragment" refers to a portion of an immunoglobulin molecule. Examples of antibody fragments include , Fab, Fab', F(ab')2, and Fv fragments, single chain antibodies (scFv) , linear antibodies, single domain antibodies (either VL or VH) such as sdAb, camelid VH H domains, as well as multispecific antibodies formed from antibody fragments. Not limited to these.

[0100] The term "scFv" refers to an antibody fragment that contains at least one variable region of a light chain. and at least one antibody fragment containing a variable region of a heavy chain. In some embodiments, the light chain variable region and the heavy chain variable region are short flexible polypeptides. can be linked sequentially via a linker and expressed as a single polypeptide chain, cFvs retain the specificity of the intact antibody from which they are derived. When used in the scFv, the VL and VH variable regions may be in either order, For example, with respect to the N-terminal and C-terminal ends of the polypeptide, scFvs may be provided with VL-linkers -VH, or may comprise VH-linker-VL.

[0101] One embodiment of the present invention also provides an antigen-binding portion of any of the antibodies described herein. Antigen-binding portions include Fab, F(ab')2, dsFv, sFv, diabodies, and It can be any moiety that has at least one antigen binding site, such as a triabody.

[0102] In some embodiments, the antigen-binding fragment comprises a heavy chain complementarity determining region (CDR). n complementarity determining regions (HCDR) 1, 2, and / or 3, light chain Complementarity determining region (LCDR) 1, 2, and / or 3, heavy chain variable region (VH), or light chain variable region (VL), Fab, F (ab')2, Fd, and Fv fragments, and one VH domain or one VL A domain antibody (dAb) comprises (e.g., consists of) any of the domains. The VH domain and the VL domain are linked together via a linker, e.g., a synthetic linker. It can be done.

[0103] "Complementarity determining regions (CDRs)" are the regions in an antibody that CDRs are antigen-binding sites within VH. CDRs can be defined using various terms: (i) within VH Three in the VL (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR The complementarity-determining regions (CDRs) of the ribosomal DNA (LCDR2, LCDR3) are based on sequence diversity (Wu and Kabat,J Exp Med 132:211-50,1970;Kaba t et al.,Sequences of Proteins of Immuno logical Interest,5th Ed.Public Health Se rvice,National Institutes of Health,Beth (esda, Md., 1991). (ii) Three in VH (H1, H2, H3) and one in VL The three "hypervariable regions," "HVRs," or "HVs" (L1, L2, and L3) in thia and Lesk(Chothia and Lesk Mol Biol 196 :901-17, 1987), which are hypervariable in structure. This refers to the region of the antibody variable domain. The cs(IMGT) database (http: / / www_imgt_org) Standard numbering and definitions for binding sites are provided. CDR, HV, and IMGT Overview The correspondence of Lefranc et al.,Dev Comparative Immunol 27:55-77, 2003. As used herein, "CDR" , "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and the term "LCDR3" refers to the same as Ka listed above, unless otherwise specified herein. bat, Chothia, or as defined by IMGT The CDRs are

[0104] Antibodies or antigen-binding portions thereof may also be conjugated to, for example, radioisotopes, fluorophores (e.g. , fluorescein isothiocyanate (FITC), FITC phycoerythrin (PE), enzymes (e.g., alkaline phosphatase, detectable labels such as chromatin (e.g., horseradish peroxidase), and elemental particles (e.g., gold particles) It may be modified to include

[0105] The present disclosure provides a method for producing a CAR, polypeptide, or protein as described herein. Nucleotide sequences encoding any of the proteins (including functional portions and functional variants thereof) It is a nucleic acid containing a sequence.

[0106] The portion of the CAR comprising the antibody or antibody fragment thereof may exist in various forms: In that case, the antigen-binding domain may be, for example, a single domain antibody fragment (sdAb), As part of a continuous polypeptide chain, such as scFv and human chimeric or humanized antibodies (Harlow et al., 1999, In:Using Antibodies dies:A Laboratory Manual,Cold Spring Har Bor Laboratory Press, NY; Harlow et al. 1989, In: Antibodies: A Laboratory Manual, C old Spring Harbor,NY;Houston et al.,19 88, Proc. Natl. Acad. Sci. USA 85:5879-5883;B ird et al., 1988, Science 242:423-426). One aspect In one embodiment, the antigen-binding domain of the CAR composition of the invention comprises an antibody fragment. In the present invention, the CAR comprises an antibody fragment, including an scFv.

[0107] The term "recombinant antibody" refers to an antibody produced by, for example, a bacteriophage or yeast expression system. The term refers to antibodies produced using recombinant DNA technology, such as antibodies expressed in It is produced by the synthesis of a DNA molecule that encodes the antibody protein, or an antibody expressing an amino acid sequence that specifies the antibody, In this case, the DNA or amino acid sequences may be obtained from any of the compositions available and known in the art. It may be obtained using recombinant DNA or amino acid sequencing techniques.

[0108] The term "antigen" refers to a molecule that provokes an immune response. This immune response includes antibody production. This may involve the activation of specific immunologically competent cells, or both. The supplier claims that virtually any macromolecule, including any protein or peptide, can function as an antigen. It will be understood that antigens may be derived from recombinant or genomic DNA. Those skilled in the art will appreciate that any DNA can be a nucleotide sequence that encodes a protein that elicits an immune response. The term is used herein to include a nucleotide sequence or partial nucleotide sequence. It will be understood that the antigen will be encoded as such. It is understood that the sequence need not be encoded solely by the full-length nucleotide sequence of a gene. While the present disclosure includes the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences may be arranged in various combinations. It is clear that the polypeptides that encode the desired immune response will be those that elicit the desired immune response.

[0109] In one aspect, the present disclosure provides a method for detecting a cellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The CAR includes a transduction domain, and the extracellular antigen-binding domain binds to the GPRC5D antigen. Combine.

[0110] In one aspect, the present disclosure provides a method for detecting a cellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a transduction domain, wherein the extracellular antigen-binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 66, SEQ ID NO: 6 7, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 68. CDR3, Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 5 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 9, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 60 CDR3, Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 4 0, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 41. CDR3, heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 42, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 43 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 44, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 45; heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 45, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 46 and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 47, The extracellular antigen-binding domain binds to the GPRC5D antigen.

[0111] In one aspect, the present disclosure provides a method for detecting a cellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a transduction domain, wherein the extracellular antigen-binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 66, SEQ ID NO: 6 7, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 68. The extracellular antigen-binding domain, which includes CDR3, binds to the GPRC5D antigen.

[0112] In one aspect, the present disclosure provides a method for detecting a cellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a transduction domain, wherein the extracellular antigen-binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 5 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 9, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 60 The extracellular antigen-binding domain, which includes CDR3, binds to the GPRC5D antigen.

[0113] In one aspect, the present disclosure provides a method for detecting a cellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a transduction domain, wherein the extracellular antigen-binding domain comprises: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 4 0, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 41. The extracellular antigen-binding domain, which includes CDR3, binds to the GPRC5D antigen.

[0114] In one aspect, the present disclosure provides a method for detecting a cellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a transduction domain, wherein the extracellular antigen-binding domain comprises: heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 42, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 43 and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 44, The extracellular antigen-binding domain binds to the GPRC5D antigen.

[0115] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The CAR comprises a CAR transduction domain, and the extracellular antigen binding domain is the CAR of SEQ ID NO: 45. heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 46; 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, The extracellular antigen-binding domain binds to the GPRC5D antigen.

[0116] In one embodiment, the extracellular antigen binding domain comprises: heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 66, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 67 and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 68, light chain CDR1 having the amino acid sequence of SEQ ID NO: 69, light chain CDR2 having the amino acid sequence of SEQ ID NO: 70 an extracellular antibody further comprising a light chain CDR2 and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 71; an antigen-binding domain, heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 58, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 59 and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 60, light chain CDR1 having the amino acid sequence of SEQ ID NO: 61, light chain CDR2 having the amino acid sequence of SEQ ID NO: 62 an extracellular antibody further comprising a light chain CDR2 and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 63; an antigen-binding domain, heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 39, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 40 and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 41, light chain CDR1 having the amino acid sequence of SEQ ID NO: 48, light chain CDR2 having the amino acid sequence of SEQ ID NO: 49 an extracellular antibody further comprising a light chain CDR2 and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 50; an antigen-binding domain, heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 42, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 43 and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 44, light chain CDR1 having the amino acid sequence of SEQ ID NO: 51, light chain CDR2 having the amino acid sequence of SEQ ID NO: 52 an extracellular antibody further comprising a light chain CDR2 and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 53; an antigen-binding domain, or heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 45, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 46 and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 47, light chain CDR1 having the amino acid sequence of SEQ ID NO: 54, light chain CDR2 having the amino acid sequence of SEQ ID NO: 55 an extracellular antibody further comprising a light chain CDR2 and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 56; an antigen-binding domain.

[0117] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a cellular transduction domain, wherein the extracellular antigen-binding domain is SEQ ID NO: 66, SEQ ID NO: 58, SEQ ID NO: 39, SEQ ID NO: 42, and SEQ ID NO: 45, and and a heavy chain CDR1 having an amino acid sequence selected from the group consisting of these conservative modifications. The extracellular antigen-binding domain binds to the GPRC5D antigen.

[0118] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a cellular transduction domain, wherein the extracellular antigen-binding domain is SEQ ID NO: 67, SEQ ID NO: 59, SEQ ID NO: 40, SEQ ID NO: 43, and SEQ ID NO: 46, and and a heavy chain CDR2 having an amino acid sequence selected from the group consisting of these conservative modifications. The extracellular antigen-binding domain binds to the GPRC5D antigen.

[0119] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a cellular transduction domain, wherein the extracellular antigen-binding domain is SEQ ID NO: 68, SEQ ID NO: 60, SEQ ID NO: 41, SEQ ID NO: 44, and SEQ ID NO: 47, and and a heavy chain CDR3 having an amino acid sequence selected from the group consisting of these conservative modifications. The extracellular antigen-binding domain binds to the GPRC5D antigen.

[0120] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a cellular transduction domain, wherein the extracellular antigen-binding domain is SEQ ID NO: 66, SEQ ID NO: 58, SEQ ID NO: 39, SEQ ID NO: 42, and SEQ ID NO: 45, and a heavy chain CDR1 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 a heavy chain CDR2 having an amino acid sequence selected from the group consisting of these conservative modifications; Beauty, SEQ ID NO: 68, SEQ ID NO: 60, SEQ ID NO: 41, SEQ ID NO: 44, and SEQ ID NO: 47, and a heavy chain CDR3 having an amino acid sequence selected from the group consisting of: Including, The extracellular antigen-binding domain binds to the GPRC5D antigen.

[0121] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a cellular transduction domain, wherein the extracellular antigen-binding domain is SEQ ID NO: 69, SEQ ID NO: 54, SEQ ID NO: 48, SEQ ID NO: 51, and SEQ ID NO: 54, and and a light chain CDR1 having an amino acid sequence selected from the group consisting of these conservative modifications. The extracellular antigen-binding domain binds to the GPRC5D antigen.

[0122] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a cellular transduction domain, wherein the extracellular antigen-binding domain is SEQ ID NO: 70, SEQ ID NO: 55, SEQ ID NO: 49, SEQ ID NO: 52, and SEQ ID NO: 55, and and a light chain CDR2 having an amino acid sequence selected from the group consisting of these conservative modifications. The extracellular antigen-binding domain binds to the GPRC5D antigen.

[0123] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a cellular transduction domain, wherein the extracellular antigen-binding domain is SEQ ID NO: 71, SEQ ID NO: 56, SEQ ID NO: 50, SEQ ID NO: 53, and SEQ ID NO: 56, and and a light chain CDR3 having an amino acid sequence selected from the group consisting of these conservative modifications. The extracellular antigen-binding domain binds to the GPRC5D antigen.

[0124] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a cellular transduction domain, wherein the extracellular antigen-binding domain is SEQ ID NO: 69, SEQ ID NO: 54, SEQ ID NO: 48, SEQ ID NO: 51, and SEQ ID NO: 54, and a light chain CDR1 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 a light chain CDR2 having an amino acid sequence selected from the group consisting of these conservative modifications, Beauty, SEQ ID NO: 71, SEQ ID NO: 56, SEQ ID NO: 50, SEQ ID NO: 53, and SEQ ID NO: 56, and and a light chain CDR3 having an amino acid sequence selected from the group consisting of these conservative modifications. The extracellular antigen-binding domain binds to the GPRC5D antigen.

[0125] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a cellular transduction domain, wherein the extracellular antigen-binding domain is SEQ ID NO: 66, SEQ ID NO: 58, SEQ ID NO: 39, SEQ ID NO: 42, and SEQ ID NO: 45, and a heavy chain CDR1 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 a heavy chain CDR2 having an amino acid sequence selected from the group consisting of these conservative modifications; Beauty, SEQ ID NO: 68, SEQ ID NO: 60, SEQ ID NO: 41, SEQ ID NO: 44, and SEQ ID NO: 47, and a heavy chain CDR3 having an amino acid sequence selected from the group consisting of these conservative modifications, Beauty, SEQ ID NO: 69, SEQ ID NO: 54, SEQ ID NO: 48, SEQ ID NO: 51, and SEQ ID NO: 54, and a light chain CDR1 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 a light chain CDR2 having an amino acid sequence selected from the group consisting of these conservative modifications, Beauty, SEQ ID NO: 71, SEQ ID NO: 56, SEQ ID NO: 50, SEQ ID NO: 53, and SEQ ID NO: 56, and a light chain CDR3 having an amino acid sequence selected from the group consisting of: Including, The extracellular antigen-binding domain binds to the GPRC5D antigen.

[0126] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a cellular transduction domain, wherein the extracellular antigen-binding domain is heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 66, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 67 and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 68, light chain CDR1 having the amino acid sequence of SEQ ID NO: 69, light chain CDR2 having the amino acid sequence of SEQ ID NO: 70 The antibody further comprises a light chain CDR2 and a light chain CDR3 having the amino acid sequence of SEQ ID NO:71.

[0127] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a cellular transduction domain, wherein the extracellular antigen-binding domain is heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 58, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 59 and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 60, light chain CDR1 having the amino acid sequence of SEQ ID NO: 61, light chain CDR2 having the amino acid sequence of SEQ ID NO: 62 The antibody further comprises a light chain CDR2 and a light chain CDR3 having the amino acid sequence of SEQ ID NO:63.

[0128] In one aspect, the present disclosure provides a method for detecting a cellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a transduction domain, wherein the extracellular antigen-binding domain comprises: heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 39, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 40 and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 41, light chain CDR1 having the amino acid sequence of SEQ ID NO: 48, light chain CDR2 having the amino acid sequence of SEQ ID NO: 49 The antibody further comprises a light chain CDR2 and a light chain CDR3 having the amino acid sequence of SEQ ID NO:50.

[0129] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a cellular transduction domain, wherein the extracellular antigen-binding domain is heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 42, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 43 and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 44, light chain CDR1 having the amino acid sequence of SEQ ID NO: 51, light chain CDR2 having the amino acid sequence of SEQ ID NO: 52 The antibody further comprises a light chain CDR2 and a light chain CDR3 having the amino acid sequence of SEQ ID NO:53.

[0130] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. and a CAR comprising a cellular transduction domain, wherein the extracellular antigen-binding domain is heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 45, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 46 and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 47, light chain CDR1 having the amino acid sequence of SEQ ID NO: 54, light chain CDR2 having the amino acid sequence of SEQ ID NO: 55 The antibody further comprises a light chain CDR2 and a light chain CDR3 having the amino acid sequence of SEQ ID NO:56.

[0131] In one embodiment, the extracellular antigen binding domain is selected from SEQ ID NOs: 73, 65, 1, 3, and 5. a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: 2, 64, 2, 4, and 6 HCVR), or an amino acid selected from the group consisting of SEQ ID NOs: 73, 65, 1, 3, and 5 and a LCVR comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 72, 64, 2, 4, and 6. The amino acid sequence of the HCVR may be a sequence of the nucleotide ...

[0132] In some embodiments, the extracellular antigen binding domain is selected from the group consisting of SEQ ID NOs: 73, 65, 1, 3 or Each of the five amino acid sequences comprises a light chain variable region (LCVR). In embodiments, the extracellular antigen binding domain is any of SEQ ID NOs: 73, 65, 1, 3, or 5. one of them and at least 50%, at least 55%, at least 60%, at least 65% , at least 70%, at least 75%, at least 80%, at least 85%, less At least 90%, at least 91%, at least 92%, at least 93%, at least 94% %, at least 95%, at least 96%, at least 97%, at least 98%, or Light chain variable region (LCVR) containing amino acid sequences with at least 99% sequence identity In some embodiments, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the extracellular antigen binding The domain may have at least 50%, at least 55%, at least 60%, or a similar sequence to SEQ ID NO: 73. At least 65%, at least 70%, at least 75%, at least 80%, at least At least 85%, at least 90%, at least 91%, at least 92%, at least 93% , at least 94%, at least 95%, at least 96%, at least 97%, at least and a light chain variable region comprising an amino acid sequence that has 98% or at least 99% sequence identity with the In some embodiments, the extracellular antigen-binding domain comprises a region (LCVR) having SEQ ID NO: In some embodiments, the light chain variable region (LCVR) comprises the amino acid sequence of SEQ ID NO:65. The extracellular antigen-binding domain may have at least 50%, at least 55%, or at least a partial identity with SEQ ID NO: 65. at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, At least 93%, at least 94%, at least 95%, at least 96%, at least amino acid sequences having at least 97%, at least 98%, or at least 99% sequence identity In some embodiments, the extracellular antigen-binding domain comprises a light chain variable region (LCVR) comprising a nucleotide sequence. The main antibody contains a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1. In this embodiment, the extracellular antigen-binding domain has a sequence identity at least 50%, at least 10% identical to SEQ ID NO:1. At least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 91%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% sequence identity In some embodiments, the cell comprises a light chain variable region (LCVR) comprising the amino acid sequence The 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 has at least 50 amino acids identical to SEQ ID NO:3. %, at least 55%, at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 85%, at least 90%, at least 9 1%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity In some embodiments, the light chain variable region (LCVR) comprises an amino acid sequence having the same identity as the LCVR. In this embodiment, the extracellular antigen-binding domain comprises a light chain variable region (L) comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the extracellular antigen-binding domain comprises at least one of SEQ ID NO: 5 and SEQ ID NO: 6. at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, or at least 9 It contains a light chain variable region (LCVR) containing amino acid sequences with 9% sequence identity.

[0133] In some embodiments, the extracellular antigen binding domain is selected from the group consisting of SEQ ID NOs: 72, 64, 2, 4, or a light chain variable region (LCVR) comprising any one of the amino acid sequences of In this embodiment, the extracellular antigen binding domain is selected from the group consisting of SEQ ID NOs: 72, 64, 2, 4, and 6. Any one of them and at least 50%, at least 55%, at least 60%, at least 65 %, at least 70%, at least 75%, at least 80%, at least 85%, at least at least 90%, at least 91%, at least 92%, at least 93%, at least 9 4%, at least 95%, at least 96%, at least 97%, at least 98%, or heavy chain variable region (HCVR) containing amino acid sequences with at least 99% sequence identity In some embodiments, the extracellular antigen-binding domain comprises amino acid sequence of SEQ ID NO: 72. In some embodiments, the antibody binds to an extracellular antigen. The binding domain has at least 50%, at least 55%, or at least 60% similarity to SEQ ID NO: 72. , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 91%, at least 92%, at least 93% %, at least 94%, at least 95%, at least 96%, at least 97%, at least heavy chains containing amino acid sequences with at least 98%, or at least 99%, sequence identity; In some embodiments, the extracellular antigen-binding domain comprises a sequence In some embodiments, the antibody comprises a light chain variable region (LCVR) comprising the amino acid sequence of number 2. The extracellular antigen-binding domain may have at least 50%, at least 55%, or at least a small fraction identical to SEQ ID NO: 64. at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, At least 93%, at least 94%, at least 95%, at least 96%, at least amino acid sequences having at least 97%, at least 98%, or at least 99% sequence identity In some embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region (HCVR) comprising a nucleotide sequence. The main sequence is at least 50%, at least 55%, at least 60%, or at least at least 65%, at least 70%, at least 75%, at least 80%, at least 8 5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least A heavy chain variable region comprising an amino acid sequence having 98% or at least 99% sequence identity (HCVR). In some embodiments, the extracellular antigen binding domain comprises SEQ ID NO:6 In some embodiments, the cell comprises a light chain variable region (LCVR) comprising the amino acid sequence of The antigen-binding domain may have at least 50%, at least 55%, or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 91%, at least 92%, at least At least 93%, at least 94%, at least 95%, at least 96%, at least 97% , or at least 98%, or at least 99% sequence identity to In some embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region (HCVR). , at least 50%, at least 55%, at least 60%, at least 6 5%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or a heavy chain variable region (HCV) comprising an amino acid sequence having at least 99% sequence identity R).

[0134] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 73, or At least 50%, at least 55%, at least 60%, at least 65%, At least 70%, at least 75%, at least 80%, at least 85%, at least At least 90%, at least 91%, at least 92%, at least 93%, at least 94% , at least 95%, at least 96%, at least 97%, at least 98% or a light chain variable region comprising an amino acid sequence having at least 99% sequence identity with SEQ ID NO:7; 2, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 91%, at least 92%, at least At least 93%, at least 94%, at least 95%, at least 96%, at least 97% , including amino acid sequences with at least 98% or at least 99% sequence identity and a heavy chain variable region.

[0135] In one embodiment, the extracellular antigen-binding domain has the amino acid sequence of SEQ ID NO: 65, or At least 50%, at least 55%, at least 60%, at least 65%, At least 70%, at least 75%, at least 80%, at least 85%, at least At least 90%, at least 91%, at least 92%, at least 93%, at least 94% , at least 95%, at least 96%, at least 97%, at least 98% or a light chain variable region comprising an amino acid sequence having at least 99% sequence identity with SEQ ID NO:6; 4, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 91%, at least 92%, at least At least 93%, at least 94%, at least 95%, at least 96%, at least 97% , including amino acid sequences with at least 98% or at least 99% sequence identity and a heavy chain variable region.

[0136] In one embodiment, the extracellular antigen-binding domain has the amino acid sequence of SEQ ID NO: 1, or At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, At least 95%, at least 96%, at least 97%, at least 98% or less a light chain variable region comprising an amino acid sequence having at least 99% sequence identity with a sequence of SEQ ID NO: 2; an amino acid sequence, or at least 50%, at least 55%, at least 60% thereof; %, at least 65%, at least 70%, at least 75%, at least 80%, at least at least 85%, at least 90%, at least 91%, at least 92%, at least 9 3%, at least 94%, at least 95%, at least 96%, at least 97%, heavy chains comprising amino acid sequences with at least 98% or at least 99% sequence identity and a variable region.

[0137] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 3, or At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, At least 95%, at least 96%, at least 97%, at least 98% or less a light chain variable region comprising an amino acid sequence having at least 99% sequence identity with a sequence of SEQ ID NO: 4; an amino acid sequence, or at least 50%, at least 55%, at least 60% thereof; %, at least 65%, at least 70%, at least 75%, at least 80%, at least at least 85%, at least 90%, at least 91%, at least 92%, at least 9 3%, at least 94%, at least 95%, at least 96%, at least 97%, heavy chains comprising amino acid sequences with at least 98% or at least 99% sequence identity and a variable region.

[0138] In one embodiment, the extracellular antigen binding domain has the amino acid sequence of SEQ ID NO: 5, or At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, At least 95%, at least 96%, at least 97%, at least 98% or less a light chain variable region comprising an amino acid sequence having at least 99% sequence identity with a sequence of SEQ ID NO: 6; an amino acid sequence, or at least 50%, at least 55%, at least 60% thereof; %, at least 65%, at least 70%, at least 75%, at least 80%, at least at least 85%, at least 90%, at least 91%, at least 92%, at least 9 3%, at least 94%, at least 95%, at least 96%, at least 97%, heavy chains comprising amino acid sequences with at least 98% or at least 99% sequence identity and a variable region.

[0139] In one embodiment, the extracellular antigen-binding domain comprises a light chain 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.

[0140] In one embodiment, the extracellular antigen-binding domain comprises a light chain 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.

[0141] In one embodiment, the extracellular antigen-binding domain comprises a light chain polypeptide 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.

[0142] In one embodiment, the extracellular antigen-binding domain comprises a light chain polypeptide 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.

[0143] In one embodiment, the extracellular antigen-binding domain comprises a light chain polypeptide 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.

[0144] As used herein, a VH domain whose amino acid sequence is less than 100% identical to a given reference VH sequence. In embodiments showing sequence identity of HCDR1, HCDR2, and It may contain heavy chain CDRs that are identical to HCDR3, while amino acids within the framework regions Similarly, if the amino acid sequence of the VL domain is not 100% identical to a given reference sequence, In embodiments showing less than 100% sequence identity, the LCDR1, LCDR2, and and LCDR3, while the amino acids in the framework regions are identical to those in the light chain. Similarly, the scFv or extracellular domain of the CAR of the present disclosure may be modified by a given If the VH sequence shows less than 100% sequence identity with the reference VH sequence, it is still possible to identify the HCDR1 of the reference sequence. , HCDR2 and HCDR3, and LCDR1, LC The light chain CDRs may comprise CDRs that are identical to LCDR2, LCDR3, and LCDR4, while the framework regions The amino acid sequence mutations within the

[0145] In one embodiment, the extracellular antigen-binding domain comprises an scFv. In general, scFvs contain a linker polypeptide between the light chain variable region and the heavy chain variable region. In certain embodiments, the extracellular antigen binding domain is selected from the group consisting of SEQ ID NOs: 78, 77, 75, 76, scF comprising an amino acid sequence selected from the group consisting of 8, 9, 10, 24, 25 and 26 v, and a GPRC5D polypeptide (e.g., a human GPRC5D polypeptide having the amino acid sequence of SEQ ID NO: 57). GPRC5D polypeptide or a fragment thereof) .

[0146] In some embodiments, the scFv comprises, from N-terminus to C-terminus, a VH, a linker, and Contains VL (VH-linker-VL).

[0147] In some embodiments, the scFv comprises, from N-terminus to C-terminus, a VL, a linker, and Contains VH (VL-linker-VH).

[0148] In recombinant expression systems, the linker is a peptide linker and may be any naturally occurring Exemplary amino acids that can be included in the linker include Gly, Ser, Pr, o, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. Cars are required to be in the correct conformation with each other to retain the desired activity, such as binding to GPRC5D. The length must be sufficient to link VH and VL to form the structure.

[0149] The linker can be about 5 to 50 amino acids in length. In some embodiments, the linker In some embodiments, the linker is about 10 to 40 amino acids in length. In some embodiments, the linker is about 10 to 30 amino acids in length. In some embodiments, the linker is about 10 to 25 amino acids in length. In some embodiments, the linker is about 10 to 20 amino acids in length. In some embodiments, the linker is about 15-20 amino acids in length. In embodiments, the linker is 6 amino acids in length. In some embodiments, the linker is 7 amino acids in length. In some embodiments, the linker is 8 amino acids in length. In some embodiments, the linker is 9 amino acids in length. In some embodiments, the linker is 10 amino acids long. In some embodiments, the linker is 12 amino acids long. In some embodiments, the linker is 13 amino acids in length. In some embodiments, the linker is 14 amino acids long. In some embodiments, the linker is 16 amino acids long. In some embodiments, the linker is 17 amino acids in length. In some embodiments, the linker is 18 amino acids long. In some embodiments, the linker is 20 amino acids long. In some embodiments, the linker is 21 amino acids in length. In some embodiments, the linker is 22 amino acids long. In some embodiments, the linker is 24 amino acids long. In some embodiments, the linker is 25 amino acids in length. In some embodiments, the linker is 26 amino acids long. In some embodiments, the linker is 28 amino acids long. In some embodiments, the linker is 29 amino acids in length. In some embodiments, the linker is 30 amino acids long. In some embodiments, the linker is 32 amino acids long. In some embodiments, the linker is 33 amino acids in length. In some embodiments, the linker is 34 amino acids long. In some embodiments, the linker is 36 amino acids long. In some embodiments, the linker is 37 amino acids in length. In some embodiments, the linker is 38 amino acids long. In some embodiments, the linker is 40 amino acids long. Exemplary linkers that can be used include Gly-rich linkers, Gly- and Ser-containing linkers, and linkers, Gly and Ala-containing linkers, Ala and Ser-containing linkers, and other It is a flexible linker.

[0150] In one embodiment, the linker polypeptide comprises the amino acid sequence of SEQ ID NO:7.

[0151] Other linker sequences may be used with immunoglobulins derived from immunoglobulin heavy or light chain isotypes. The linker may comprise a portion of the phospho-hinge region, CL, or CH1. 1. Additional linkers are described, for example, in International Publication No. WO2019 / 060695. and is incorporated herein by reference in its entirety.

[0152] In one embodiment, the linker polypeptide comprises any one of SEQ ID NOs: 91-123. It contains the amino acid sequence.

[0153] [Table 1]

[0154] In one embodiment, the scFv is selected from the group consisting of SEQ ID NOs: 78, 77, 75, 76, 8, 9, 10, 24 In some embodiments, the amino acid sequence is selected from the group consisting of: The scFvs are SEQ ID NOs: 78, 77, 75, 76, 8, 9, 10, 24, 25 and 26 and at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, At least 90%, at least 91%, at least 92%, at least 93%, at least At least 94%, at least 95%, at least 96%, at least 97%, at least 98% or an amino acid sequence having at least 99% sequence identity. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 78. The cFv may have at least 50%, at least 55%, at least 60%, or at least a small affinity to SEQ ID NO: 78. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least Some amino acid sequences have at least 98% or at least 99% sequence identity. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 77. In this case, the scFv has at least 50%, at least 55%, or at least 6% identity with SEQ ID NO: 77. 0%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, It includes amino acid sequences with at least 98%, or at least 99% sequence identity. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 75. In embodiments, the scFv has at least 50%, at least 55%, at least one identical sequence to SEQ ID NO: 75. at least 60%, at least 65%, at least 70%, at least 75%, at least 8 0%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least Amino acid sequences with 97%, at least 98%, or at least 99% sequence identity In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the scFv has at least 50%, at least 55%, or both a homologue of SEQ ID NO: 76. %, at least 60%, at least 65%, at least 70%, at least 75%, at least at least 80%, at least 85%, at least 90%, at least 91%, at least 9 2%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the scFv has at least 50% identical or at least 50% identical to SEQ ID NO:8. 55%, at least 60%, at least 65%, at least 70%, at least 75%, At least 80%, at least 85%, at least 90%, at least 91%, at least At least 92%, at least 93%, at least 94%, at least 95%, at least 96% , having at least 97%, at least 98%, or at least 99% sequence identity; In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the scFv has at least 50% identical sequence to SEQ ID NO:9, At least 55%, at least 60%, at least 65%, at least 70%, at least 75% %, at least 80%, at least 85%, at least 90%, at least 91%, at least at least 92%, at least 93%, at least 94%, at least 95%, at least 9 6%, at least 97%, at least 98%, or at least 99% sequence identity In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the scFv comprises the sequence SEQ ID NO: 10. , at least 55%, at least 60%, at least 65%, at least 70%, less At least 75%, at least 80%, at least 85%, at least 90%, at least 91% %, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% sequence identity In some embodiments, the scFv comprises an amino acid sequence having the following structure: In some embodiments, the scFv comprises an amino acid sequence at least as long as SEQ ID NO: 24. At least 50%, at least 55%, at least 60%, at least 65%, at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, less At least 91%, at least 92%, at least 93%, at least 94%, at least 95% %, at least 96%, at least 97%, at least 98%, or at least 99% In some embodiments, the scFv comprises an amino acid sequence having sequence identity to the sequence In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 25. At least 50%, at least 55%, at least 60%, at least 65%, at least At least 70%, at least 75%, at least 80%, at least 85%, at least 90% , at least 91%, at least 92%, at least 93%, at least 94%, at least At least 95%, at least 96%, at least 97%, at least 98%, or at least In some embodiments, the sc The Fv comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the scFv comprises the sequence Column number 26 and at least 50%, at least 55%, at least 60%, at least 65 %, at least 70%, at least 75%, at least 80%, at least 85%, at least at least 90%, at least 91%, at least 92%, at least 93%, at least 9 4%, at least 95%, at least 96%, at least 97%, at least 98%, or comprises an amino acid sequence that has at least 99% sequence identity.

[0155] In one embodiment, the extracellular antigen-binding domain comprises a signal polypeptide. In this embodiment, the signal polypeptide comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the extracellular antigen-binding domain is at least 50% identical to SEQ ID NO: 11, at least 50% identical to SEQ ID NO: 12. at least 55%, at least 60%, at least 65%, at least 70%, at least 7 5%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity It includes an amino acid sequence.

[0156] In one aspect, the present disclosure provides a method for producing a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen-binding domain is a CAR comprising a nucleic acid sequence encoding a nucleic acid sequence of SEQ ID NO: 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 10 A compound selected from the group consisting of 1, 80, 79, 17, 18, 19, 20, 21, and 22 amino acid sequence, or at least 50%, at least 55%, or at least 60% thereof , at least 65%, at least 70%, at least 75%, at least 80%, less At least 85%, at least 90%, at least 91%, at least 92%, at least 93% %, at least 94%, at least 95%, at least 96%, at least 97%, at least SEQ ID NO: 8 includes sequences with at least 98%, or at least 99% sequence identity. 2, 81, 80, 79, 17, 18, 19, 20, 21 and 22 Another feature of CARs having an extracellular antigen-binding domain containing an amino acid sequence that binds to the extracellular antigen is that the The binding domain binds to the GPRC5D antigen.

[0157] In one embodiment, the intracellular signaling domain is a TNF receptor superfamily member. member 9 (CD137) component, T cell surface glycoprotein CD3 zeta chain (CD3z) component , cluster of differentiation (CD27) component, cluster of differentiation superfamily member (e.g., CD28 or inducible T cell costimulator (ICOS), and combinations thereof The polypeptide component comprises a polypeptide component selected from the group consisting of:

[0158] In one embodiment, the CD137 component comprises the amino acid sequence of SEQ ID NO: 12. In embodiments, the CD137 component is at least 50%, at least 55%, identical to SEQ ID NO: 12. , at least 60%, at least 65%, at least 70%, at least 75%, less At least 80%, at least 85%, at least 90%, at least 95%, at least 98% %, or at least 99% sequence identity.

[0159] In one embodiment, the CD3z component comprises the amino acid sequence of SEQ ID NO: 13. In embodiments, the CD3z component is at least 50%, at least 55%, or at least 100% identical to SEQ ID NO:13. 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 an amino acid sequence having at least 99% sequence identity.

[0160] In some embodiments, the intracellular signaling domain comprises a CD137 component and a CD3 Includes the z component.

[0161] In one embodiment, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the intracellular signaling domain has at least one sequence identical to SEQ ID NO: 14. 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least amino acid sequences having at least 95%, at least 98%, or at least 99% sequence identity Contains columns.

[0162] The transmembrane domain of CAR is similar to that of CD8, α, β, or other T cell receptors. ζ chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, C D16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1(C DI la, CD18), ICOS (CD278), 4-1 BB (CD137), 4- 1 BBL, GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF 7. NKp80 (KLRFI), CD160, CD1 9. IL2R beta, IL2R gas umma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD4 9D, ITGA6, VLA-6, CD49f, ITGAD, CDI ID, ITGAE, CD103, ITGAL, CDI la, LFA-1, ITGAM, CDI lb, IT GAX, CDI lc, ITGB1, CD29, ITGB2, CD1 8, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B 4), CD84, CD96 (antennary), CEACAM1, CRT AM, Ly9 (CD22 9), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF 6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp , NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. do.

[0163] In one embodiment, the transmembrane domain is a CD8a transmembrane region (CD8A-TM) polypeptide. In some embodiments, the CD8a-TM polypeptide comprises the sequence of SEQ ID NO: 15. In some embodiments, the CD8a-TM polypeptide comprises the amino acid sequence SEQ ID NO: No. 15 and at least 50%, at least 55%, at least 60%, at least 65%, At least 70%, at least 75%, at least 80%, at least 85%, at least at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. The amino acid sequence includes:

[0164] In one embodiment, the transmembrane domain is a domain selected from the group consisting of the α, β, or ζ chain of a T cell receptor, CD28, CD 3 epsilon, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD 22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, C In another embodiment, the transmembrane domain comprises at least the transmembrane domain of CD137 or CD154. The main ones are ζ, η, or FcεR1γ, and -β, MB1 (Igα), B29, or CD3 In another embodiment, the transmembrane domains are selected from the group consisting of γ, ζ, and η. The amino acid is synthetic, e.g., a triplet of leucine and valine, phenylalanine, or It contains mainly hydrophobic residues such as tryptophan.

[0165] In one embodiment, the CAR comprises a cysteine-binding domain linking a transmembrane domain to an extracellular antigen-binding domain. In some embodiments, the hinge region is a CD8a hinge region. In some embodiments, the CD8a hinge region comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the CD8a hinge region is at least 50% identical to SEQ ID NO: 16. At least 55, at least 60, at least 65, at least 70, at least 75, at least at least 80, at least 85, at least 90, at least 95, at least 98, or comprises an amino acid sequence with at least 99% sequence identity. , wherein the hinge region comprises the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 124), or EPKSCDKTHTCPPCP (SEQ ID NO: 124) and at least 50, at least 55 , at least 60, at least 65, at least 70, at least 75, at least 80 , at least 85, at least 90, at least 95, at least 98, or less In some embodiments, the hinge region comprises an amino acid sequence that has 99% sequence identity with the amino acid sequence of the target protein. The region comprises the sequence ERKCCVECPPCP (SEQ ID NO: 125), or CPPCP (SEQ ID NO: 125) and at least 50, at least 55, at least 60, At least 65, at least 70, at least 75, at least 80, at least 85, at least at least 90, at least 95, at least 98, or at least 99% sequence identity In some embodiments, the hinge region comprises an amino acid sequence having the sequence ELKT PLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO: 126) Includes or ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP) 3 (SEQ ID NO: 126) and at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99% sequence identity In some embodiments, the hinge region comprises an amino acid sequence comprising the sequence ESKYGPPC PSCP (SEQ ID NO: 127) or ESKYGPPCPSCP (SEQ ID NO: 127) and at least 50, at least 55, at least 60, at least 65, at least 70 , at least 75, at least 80, at least 85, at least 90, at least 95 , containing amino acid sequences with at least 98, or at least 99% sequence identity .

[0166] In one embodiment, a 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 comprises a CD8α The hinge region and transmembrane domain are CD8α-TM domains, which are involved in intracellular signal transduction. The delivery domain includes the CD3-zeta domain and the 4-1BB / CD137 domain. In one such embodiment, the hinge region comprises a CD8α hinge comprising the amino acid sequence of SEQ ID NO: 16. and the transmembrane domain is a CD8α-TM domain comprising the amino acid sequence of SEQ ID NO: 15. The intracellular signaling domain is the CD3-zeta domain and 4-1BB / The intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 14. Contains columns.

[0167] In one embodiment, the extracellular antigen binding domain is selected from the group consisting of SEQ ID NOs: 82, 81, 80, 79, 17 , 18, 19, 20, 21, and 22. In some embodiments, the extracellular antigen binding domain is selected from the group consisting of SEQ ID NOs: 82, 81, 80, 79, At least 50% of any one of 17, 18, 19, 20, 21, and 22 At least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 91%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% sequence identity , including amino acid sequences.

[0168] 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 at least one sequence selected from the group consisting of SEQ ID NO: 82 and SEQ ID NO: 83. 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least At least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% It contains amino acid sequences that have sequence identity.

[0169] 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 at least one sequence selected from the group consisting of SEQ ID NO: 81 and SEQ ID NO: 82. 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least At least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% It contains amino acid sequences that have sequence identity.

[0170] 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 at least one sequence selected from the group consisting of SEQ ID NO: 80 and SEQ ID NO: 90. 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least At least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% It contains amino acid sequences that have sequence identity.

[0171] 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 at least one sequence selected from the group consisting of SEQ ID NO: 79 and SEQ ID NO: 80. 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least At least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% It contains amino acid sequences that have sequence identity.

[0172] 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 at least one sequence selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 18. 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least At least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% It contains amino acid sequences that have sequence identity.

[0173] 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 at least one sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 19. 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least At least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% It contains amino acid sequences that have sequence identity.

[0174] 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 at least one sequence selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 19. 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least At least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% It contains amino acid sequences that have sequence identity.

[0175] 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 at least one sequence selected from the group consisting of SEQ ID NO: 20 and SEQ ID NO: 21. 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least At least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% It contains amino acid sequences that have sequence identity.

[0176] 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 at least one sequence selected from the group consisting of SEQ ID NO: 21 and SEQ ID NO: 22. 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least At least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% It contains amino acid sequences that have sequence identity.

[0177] 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 at least one sequence selected from the group consisting of SEQ ID NO: 22 and SEQ ID NO: 32. 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least At least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% It contains amino acid sequences that have sequence identity.

[0178] In one embodiment, the CAR of the present disclosure is selected from the group consisting of SEQ ID NOs: 86, 85, 83, 84, 27, 28, Some embodiments include an amino acid sequence selected from the group consisting of 29, 30, 31, and 32. In embodiments, the CAR of the present disclosure is selected from the group consisting of SEQ ID NOs: 86, 85, 83, 84, 27, 28, 29, Any one of 30, 31, and 32 and at least 50%, at least 55%, or At least 60%, at least 65%, at least 70%, at least 75%, at least 80% %, at least 85%, at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 9 7%, at least 98%, or at least 99% sequence identity with the amino acid sequence include.

[0179] In some embodiments, a CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, the CAR of the present disclosure is at least 50% identical to SEQ ID NO: 86. At least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 91%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% sequence identity , including amino acid sequences.

[0180] In some embodiments, a CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the CAR of the present disclosure is at least 50% identical to SEQ ID NO: 85. At least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 91%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% sequence identity , including amino acid sequences.

[0181] In some embodiments, a CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the CAR of the present disclosure is at least 50% identical to SEQ ID NO: 83. At least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 91%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% sequence identity , including amino acid sequences.

[0182] In some embodiments, a CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the CAR of the present disclosure is at least 50% identical to SEQ ID NO: 84. At least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 91%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% sequence identity , including amino acid sequences.

[0183] In some embodiments, a CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CAR of the present disclosure is at least 50% identical to SEQ ID NO:27. At least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 91%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% sequence identity , including amino acid sequences.

[0184] In some embodiments, a CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the CAR of the present disclosure is at least 50% identical to SEQ ID NO:28. At least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 91%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% sequence identity , including amino acid sequences.

[0185] In some embodiments, a CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the CAR of the present disclosure is at least 50% identical to SEQ ID NO:29. At least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 91%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% sequence identity , including amino acid sequences.

[0186] In some embodiments, a CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the CAR of the present disclosure is at least 50% identical to SEQ ID NO: 30. At least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 91%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% sequence identity , including amino acid sequences.

[0187] In some embodiments, a CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the CAR of the present disclosure is at least 50% identical to SEQ ID NO: 31. At least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 91%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% sequence identity , including the amino acid sequence.

[0188] In some embodiments, a CAR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CAR of the present disclosure is at least 50% identical to SEQ ID NO: 32. At least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 91%, less At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% sequence identity , including amino acid sequences.

[0189] In certain embodiments, the CAR may comprise a signal peptide. In certain embodiments, the signal peptide is at the N-terminus of the CAR sequence. The signal peptide is at the C-terminus of the CAR sequence. The peptide comprises or consists of the amino acid sequence of SEQ ID NO:11.

[0190] CAR constructs and CAR-expressing immunoresponsive cells In one aspect, the disclosure provides an isolated nucleic acid molecule encoding a CAR described herein. In some embodiments, the nucleic acid molecule is selected from the group consisting of SEQ ID NOs: 90, 89, 87, 88, 33 , 34, 35, 36, 37, and 38. In some embodiments, the isolated nucleic acid molecule is selected from the group consisting of SEQ ID NOs: 90, 89, 87, 88, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 4, 35, 36, 37 or 38 nucleotide sequences and 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 nucleotides Contains code arrays.

[0191] In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 90. In embodiments, the isolated nucleic acid molecule has at least about 8 sequences identical to the nucleotide sequence of SEQ ID NO:90. 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is identical to SEQ ID NO: 89. In some embodiments, the isolated nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 89. Nucleotide sequence and 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 the first embodiment. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 87. The nucleic acid molecule has at least about 80%, 81%, 82%, or 90% identity with the nucleotide sequence of SEQ ID NO:87. ,83%,84%,85%,86%,87%,88%,89%,90%,91%,92% , 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleotides 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 has at least the nucleotide sequence of SEQ ID NO:88. At most about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is 99% identical. In some embodiments, the isolated nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 33. nucleotide sequence of No. 33 at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9 Some contain nucleotide sequences that are 5%, 96%, 97%, 98% or 99% identical. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 34. The isolated nucleic acid molecule has at least about 80%, 81%, or 90% identical to the nucleotide sequence of SEQ ID NO:34. %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical 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 the nucleotide sequence of SEQ ID NO: 35. Sequence and at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% ,88%,89%,90%,91%,92%,93%,94%,95%,96%,97% In some embodiments, the nucleic acid The molecule comprises the nucleic acid sequence of SEQ ID NO: 36. In some embodiments, the isolated nucleic acid molecule has at least about 80%, 81%, 82%, 83%, or 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Contains a nucleotide sequence that is 94%, 95%, 96%, 97%, 98% or 99% identical In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 37. In embodiments, the isolated nucleic acid molecule has at least about 8 sequences identical to the nucleotide sequence of SEQ ID NO:37. 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is identical to SEQ ID NO: 38. In some embodiments, the isolated nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 38. Nucleotide sequence and 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 the present invention.

[0192] The term "encoding" refers to the encoding of a defined sequence of nucleotides (e.g., rR an organism with either a defined sequence of amino acids (RNA, tRNA, and mRNA) or They act as templates for the synthesis of other polymers and macromolecules in biological processes. The specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA. It refers to the inherent properties of a sequence and the biological properties that result from it. In biological systems, transcription and translation of mRNA corresponding to a gene produces a protein. In this case, the gene, cDNA, or RNA encodes a protein. the coding strand, whose sequence is identical to the mRNA sequence, and the Both the non-coding strand and the non-coding strand used in the present invention are used to identify the protein or other product of that gene or cDNA. may be referred to as encoding

[0193] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" are degenerate sequences of each other. All nucleotide sequences encoding the same amino acid sequence are The phrase "nucleotide sequence encoding a protein or RNA" also includes In some cases, the nucleotide sequence encoding the protein may contain introns. It may include introns to the extent necessary.

[0194] The term "isolated" refers to a molecule that is free from other components of the system in which it is produced, such as a recombinant cell. A homogeneous population of substantially separated and / or purified molecules (e.g., synthetic polynucleotides or or synthetic polypeptides), as well as proteins that have been subjected to at least one purification or isolation step. "Isolated" refers to a molecule that is substantially free from other cellular material and / or chemicals. refers to higher purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86% %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% purity are encompassed by molecules isolated to that degree.

[0195] In some embodiments, the present disclosure provides a method for the production of a nucleic acid molecule as described (e.g., SEQ ID NO: 90, 8 9, 87, 88, 33, 34, 35, 36, 37, or 38) is provided. In some embodiments, the expression vector comprises a sequence selected from the group consisting of SEQ ID NOs: 90, 89, 87, 88, 3 nucleotide sequence of 3, 34, 35, 36, 37 or 38 and 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 The present invention includes nucleic acid molecules containing the nucleic acid sequence.

[0196] In some embodiments, the expression vector comprises the nucleic acid molecule of SEQ ID NO: 90. In this embodiment, the expression vector comprises at least about 80 copies of the nucleotide sequence of SEQ ID NO: 90. %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical In some embodiments, the expression vector comprises a nucleic acid molecule comprising a nucleotide sequence comprises the nucleic acid molecule of SEQ ID NO: 89. In some embodiments, the expression vector comprises the nucleic acid molecule of SEQ ID NO: nucleotide sequence of No. 89 at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 13 5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9 Nucleic acid molecules containing nucleotide sequences that are 5%, 96%, 97%, 98% or 99% identical to In some embodiments, the expression vector comprises the nucleic acid molecule of SEQ ID NO: 87. In some embodiments, the expression vector comprises at least about the nucleotide sequence of SEQ ID NO:87. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% In some embodiments, the expression vector comprises a nucleic acid molecule that contains a nucleotide sequence that is identical to the nucleic acid molecule. In some embodiments, the expression vector comprises the nucleic acid molecule of SEQ ID NO: 88. At least about 80%, 81%, 82%, 83%, and 84% of the nucleotide sequence of sequence number 88 ,85%,86%,87%,88%,89%,90%,91%,92%,93%,94% , 95%, 96%, 97%, 98% or 99% identical nucleotide sequence In some embodiments, the expression vector comprises the nucleic acid molecule of SEQ ID NO:33. In some embodiments, the expression vector comprises at least the nucleotide sequence of SEQ ID NO:33. Also about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 9 In some embodiments, the expression vectors include nucleic acid molecules comprising nucleotide sequences that are 9% identical to each other. The vector comprises the nucleic acid molecule of SEQ ID NO: 34. In some embodiments, the expression vector , and a sequence identical to the nucleotide sequence of SEQ ID NO: 34, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 10 4%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 Nucleotides containing nucleotide sequences that are 4%, 95%, 96%, 97%, 98% or 99% identical In some embodiments, the expression vector comprises a nucleic acid molecule of SEQ ID NO: 35. In some embodiments, the expression vector contains at least the nucleotide sequence of SEQ ID NO:35. At most about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or In some embodiments, the nucleic acid molecules include those that contain nucleotide sequences that are 99% identical to each other. The expression vector comprises the nucleic acid molecule of SEQ ID NO: 36. In some embodiments, the expression vector - has at least about 80%, 81%, 82%, 83% identical to the nucleotide sequence of SEQ ID NO: 36 ,84%,85%,86%,87%,88%,89%,90%,91%,92%,93% , 94%, 95%, 96%, 97%, 98% or 99% identical nucleotide sequence. In some embodiments, the expression vector comprises a nucleic acid molecule comprising SEQ ID NO: 37. In some embodiments, the expression vector comprises the nucleotide sequence of SEQ ID NO: 37. At least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is 100% or 99% identical to the nucleic acid molecule. In some embodiments, the expression vector comprises a nucleic acid molecule of SEQ ID NO: 38. The vector has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 3%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 9 nucleotide sequences that are 3%, 94%, 95%, 96%, 97%, 98% or 99% identical The nucleic acid molecule comprises:

[0197] The nucleic acid molecules described are SEQ ID NOs: 86, 85, 83, 84, 27, 28, 29, 30, 31 and 32, or variants thereof. In some embodiments, the expression vector encodes 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 and at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, These include nucleic acid molecules that encode amino acid sequences that are 97%, 98%, or 99% identical. In some embodiments, the expression vector contains a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:85. In some embodiments, the expression vector comprises at least the amino acid sequence of SEQ ID NO:85. At most about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or In some embodiments, the nucleic acid molecules encoding the amino acid sequences are 99% identical. The expression vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 83. In one embodiment, the expression vector contains at least about 80% of the amino acid sequence of SEQ ID NO: 83. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical In some embodiments, the expression vector comprises a nucleic acid molecule encoding an amino acid sequence that In some embodiments, the nucleic acid molecule comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 84. The expression vector may contain at least about 80%, 81%, 82%, or 83% of the amino acid sequence of SEQ ID NO: 84. 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences In some embodiments, the expression vector comprises a nucleic acid molecule encoding the In some embodiments, the expression vector comprises a nucleic acid molecule encoding the amino acid sequence. at least about 80%, 81%, 82%, 83%, 84%, or the amino acid sequence of SEQ ID NO: 27; 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Nucleic acid molecules encoding amino acid sequences that are 95%, 96%, 97%, 98% or 99% identical In some embodiments, the expression vector encodes the amino acid sequence of SEQ ID NO:28. In some embodiments, the expression vector comprises a nucleic acid molecule encoding the and 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 nucleic acid molecule encoding the amino acid sequence. In some embodiments, the expression vector contains a nucleic acid encoding the amino acid sequence of SEQ ID NO:29. In some embodiments, the expression vector comprises the amino acid sequence of SEQ ID NO: 29. At least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 Some embodiments include nucleic acid molecules that encode amino acid sequences that are 100% or 99% identical to the amino acid sequence of the nucleic acid molecule. 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 contains at least about the amino acid sequence of SEQ ID NO: 30. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% In some embodiments, the expression vector includes a nucleic acid molecule encoding an amino acid sequence that is identical to the expression vector. The vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 31. In some embodiments, the expression vector contains at least about 80%, 81%, or 90% identical to the amino acid sequence of SEQ ID NO: 31. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical In some embodiments, the expression vector comprises a nucleic acid molecule encoding a sequence of SEQ ID NO: In some embodiments, the expression vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 32. The target has at least about 80%, 81%, 82%, 83%, or 84% identical to the amino acid sequence of SEQ ID NO: 32. 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Encoding amino acid sequences that are 94%, 95%, 96%, 97%, 98% or 99% identical The present invention includes nucleic acid molecules.

[0198] As used herein, the structure of a nucleic acid molecule is defined as a sequence of a given reference sequence (having a given SEQ ID NO). In this context, the % sequence identity of a nucleic acid molecule is appropriately defined based on the % sequence identity. Sequence identity refers to the similarity between at least two different nucleic acid sequences. If both positions of two DNA molecules are occupied by the same base, e.g. If each position is occupied by an adenine, the molecules are identical at that position and the two The percent identity between sequences is the number of matches or homologies at positions shared by the two sequences. is a function of the number of positions compared divided by the number of positions compared and multiplied by 100. For example, if 6 of the 10 positions in two sequences are identical or homologous, the two sequences are 0% identical (or homologous). As an example, the DNA sequences ATTGCC and TATGGC are 50% identity (or homology). Generally, two sequences are aligned to find a maximum of The comparison is made when homology is given. The percentage of each identity is calculated based on the standard alignment. Altshul et al. sic Local Alignment Search Tool(BLAST)(( 1990)J.MoI.Biol.215:403-10);Needleman et al.'s algorithm ((1970) J.MoI.Biol.48:444-53); or the algorithm of Meyers et al. ((1988) Comput. Appl .Biosci.4:11-17). has a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap. It can be a Blosum62 scoring matrix with a penalty of 5. The percent identity between the octide sequences was calculated using the ALIGN program (version 2.0). Meyers and Miller (1989) CABIOS 4:1 1-17) algorithm and gapped using the PAM120 weighted residue table. A length penalty of 12 and a gap penalty of 4 can be used to determine the percent identity. The nucleotide sequence is usually calculated by comparing sequences of similar length.

[0199] The term "expression vector" refers to a vector that contains a nucleotide sequence operably linked to it to be expressed. An expression vector refers to a vector containing a recombinant polynucleotide that includes an expression control sequence. It contains sufficient cis-acting elements for expression, and other elements for expression are required by the host cell. The expression vector can be provided in a recombinant form or in an in vitro expression system. Cosmids, plasmids (e.g., naked or liposomes) incorporating recombinant polynucleotides viruses (e.g., lentiviruses, retroviruses, adenoviruses, etc.); All vectors known in the art, including avian viruses (e.g., endoviruses, and adeno-associated viruses), Examples include:

[0200] In one embodiment, the present disclosure provides a cell that expresses a nucleic acid molecule described herein. In embodiments, the present disclosure provides a cell expressing a CAR of the present disclosure. The CARs of the present disclosure are represented by SEQ ID NOs: 86, 85, 83, 84, 27, 28, 29, 30, 31 and In some embodiments, the present disclosure provides an amino acid sequence selected from the group consisting of: CARs of SEQ ID NOs: 86, 85, 83, 84, 27, 28, 29, 30, 31 and 32 and at least 50%, at least 55%, at least 60%, at least At least 65%, at least 70%, at least 75%, at least 80%, at least 85% , at least 90%, at least 91%, at least 92%, at least 93%, at least At least 94%, at least 95%, at least 96%, at least 97%, at least 98% %, or at least 99% sequence identity.

[0201] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 86. In embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 31. AR comprises the amino acid sequence of SEQ ID NO:32.

[0202] In some embodiments, the CAR has at least 50% and at least 5% identical sequences to SEQ ID NO: 86. 5%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, having at least 97%, at least 98%, or at least 99% sequence identity In some embodiments, the CAR comprises a sequence identical to SEQ ID NO: 85 and at least 50 amino acids. %, at least 55%, at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 85%, at least 90%, at least 9 1%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity In some embodiments, the CAR comprises an amino acid sequence having the sequence SEQ ID NO: 83 and at least 50%, at least 55%, at least 60%, at least 65%, or less At least 70%, at least 75%, at least 80%, at least 85%, at least 90% %, at least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, or at least In some embodiments, the CAR comprises an amino acid sequence that has at least 99% sequence identity with the CAR. has at least 50%, at least 55%, at least 60%, or at least At least 65%, at least 70%, at least 75%, at least 80%, at least 85% , at least 90%, at least 91%, at least 92%, at least 93%, at least At least 94%, at least 95%, at least 96%, at least 97%, at least 98% %, or at least 99% sequence identity. In some embodiments, the CAR has at least 50%, at least 55%, or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 91%, at least 92%, at least At least 93%, at least 94%, at least 95%, at least 96%, at least 97% , or at least 98%, or at least 99% sequence identity to In some embodiments, the CAR has at least 50% and at least 5% homology with SEQ ID NO: 28. 5%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, having at least 97%, at least 98%, or at least 99% sequence identity In some embodiments, the CAR comprises a sequence identical to SEQ ID NO: 29 and at least 50 amino acid sequences. %, at least 55%, at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 85%, at least 90%, at least 9 1%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity In some embodiments, the CAR comprises an amino acid sequence having the sequence of SEQ ID NO: 30. and at least 50%, at least 55%, at least 60%, at least 65%, or less At least 70%, at least 75%, at least 80%, at least 85%, at least 90% %, at least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, or at least In some embodiments, the CAR comprises an amino acid sequence that has at least 99% sequence identity with the CAR. has at least 50%, at least 55%, at least 60%, or at least At least 65%, at least 70%, at least 75%, at least 80%, at least 85% , at least 90%, at least 91%, at least 92%, at least 93%, at least At least 94%, at least 95%, at least 96%, at least 97%, at least 98% %, or at least 99% sequence identity. In some embodiments, the CAR has at least 50%, at least 55%, or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 91%, at least 92%, at least At least 93%, at least 94%, at least 95%, at least 96%, at least 97% , or at least 98%, or at least 99% sequence identity to .

[0203] In one aspect, the present disclosure provides an isolated immunoresponsive cell comprising a CAR described herein. In some embodiments, the isolated immunoresponsive cells are transduced with a CAR. For example, the CAR is constitutively expressed on the surface of an immunoresponsive cell. In one embodiment, the isolated immunoresponsive cells are further transduced with at least one costimulatory ligand. The immunocompetent cells are transfected with the IgG4-dependent IL-1 receptor to induce the expression of at least one co-stimulatory ligand. In certain embodiments, the at least one costimulatory ligand is 4-1BBL, CD48, C D70, CD80, CD86, OX40L, TNFRSF14, and combinations thereof In certain embodiments, the isolated immunoresponsive cells are selected from the group consisting of: and further transduced with at least one cytokine, and immunoresponsive cells are transduced into at least one subpopulation. In certain embodiments, at least one cytokine is secreted. , IL-2, IL-3, IL-6, IL-7, IL-11, IL-12, IL-15, I Some of the antibodies are selected from the group consisting of IL-17, IL-21, and combinations thereof. In embodiments, the isolated immunoresponsive cells are T lymphocytes (T cells), natural killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, human embryonic stem cells, lymph Pluripotent stem cells from which lymphoid progenitor cells, T cell progenitor cells, and lymphoid cells can differentiate. is selected from the group consisting of:

[0204] The nucleic acid sequence of the present invention encoding a CAR can be "transfected," "transformed," or "transduction." As used herein, "transfection" refers to the process by which a gene or gene product is introduced into a cell. "Transfection," "transformation," or "transduction" can be achieved using physical or chemical methods. and introducing one or more exogenous polynucleotides into a host cell. Many transfection techniques are known in the art, for example, Calcium phosphate DNA coprecipitation (see, e.g., Murray EJ (ed.), Met. hods in Molecular Biology,Vol.7,Gene Tra nsfer and Expression Protocols,Humana Pr ess (1991); DEAE-dextran method; electroporation method; Cationic liposome-mediated transfection: tungsten particle-facilitated microparticle bombardment. Strike method (Johnston, Nature, 346:776-777 (1990)); and Strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)). The viral vector is then transduced into a host cell after growth of the infectious particle in a suitable packaging cell. can be introduced, many of which are commercially available.

[0205] In one embodiment, the CAR T cells of the present disclosure are directed to a desired CAR, e.g., anti-GPRC5D , CD8α hinge and transmembrane domain, and human 4-1BB and CD3-zeta signaling By introducing a lentiviral vector containing a CAR containing a CAR transduction domain into cells, The CAR T cells of the present invention can be generated by: Providing long-term durability that may lead to sustained tumor control.

[0206] Embodiments of the present invention include host cells containing any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to a cell that is capable of expressing a recombinant gene. A host cell refers to any type of cell that can contain a vector. A host cell is a eukaryotic cell, such as a plant cell. , animal, or algae, fungi, or prokaryotic cells, e.g., bacteria or protists. The host cell may be a cultured cell or a primary cell, i.e., a living organism, e.g., an animal. For example, the host cells can be isolated directly from a human. The host cells can be adherent cells or suspension cells, i.e., cells in suspension. Suitable host cells are known in the art, e.g., cells that grow in For example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, CO S cells, HEK293 cells, etc. Amplify or replicate recombinant expression vectors For purposes of this invention, the host cell may be a prokaryotic cell, such as a DH5α cell. For purposes of producing a polypeptide or protein, the host cell may be a mammalian cell. The host cell can be a human cell. The host cell can be of any cell type, including any The host cells may be derived from any tissue type and may be at any stage of development, but the host cells may be derived from peripheral blood lymphocytes. The host cell may be a peripheral blood lymphocyte (PBL). The host cell may be a T cell. .

[0207] For purposes herein, T cells may refer to cultured T cells (e.g., primary T cells), or cultured T T cells from cell lines (e.g., Jurkat, SupT1, etc.) or mammalian T cells When obtained from a mammal, the T cells may be any T cells, such as T cells isolated from mammals. sources of the disease (including, but not limited to, bone marrow, blood, lymph nodes, thymus, or other tissues or fluids) T cells can also be enriched or purified. The T cells may be T cells isolated from a human. T cells can be of any type and at any stage of development (CD4 + / CD8 + double positive T cells, CD8 + T cells (e.g., cytotoxic T cells), CD4 + Helper T cells (e.g. , Th1 and Th2 cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumors (including, but not limited to, infiltrating cells, memory T cells, and naive T cells) T cells are CD8 + T cells or CD4 + It may be a T cell.

[0208] T cells may also include "NKT cells." NKT cells are cells that express the semi-invariant αβ T cell receptor It refers to a special population of T cells that express various NK cell types, such as NK1.1, which are typically associated with NK cells. NKT cells also express various molecular markers. These include CD4+, CD4-, CD8+, and CD8 cells. The TCR of NKT cells is , which is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells produce cytokines that promote either inflammation or immune tolerance. Depending on their ability, they can have either protective or harmful effects. Gamma delta T cells also contain different TCRs on their surface. It refers to a special population of a small subset of T cells that express TCR, which is represented by α and β TCR chains. Unlike most T cells, which consist of two glycoprotein chains, The TCR in γδ T cells is composed of γ and δ chains. γδ T cells play a key role in immune surveillance and immune regulation. It is believed that the IL-17 receptor plays a role in the regulation of IL-17 production and is an important source of IL-17, and that it stimulates active CD8 It has also been found to induce cytotoxic T cell responses and suppress abnormal or excessive immune responses. "Regulatory T cells" or "Tregs" refer to T cells that suppress and play a role in immune tolerance Treg cells are typically CD4+ T cells that express the transcription factor Foxp3. In addition, IL-10-producing CD4+ T cells, transcription factor Foxp3-negative regulatory T cells, were also identified. It may include.

[0209] T cell lines are available from, for example, the American Type Culture Collection ion (ATCC, Manassas, VA), and German Collection n of Microorganisms and Cell Cultures(DS MZ), for example, Jurkat cells (ATCC TIB-152), Sup-Tl cells (ATCC CRL-1942), RPMI 8402 cells (DSMZ ACC-290), Karpas 45 cells (DSMZ ACC-545), and Derivatives thereof include:

[0210] In another embodiment, the host cells are natural killer (NK) cells. NK cells are NK cells are a type of cytotoxic lymphocyte that play a role in the innate immune system. Defined as granular lymphocytes, they are common lymphoid precursors that further give rise to B and T lymphocytes. Constitute a third type of cell differentiated from progenitor cells (e.g., immunoglobulins). gy, 5th ed., Janeway et al., eds., Garland P. Publishing, New York, NY (2001). NK cells NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. After maturation, NK cells The cells circulate as large lymphocytes with characteristic cytotoxic granules. Recognizes and kills certain abnormal cells (e.g., certain tumor cells and virus-infected cells). and are thought to be important in innate immune defense against intracellular pathogens. As described above for T cells, NK cells can be any NK cell (cultured NK cells, e.g. NK cells from primary NK cells, or cultured NK cell lines, or NK cells obtained from mammals. When obtained from mammals, NK cells can be derived from a number of sources (blood, bone marrow, etc.). from tissues or fluids, including but not limited to the spinal cord, lymph nodes, thymus, or other tissues or fluids NK cells can also be enriched or purified. NK cells are preferably obtained from human NK cell lines are, for example, American NK cells (e.g., isolated from humans). can Type Culture Collection(ATCC,Manassa s, VA), for example, NK-92 cells (ATCC CRL-2407 ), NK92MI cells (ATCC CRL-2408), and their derivatives. do.

[0211] Also provided is a cell population comprising at least one host cell described herein. , at least one other cell, e.g., a host cell that does not contain any of the recombinant expression vectors. cells (e.g., T cells), or cells other than T cells (e.g., B cells, macrophages, red blood cells, etc.) leukocytes, neutrophils, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc.) It may be a heterogeneous population containing host cells containing any of the recombinant expression vectors. Alternatively, the cell population may be a substantially homogenous population, in which case the population contains a recombinant expression vector. A population may also be primarily comprised of (e.g., consisting essentially of) host cells containing the vector. The recombinant expression vector may be a clonal population of cells, where all cells in the population contain the recombinant expression vector. A clone of a single host cell, so that all cells in the population contain the recombinant expression vector In one embodiment, the cell population comprises a host cell comprising a recombinant expression vector described herein. It is a clonal population that contains the chief cells.

[0212] Pharmaceutical Composition / Administration In embodiments of the present disclosure, the CAR-expressing cells are combined with a composition, e.g., a CAR-expressing cell and a pharmaceutical In one aspect, the present disclosure provides a method for treating a rheumatoid arthritis, comprising administering to a subject a rheumatoid arthritis patient a rheumatoid arthritis, or a rheumatoid arthritis patient, the method comprising administering to a subject ... an effective amount of lymphocytes expressing one or more CARs and a pharmaceutically acceptable carrier; The pharmaceutical compositions of the present disclosure are provided in a pharmaceutical composition comprising a CAR-expressing cell, e.g., A plurality of CAR-expressing cells as described herein can be administered to one or more pharmaceutical or biological It may be included in combination with a pharmaceutically acceptable carrier, excipient, or diluent. The carrier used can be an ingredient in a pharmaceutical composition other than the active ingredient that is non-toxic to a subject.

[0213] Pharmaceutically acceptable carriers may include buffers, excipients, stabilizers, or preservatives. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiologically compatible solvents. dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. For example, salts, buffers, antioxidants, sugars, aqueous or non-aqueous carriers, preservatives, humectants, surfactants The pharmaceutically acceptable carrier in the pharmaceutical composition is a soluble or insoluble carrier, or an emulsifying agent, or a combination thereof. The amount of carrier used will depend on the activity of the carrier and the desired properties of the formulation, e.g., stability and / or minimal oxidation. This can be determined experimentally based on

[0214] Such compositions may contain acetic acid, citric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, Lactic acid, histidine, boric acid, Tris buffer, HEPPSO, HEPES, neutral buffer Buffer solutions such as saline, phosphate buffered saline; glucose, mannose, sucrose or carbohydrates such as dextran and mannitol; proteins; polypeptides such as glycine or amino acids; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); antibacterial and antifungal agents; and preservatives.

[0215] The compositions of the present disclosure can be administered parenterally or by various means of non-parenteral administration. In one embodiment, the composition may be formulated for injection or intravenous administration. The compositions disclosed herein can be prepared, for example, as sterile liquid preparations (e.g., isotonic aqueous solutions, emulsions, etc.). , suspensions, dispersions, or viscous compositions), which may be used to achieve the desired p Formulations suitable for oral administration include liquid solutions, capsules, sachets, and the like. , tablets, drops, and lozenges, powder liquid suspensions in suitable liquids and emulsions Examples include:

[0216] As used herein with respect to pharmaceutical compositions, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for animal and / or human use approved or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia This means that

[0217] In one aspect, the present disclosure provides a method for treating a subject having or at risk of having cancer. , relating to administering genetically modified T cells expressing a CAR using lymphocyte infusion. In at least one embodiment, autologous lymphocyte infusion is used for treatment. Autologous PBMCs is collected from a subject in need of treatment and is administered using methods described herein and known in the art. Using this method, T cells are activated and expanded and then infused back into the subject.

[0218] In one aspect, the present disclosure relates generally to treating subjects at risk of developing cancer. It is also possible that chemotherapy and / or immunotherapy in a subject results in significant immunosuppression. and treating malignant tumors or autoimmune diseases, thereby increasing a subject's risk of developing cancer. In one aspect, the disclosure provides a method for preventing cancer, the method comprising: and administering to a subject in need thereof an amount of lymphocytes expressing one or more CARs to be administered. This includes giving.

[0219] In one aspect, the disclosure provides a method of treating a subject having cancer, the method comprising administering to a subject a therapeutically effective amount of a compound selected from the group consisting of benzodiazepines, ... and administering to a subject in need thereof a therapeutically effective amount of lymphocytes expressing one or more CARs. to the subject, whereby lymphocytes induce or mediate the killing of cancer cells in the subject. To reduce.

[0220] In another aspect, the present disclosure provides a method for reducing tumor burden in a subject having cancer, the method comprising: The method comprises administering a therapeutically effective amount of lymphocytes expressing one or more of the described CARs to the to a subject in need thereof, whereby lymphocytes are administered to a subject in need thereof to treat cancer cells in the subject. In another aspect, the present disclosure provides a method for increasing survival time of a subject having cancer. The method provides a therapeutically effective amount of a phospholipase A1 expressing one or more of the described CARs. and administering the immune system cells to a subject in need thereof, thereby prolonging the survival of the subject. Generally, lymphocytes expressing CAR induce the death of cancer cells in a subject. This results in the reduction or eradication of tumor / cancer cells in the subject. A non-limiting list of cancers includes lung cancer, stomach cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urinary Road cell carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, neuro Glioblastoma, Non-Hodgkin's lymphoma (NHL), Acute lymphoblastic leukemia (ALL), Chronic lymphocytic Leukemia (CLL), chronic myeloid leukemia (CML), smoldering multiple myeloma (SMM), Includes multiple myeloma (MM), acute myeloid leukemia (AML), and combinations thereof In one embodiment, the cancer being treated in the subject is multiple myeloma.

[0221] In one aspect, the methods described herein are directed to treating a cancerous condition (e.g., a hematologic condition such as MM or lymphoma). Monoclonal gammopathy of undetermined significance (monoclonal gammopathy) with risk of developing into hemoglobinoma development of MGUS (metastasis of undetermined significance) and blood cancers such as leukemia Suitable for the treatment of non-cancerous conditions that are at risk of developing into cancer (such as myelodysplastic syndromes, which are at risk of developing into cancer). It is available.

[0222] In one aspect, a method of treating a subject having cancer is provided, the method comprising administering a GPRC5D antibody to a subject. A therapeutically effective amount of lymphocytes expressing a CAR having an extracellular antigen-binding domain that binds to the antigen is administered. to a subject in need thereof, whereby lymphocytes are administered to a subject in need thereof to treat cancer In some embodiments, at least one CAR is selected from the group consisting of SEQ ID NO: 86, 85, 83, 84, 27, 28, 29, 30, 31 and 32 It includes the amino acid sequence.

[0223] In one aspect, a method for targeted killing of cancer cells is disclosed. The method comprises treating the cancer cells with a method as described above. with lymphocytes expressing one or more CARs, thereby Lymphocytes induce the death of cancer cells. Non-limiting examples of cancer cells that can be targeted include metastatic cancer cells. A typical list includes lung cancer cells, stomach cancer cells, colon cancer cells, hepatocellular carcinoma cells, renal cell carcinoma cells, bladder cancer cells, Urothelial cancer cells, metastatic melanoma cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, head and neck cancer cells cells, pancreatic cancer cells, glioma cells, glioblastoma cells, and non-Hodgkin's lymphoma (NHL) cells cells, acute lymphocytic leukemia (ALL) cells, chronic lymphocytic leukemia (CLL) cells, chronic myeloid leukemia leukemia (CML) cells, smoldering multiple myeloma (SMM) cells, multiple myeloma (MM ) cells, acute myeloid leukemia (AML) cells, and combinations thereof. In morphology, the cancer cells are multiple myeloma cells.

[0224] The pharmaceutical compositions of the present disclosure may be administered in a manner appropriate to the disease being treated (or prevented). The amount and frequency of administration will depend on factors such as the condition of the subject and the type and severity of the subject's disease. However, appropriate dosages can be determined through clinical trials.

[0225] The terms "treat" or "treatment" refer to the delay of an undesired physiological change or disease. to reduce or produce a beneficial or desired clinical result during treatment A beneficial or desired clinical outcome is a detectable or Whether or not the disease is undetectable, it may be accompanied by a reduction in symptoms, a reduction in the extent of the disease, or a stable ( i.e., not worsening) the disease state, delaying or slowing the progression of the disease, improving or alleviating the disease state "Treatment" includes the treatment of the rheumatoid arthritis, rheumatoid arthritis, and remission (whether partial or total). and prolonging survival compared with the expected survival if the subject did not receive treatment. A subject in need of treatment may be any person experiencing an undesired physiological change or Subjects who already have the disease, as well as subjects who are prone to physiological changes or disease, Included.

[0226] "Therapeutically effective amount" or "effective amount," as used interchangeably herein, refers to the dosage required. A therapeutically effective amount refers to an amount, at a dose and for a period of time, effective to achieve the desired therapeutic result. factors such as condition, age, sex, and weight, as well as therapeutic agents that induce the desired response in the individual. or combination of therapeutic agents. Examples of combination endpoints include improved patient health, reduced tumor burden, and tumor progression. These include an arrest or slowing of growth, and / or the absence of metastasis of cancer cells to other locations in the body.

[0227] As used herein, the term "subject" refers to an animal. "Subject" and "Patient" The term "subject" may be used interchangeably herein with respect to the subject. " includes humans who are treated as patients, either for disease or for prevention of disease.

[0228] The methods described herein can be used to treat any category of animal subject. Examples of such animals include mammals. Mammals include rodents (e.g., rodents, mammals of the order Lagomorpha (such as rabbits) and mammals of the order Pterygomorpha (such as hamsters and mice) Mammals include, but are not limited to, felines (cats) and canines (dogs). Mammals may be from the order Carnivora, including Bovidae (cattle) and Suidae. may be from the order Artiodactyla, which includes animals (pigs), or Perissodactyla, which includes animals (horses) Mammals are classified into the order Primates, Ceboids, or Simoids (monkeys) In one embodiment, the mammal is a human. do.

[0229] When a therapeutically effective amount is indicated, the precise amount of the composition of the present disclosure to be administered will depend on the age, body mass, and other factors of the subject. Considering the severity, tumor size, degree of infection or metastasis, and individual differences in condition, The pharmaceutical compositions comprising the T cells described herein can be determined by the following: Contains all integer values, approximately 10 4 ~about 10 10 cells / kg body weight, in some cases approximately 10 5 ~about 10 6 It can be generally stated that the dose of the cells / kg body weight can be administered. In some embodiments, the pharmaceutical compositions comprising the T cells described herein may be administered in a dose of about 10 6 Dosing of cells / kg body weight The T cell compositions may also be administered multiple times at these doses. , can be administered by using injection techniques commonly known in immunotherapy. (e.g., Rosenberg et al., New Eng. J. of Me d.319:1676,1988).

[0230] Delivery systems useful in the context of embodiments of the present invention include sustained release, delayed release, and sustained release. The T cell composition may comprise a rapid release delivery system, wherein delivery of the T cell composition occurs prior to sensitization of the area to be treated. The composition may be administered in the presence of other therapeutic agents or for a period of time sufficient to induce the same. Such a system avoids repeated administration of the composition. This increases convenience for patients and physicians, and the specific composition of the present invention This may be particularly suitable for the embodiment.

[0231] Many types of release delivery systems are available and known to those of ordinary skill in the art. , poly(lactide-glycolide), copolyoxalate, polyesteramide, polyol polymers such as diol esters, polycaprolactone, polyhydroxybutyric acid, and polyanhydrides The drug-containing polymer microcapsules described above are based on, for example, The delivery system is described in US Patent No. 5,075,109. sterols, such as cholesterol, cholesterol esters, and fatty acids, or mono- and di-glycerides and non-polymeric systems that are lipids, including neutral fats such as triglycerides; sylastic systems; peptide-based systems; hydrogel release systems; wax coated tablets; compressed tablets using conventional binders and excipients; partially fused filled tablets Specific examples include (a) a matrix containing an active ingredient; erosion systems (e.g., U.S. Pat. Nos. 4,452,775; 4,667, 014; 4,748,034; and 5,239,660. etc.) and (b) diffusion systems in which the active ingredient permeates through the polymer at a controlled rate (e.g. For example, those described in U.S. Patent Nos. 3,854,480 and 3,832,253. In addition, pump-based hardware Delivery systems can be used, some of which are adapted for implantation.

[0232] In certain embodiments, activated T cells are administered to a subject, after which blood is re-drawn (or afferent) and the subject is then given a second dose of activated T cells. and performing a T cell lysis to activate the T cells according to the present disclosure, and these activated and expanded T cells It may be desirable to reinject the cells into the subject. This process may be repeated multiple times every few weeks. In certain embodiments, T cells are activated from a blood draw of 10 cc to 400 cc. In certain embodiments, T cells can be activated in 20cc, 30cc, 40cc, 50cc, It is activated by drawing 60cc, 70cc, 80cc, 90cc, or 100cc of blood.

[0233] The CAR-T cells and compositions can be administered by any method, for example, parenteral or oral administration ( administered by aerosol inhalation, injection, infusion, ingestion, transfusion, implantation, or transplantation For example, the CAR-T cells and compositions described herein can be administered transdermally. by intravenous (iv) injection via intravenous, intradermal, subcutaneous, intratumoral, intramedullary, intranodal, or intramuscular routes; In one aspect, the compositions of the present disclosure can be administered to a patient by intravenous injection. In one aspect, the compositions of the present disclosure are administered by intradermal or subcutaneous injection. The composition of T cells is administered to an elephant, for example, in a tumor, lymph node, tissue, organ, or site of infection. It can be injected directly into the area.

[0234] Administration can be self-administration or non-self-administration. For example, G protein-coupled receptors (e.g., For example, immunoresponsive cells expressing a GPRC5D)-specific CAR can be obtained from one subject. The peripheral blood-derived T cells or expanded T cells of the present disclosure may be administered to the same subject or to different subjects. (e.g., of in vivo, ex vivo, or in vitro origin) may be, for example, Administered via intravenous injection, local injection, systemic injection, catheter administration, or parenteral administration can be done.

[0235] In certain embodiments, the subject may undergo leukapheresis, in which case leukapheresis Blood cells can be collected, enriched, or depleted ex vivo to generate cells of interest, e.g., T cells. These isolated T cells are selected and / or isolated using methods known in the art. and can be propagated by the method of The cells are then processed so that they can be used to generate CAR-T cells. Subjects in need can then undergo standard chemotherapy with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after or concurrently with transplantation, the subject may undergo treatment with a proliferative In one embodiment, the expanded cells are administered before or after surgery. It is given.

[0236] The dosage administered to a patient with a malignant tumor is sufficient to alleviate or at least reduce the severity of the disease being treated. A therapeutically effective amount is an amount sufficient to at least partially inhibit the growth of the tumor (a "therapeutically effective amount"). The dosage of the treatment will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for human administration is generally accepted in the art. This can be done in accordance with accepted practice.

[0237] The CART T cells of the present invention are capable of undergoing in vivo T cell proliferation and are capable of inducing T cell proliferation in the blood. Establishment of GPRC5D-specific memory cells that persist at high levels for long periods in the serum and bone marrow In some examples, the CAR T cells of the invention infused into a subject can be In subjects with advanced chemotherapy-resistant cancer, cancer cells (e.g., white blood cells) are expressed in vivo. It can eliminate blood cells.

[0238] In one embodiment, a CAR of the present disclosure is expressed in T cells, e.g., using in vitro transcription. The CAR-T cells of the present disclosure are introduced into a subject (e.g., a human) and the subject (e.g., a human) is first administered the CAR-T cells of the present disclosure to induce CAR-T cell proliferation. One or more subsequent administrations of RT cells. In this case, the one or more subsequent administrations are , less than 15 days after the last dose, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6 In one embodiment, the CAR-T cells are administered to a subject (e.g., , human) more than once a week, e.g., administering CAR-T cells 2, 3, or 4 times a week. In one embodiment, the subject receives CAR-T cells two or more times per week (e.g., For example, two, three, or four doses per week (also referred to herein as a cycle), followed by A CAR-T cell-free week followed by one or more additional CAR-T cell doses In another embodiment, the subject is administered CAR-T cells twice or more per week. CAR-T cells were administered in two or more cycles, with the time between cycles varying between 10, 9, 8, and 10 cycles. In one embodiment, the CAR-T cells are administered for less than 7, 6, 5, 4, or 3 days. In one embodiment, the CAR-T cells are administered every other day for a single dose. Both for 2, 3, 4, 5, 6, 7, 8 or more weeks. It is administered immediately.

[0239] In one embodiment, the administration is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 Weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, or The treatment may be repeated at a later date. Treatment may also be repeated, and administration may be prolonged. Repeated administrations may be of the same dose or of different doses.

[0240] CAR-T cells may be administered as a maintenance therapy, e.g., once a week for six months or longer. can be administered in the methods of the present invention by

[0241] In one embodiment, the CAR-T cells are delivered via a lentiviral virus vector, such as a lentivirus. CAR-T cells generated using such viral vectors are Generally has stable CAR expression.

[0242] In one embodiment, the CAR-T cells are 4, 5, 6, 7, 8, 9, 10, 11 Transient expression of CAR vectors was observed for 12, 13, 14, and 15 days. Expression can be affected by delivery of an RNA CAR vector. The RNA is transduced into T cells by electroporation.

[0243] During transient CAR therapy (such as those generated by RNA transduction), patients develop antibodies If there is a high risk of generating a CAR antibody response, interruption of CAR T infusion should be considered after 10–14 days. It must not be exceeded.

[0244] The CAR-expressing cells described herein can be used in combination with other known drugs and therapies. As used herein, "administered in combination" refers to a combination of During the course of treatment, two (or more) different treatments may be delivered to the subject, e.g., after a patient has been diagnosed with cancer, but before the cancer has been cured or eliminated, or for other reasons, treatment is discontinued This means that two or more treatments are delivered before being discontinued. In some embodiments, delivery of one treatment is still occurring when delivery of the second treatment begins. Therefore, there is an overlap in administration. This is referred to herein as "simultaneous" or "co-delivery." In other embodiments, the delivery of one therapy may be delayed until the delivery of another therapy is complete. In some embodiments in either case, the combination administration results in a therapeutic The second treatment is more effective, e.g., the ... The second treatment is at least seen, or the second treatment is seen without the first treatment. The symptoms are significantly less than those seen when the first treatment is administered, or the condition is similar to that of the first treatment. In some embodiments, delivery is achieved by reducing symptoms or other parameters associated with the disorder. The relief observed from one treatment delivered without the other treatment is greater than that observed from one treatment delivered without the other treatment. The effects of the two treatments are either partially additive or totally additive. The delivery may be additive or greater than additive. The effect of the first treatment delivered may be greater than the effect of the second treatment. The antibody may be such that it is still detectable upon delivery of the antibody.

[0245] In one embodiment, other therapeutic agents, such as factors, are administered before, after, or together with (or in combination with) the CAR-T cells. Sometimes, interleukins (e.g., IL-2, IL-3, IL-6) can be administered. , IL-7, IL-11, IL-12, IL-15, IL-21, and other interferons. colony-stimulating factors such as G-, M- and GM-CSF, and interferons Examples of anti-inflammatory drugs include, but are not limited to, interferons (e.g., gamma interferon).

[0246] The CAR-expressing cells described herein and at least one additional therapeutic agent are administered simultaneously in the same In the case of sequential administration, the compounds of the present invention may be administered in separate compositions or sequentially. The CAR-expressing cells described herein may be administered first, followed by the additional agent, or the order of administration may be different. The order can be reversed.

[0247] In further embodiments, the CAR-expressing cells described herein are administered after surgery, radiation, chemotherapy, Immunosuppressants, such as methotrexate, cyclosporine, azathioprine, mycophenone acid, and FK506, antibodies, or other immunoablative agents, e.g. , anti-CD3 antibodies or other antibody therapy drugs, cytoxin, fludarabine, cyclosporine Rosporin, FK506, rapamycin, mycophenolic acid, steroids, FR9012 28, can be used in therapeutic regimens in combination with cytokines and radiation.

[0248] In one embodiment, the CAR-expressing cells described herein are used in combination with a chemotherapeutic agent. For example, chemotherapeutic agents include anthracyclines (e.g., doxorubicin, rubicine (e.g., liposomal doxorubicin), vincinine alkaloids (e.g., bicarbonate vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), Immune cell antibodies (e.g., alemtuzamab, gemtuzumab, rituximab, tositumomab) , antimetabolites (e.g., antifolates, pyrimidine analogs, purine analogs, and adenosine mTOR inhibitors, including deaminase inhibitors (e.g., fludarabine), TNFR glycoproteins, Gluticoid-induced TNFR-related protein (GITR) agonists, proteasome inhibitors ( e.g., aclacinomycin A, gliotoxin or bortezomib), immunomodulatory drugs e.g. , thalidomide or a thalidomide derivative (e.g., lenalidomide), Not limited to:

[0249] A non-exhaustive list of chemotherapy agents being considered for use in combination therapy includes anastrozole. (Arimidex®), bicalutamide (Casodex®) , bleomycin sulfate (Blenoxane®), busulfan (Myle ran®), leucovorin calcium, melphalan (Alkeran®), trademark), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxatrone (Novantrone®), ), Mylotarg, paclitaxel (Taxol®), Phoenix (Ytt implants containing Calcium 90 / MX-DTPA, pentostatin, and carmustine Lifeprosan 20 (Gliadel®), dactinomycin (Actino mycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine ( Daunorubicin Citrate Liposomal Injection (DaunoXome®) trademark), dexamethasone, docetaxel (Taxotere®), doxol Bicine hydrochloride (Adriamycin®, Rubex®), etoposide Vepesid (registered trademark), Busulfan injection (Busulfex (registered trademark) Capecitabine (Xeloda®), N4-pentoxycarbonyl-5- Deoxy-5-fluorocytidine, carboplatin (Paraplatin®) ), carmustine (BiCNU®), chlorambucil (Leukeran®), (Platinol®), cisplatin (Leucine®), cladribine (Leucine®), tatin®), cyclophosphamide (Cytoxan® or Ne Cytosar®), cytarabine, cytosine arabinoside (Cytosar-U®), (Registered Trademark), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®) fludarabine phosphate (Fludara®) )), 5-fluorouracil (Adrucil®), Efudex® )), flutamide (Eulexin®), tezacitibine, gemcitabine (Dif Fluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin Idamycin (registered trademark), ifosfamide (IFEX (registered trademark), Ili Notecan (Camptosar®), L-asparaginase (ELSPAR®), tamoxifen citrate (Nolvadex®), Teniposin® Vumon®, 6-thioguanine, thiotepa, tirapazamine zone®), injectable topotecan hydrochloride (Hycamptin®) , vinblastine (Velban®), vincristine (Oncovin®), These include vinorelbine (Navelbine®), and vinorelbine (Navelbine®).

[0250] Examples of alkylating agents include nitrogen mustard, ethyleneimine derivatives, sulfur Alkyl benzoates, nitrosoureas, and triazenes: uracil mustard (Aminoura cil Mustard®, Chlorethaminacil® , Haemanthamine®, Nordopan®, Urac Nitrogen Mustard (registered trademark), Uracillost (registered trademark) trademark), Uracilmostaza (registered trademark), Uramustin (registered trademark), U ramustine (registered trademark), chlormethine (Mustargen (registered trademark)) , cyclophosphamide (Cytoxan®, Neosar®, Cl Afen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), Lupharan (Alkeran®), chlorambucil (Leukeran®) trademark), pipobroman (Amedel®, Vercyte®), Triethylene melamine (Hemel®, Hexylen®, Hex astat®), Demethyldopan®, Desmeth yldopan®, triethylenethiophosphoramine, temozolomide dar®), thiotepa (Thioplex®), busulfan (B usilvex®, Myleran®), carmustine (BiCN U®), lomustine (CeeNU®), streptozocin (Zan osar®), and dacarbazine (DTIC-Dome®). Further examples of alkylating agents include, but are not limited to, oxaliplatin. (Eloxatin®); melphalan (L-PAM, L-sarcolysin, and and phenylalanine mustard, also known as Alkeran®); Altretamine (as hexamethylmelamine (HMM), Hexylen®) carmustine (BiCNU®); bendamustine (Tre anda®); busulfan (Busulfex® and Myler®) an(R); carboplatin (Paraplatin(R)); temozolomide mide (Temodar® and Temodal®); dactinomycin Actinomycin-D (also known as Cosmegen®); cisplatin (CCNU, also known as CeeNU®); cisplatin (CDDP Also known as Platinol® and Platinol®-AQ chlorambucil (Leukeran®); cyclophosphamide (Cy toxan® and Neosar®; dacarbazine (DTIC, D IC and imidazole carboxamide, also known as DTIC-Dome® altretamine (hexamethylmelamine (HMM), Hexylen (registered trademark) Ifosfamide (Ifex®); Prednum procarbazine (Matulane®); mechlorethamine (Nitrogen®); Mustard, mustine and mechlorethamine hydrochloride, Mustargen® also known as); streptozocin (Zanosar®); thiotepa (Thiophosphoamides, TESPA and TSPA, also known as Thioplex®) known); cyclophosphamide (Endoxan®, Cytoxan®) Registered trademark), Neosar®, Procytox®, Revimm® and bendamustine HCl (Treanda®). Examples include, but are not limited to:

[0251] Examples of immunomodulatory agents useful herein include, for example, afutuzumab (Roche (trademark); pegfilgrastim (Neulasta®), Lena Lidomide (CC-5013, Revlimid®); Thalidomide (Thal omid (registered trademark), Actimid (CC4047), and IRX-2 (Interloop A mixture of human cytokines including interleukin-1, interleukin-2, and interferon-gamma , CAS 951209-71-5, available from IRX Therapeutics) These include, but are not limited to:

[0252] In one embodiment, the subject can be administered an agent that enhances the activity of the CAR-expressing cells. For example, in one embodiment, the agent can be an agent that inhibits an inhibitory molecule. For example, Programmed Death 1 (PD1) is used in some embodiments. This can reduce the ability of CAR-expressing cells to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, and VIS. TA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta Examples include:

[0253] A description of exemplary embodiments follows.

[0254] Embodiment 1. Extracellular antigen-binding domain, transmembrane domain, and intracellular signaling domain A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising: Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 66, SEQ ID NO: 6 7, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 68. CDR3, Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 5 heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 9, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 60 CDR3, Heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 4 0, and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 41. CDR3, heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 42, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 43 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 44, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 45; heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 45, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 46 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 47; and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 48. Including, The extracellular antigen-binding domain is anti-G protein receptor family C group 5 member D (GPRC5D) antigen binding, Chimeric antigen receptor.

[0255] Embodiment 2. heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 66, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 67 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 68, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 69; the outer antigen-binding domain is 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: 7 0, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 71. further comprising CDR3, heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 58, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 59 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 60; and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 60. the outer antigen-binding domain is 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: 6 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 63 further comprising CDR3, heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 39, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 40 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 41, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 42. the outer antigen-binding domain is 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: 4 a light chain CDR2 having the amino acid sequence of SEQ ID NO: 9, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 50; further comprising CDR3, heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 42, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 43 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 44, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 45. the outer antigen-binding domain is 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: 5 a light chain CDR2 having the amino acid sequence of SEQ ID NO:2, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:53 further comprising a CDR3; or heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 45, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 46 a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 47, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 48. the outer antigen-binding domain is 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: 5 a light chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:56 further comprising CDR3, CAR according to embodiment 1.

[0256] Embodiment 3. The extracellular antigen binding domain comprises SEQ ID NOs: 73, 65, 1, 3, and 5. a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of: a heavy chain variable region (HC) comprising an amino acid sequence selected from the group consisting of 64, 2, 4, and 6; VR), or an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 65, 1, 3, and 5 and an LCVR comprising the sequence of SEQ ID NO: 72, 64, 2, 4, and 6. 3. The CAR of embodiment 1 or 2, comprising a combination with an HCVR comprising an amino acid sequence.

[0257] Embodiment 4. The extracellular antigen-binding domain comprises: a light chain variable region comprising the amino acid sequence of SEQ ID NO: 73 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 72; a heavy chain variable region comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 65 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64; a heavy chain variable region comprising 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 chain variable region, A light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 chain variable region, or 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 chain variable region, A CAR according to embodiments 1 to 3.

[0258] Embodiment 5. The extracellular antigen-binding domain comprises a single-chain variable fragment (scFv). A CAR according to any one of embodiments 1 to 4.

[0259] Embodiment 6. The scFv comprises a linker polypeptide between the light chain variable region and the heavy chain variable region. 6. The CAR of embodiment 5, comprising:

[0260] Embodiment 7. Embodiment 6, wherein the linker polypeptide comprises the amino acid sequence of SEQ ID NO: 7. The CAR described in

[0261] Embodiment 8. The scFv is selected from the group consisting of SEQ ID NOs: 78, 77, 75, 76, 8, 9, 10, 24, 2 8. Any of embodiments 5 to 7, comprising an amino acid sequence selected from the group consisting of: 5, and 26. CAR as described in one.

[0262] Embodiment 9. The extracellular antigen-binding domain of any one of embodiments 1 to 10, wherein the extracellular antigen-binding domain comprises a signal polypeptide. 9. A CAR according to any one of items 8 to 9.

[0263] Embodiment 10. An embodiment in which the signal polypeptide comprises the amino acid sequence of SEQ ID NO: 11. 9. The CAR according to claim 9.

[0264] Embodiment 11. The intracellular signaling domain is a member of the TNF receptor superfamily. Bar 9 (CD137) component, T cell surface glycoprotein CD3 zeta chain (CD3z) component, Cluster of differentiation (CD27) components, cluster of differentiation superfamily member components, and

[0023] Embodiments 1 to 10 include a polypeptide component selected from the group consisting of: CAR according to any one of the preceding claims.

[0265] Embodiment 12. Embodiment 11, wherein the CD137 component comprises the amino acid sequence of SEQ ID NO: 12. The CAR described in

[0266] Embodiment 13. The method of embodiment 11, wherein the CD3z component comprises the amino acid sequence of SEQ ID NO: 13. CAR featured.

[0267] Embodiment 14. The intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 14. 12. A CAR according to embodiment 11.

[0268] Embodiment 15. The transmembrane domain is a CD8a transmembrane region (CD8A-TM) polypeptide. 15. The CAR of any one of embodiments 1 to 14, comprising:

[0269] Embodiment 16. The CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO: 15. 16. A CAR according to embodiment 15.

[0270] Embodiment 17. The polypeptide further comprises a hinge region connecting the transmembrane domain to the extracellular antigen-binding domain. 17. The CAR of any one of embodiments 1 to 16, comprising:

[0271] Embodiment 18. The CA of embodiment 17, wherein the hinge region is a CD8a hinge region. R.

[0272] Embodiment 19. The embodiment wherein the CD8a hinge region comprises the amino acid sequence of SEQ ID NO: 16. 18. The CAR according to claim 18.

[0273] Embodiment 20. The extracellular antigen-binding domain is selected from the group consisting of SEQ ID NOs: 82, 81, 80, 79, 17, 18, 19, 20, 21, and 22. 20. The CAR according to any one of aspects 1 to 19.

[0274] Embodiment 21. The CAR is selected from the group consisting of SEQ ID NOs: 86, 85, 83, 84, 27, 28, 29, 30 21. The method of any one of embodiments 1 to 20, comprising an amino acid sequence selected from the group consisting of: CAR described in one of the above.

[0275] Embodiment 22. An isolated antibody expressing a CAR according to any one of embodiments 1 to 21. Lymphocytes.

[0276] Embodiment 23. The isolated lymphocyte of embodiment 22, wherein the lymphocyte is a T lymphocyte. Pac-ball.

[0277] Embodiment 24. An isolated polypeptide encoding a CAR according to any one of embodiments 1 to 21. Nucleic acid molecules.

[0278] Embodiment 25. The nucleic acid molecule is selected from the group consisting of SEQ ID NOs: 90, 89, 87, 88, 33, 34, 35, 3 25. The method of claim 24, comprising a nucleic acid sequence selected from the group consisting of: 6, 37, and 38. Isolated nucleic acid molecules.

[0279] Embodiment 26. The nucleic acid molecule is selected from the group consisting of SEQ ID NOs: 90, 89, 87, 88, 33, 34, 35, 3 6, 37 and 38 nucleotide sequences and at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Contains a nucleotide sequence that is 94%, 95%, 96%, 97%, 98% or 99% identical 25. The isolated nucleic acid molecule of embodiment 24.

[0280] Embodiment 27. A vector comprising the nucleic acid molecule according to any one of embodiments 24 to 26. .

[0281] Embodiment 28. A cell expressing a nucleic acid molecule according to any one of embodiments 24 to 26.

[0282] Embodiment 29. A method for treating a patient with a lymphoma comprising administering an effective amount of lymphocytes according to any one of embodiments 22 to 23. Pharmaceutical compositions.

[0283] Embodiment 30: An effective amount of lymphocytes and a medicament according to any one of embodiments 22 to 23. A pharmaceutical composition comprising a physiologically acceptable excipient.

[0284] Embodiment 31. CA according to any one of embodiments 1 to 21 for use in therapy. R, or the pharmaceutical composition of embodiment 29 or 30.

[0285] Embodiment 32. A method of treating a subject with cancer according to any one of embodiments 1 to 21. A CAR according to any one of embodiments 29 or 30, or a pharmaceutical composition according to embodiment 30.

[0286] Embodiment 33. A method of treating a subject having cancer, comprising: A therapeutically effective amount of the lymphocytes according to any one of embodiments 22-23 is administered to a patient in need thereof. to a subject, whereby lymphocytes induce the death of cancer cells in the subject. How to do it.

[0287] Embodiment 34. The cancer is lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic Metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, and Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia ( CLL), chronic myeloid leukemia (CML), smoldering multiple myeloma (SMM), multiple bone marrow leukemia (MBL) From the group consisting of myeloma (MM), acute myeloid leukemia (AML), and combinations thereof Selected for use in the method according to embodiment 33 or the method according to embodiment 32 CAR or pharmaceutical composition thereof.

[0288] Embodiment 35. The method of embodiment 33 or 34, wherein the cancer is multiple myeloma, or 35. A CAR or pharmaceutical composition for use in the method of embodiment 32 or 34.

[0289] Embodiment 36. A method for targeted killing of cancer cells, comprising: 24. A method for treating a cancer cell, comprising contacting the cancer cell with a lymphocyte according to any one of embodiments 22-23. thereby inducing lymphocytes to kill cancer cells.

[0290] Embodiment 37. The cancer cells are 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 lymphoblastic leukemia (ALL), ), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), smoldering multiple bone marrow disease Myeloma (SMM), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors 37. The method of embodiment 36, wherein the tumor is selected from the group consisting of:

[0291] Embodiment 38. The method of embodiment 36 or 37, wherein the cancer cells are multiple myeloma cells. Law.

[0292] Embodiment 39. A method for detecting the presence of cancer in a subject, comprising: (a) contacting a cell sample obtained from a subject with the CAR of claim 1 forming a CAR-cell complex; and (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the subject; Methods. Preferably, the embodiments are performed in vitro. [Example]

[0293] The following examples are provided to further illustrate some of the embodiments disclosed herein. These examples are intended to be illustrative and not limiting of the embodiments of the present disclosure. It's not something like that.

[0294] Example 1: Expression of GPRC5D scFv CAR No mRNA (Mock) or α-GPRC5D scFv CAR or isoform Primary human pan-T cells were transfected with 10 μg of mRNA expressing either the CAR or control CAR. 24 hours after electroporation, biotinylated L protein was by flow cytometry after staining with streptavidin-conjugated PE. CAR surface expression was measured using the CAR staining assay (Figure 1). CAR expression was assessed by staining with protein L. The expression rates were determined by the following criteria: CAR A, CAR B, CAR C, and isotype. The results were 95, 88, 70, and 67% positive for CAR, respectively. The black T cells were electroporated only, but no mRNA was added to the cells. As expected, Mock T cells did not express any CAR constructs. These data indicated that T cells highly expressed the CAR construct. The histograms are mock, and the filled grey histograms are CAR-T populations. is.

[0295] The amino acid sequences of each component of the GPRC5D-targeting CAR construct are shown in Table 2. there were.

[0296] [Table 2-1]

[0297] [Table 2-2]

[0298] Example 2: Induction of activation markers in antigen-stimulated CAR-T cells 24 hours after transient transfection, primary pan-T cells were cultured using Cell Trace V. The cells were labeled with iolet (CTV) fluorescent proliferation dye and then co-transfected with the multiple myeloma cell line H929. After 4 days of co-culture, cells were pre-gated on the CD8+CD4 population and CAR Surface expression of activation markers CD25 and CD71 on T cells was assessed using α-CD3 / CD40 / T cells alone or in α-CD40 / T cells. This was compared with T cells cultured in the presence of 28 beads (Figure 2). The same CAR T cells as in Figure 1 were analyzed for T cell activation responses to GPRC5D antigen-expressing H929 cells. Four days after electroporation, CAR A and CAR B showed increased T cell activation responses. This was due to increased CD25 and CD71 expression in response to incubation with H929 cells. In comparison, Mock and CAR C-expressing cells responded to H929 cells. As a negative control, GPRC5D or T cell antigen was administered. The same cells were grown in the absence of either and showed minimal increases in CD25 or CD71. As positive controls, Mock, CAR A, CAR B, and CAR C cells were cultured at C Each of the four cell populations was co-cultured with CD25 and CD7 / CD28 beads. Overall, these data demonstrate a similar increase in CAR A and CAR 1 expression. RB cells showed the greatest T cell activation response to GPRC5D-expressing H929 cells This indicates that...

[0299] Example 3: Cytokine profiling of antigen-stimulated CAR-T cells Primary pan-T cells transiently transfected with CAR-expressing mRNA 24 hours prior , high (+++) at a 1:1 ratio with various myeloma cell lines expressing the target antigen GPRC5D, Co-culture was performed at medium (++), low (+) or negative (-) levels. After 16 hours of co-culture, the supernatant was collected. The supernatant from the co-culture was analyzed by Meso Scale Discovery (MSD). Cytokine profile analysis was performed (Figure 3). GPRC5D CAR-T cells The supernatants of the 1000 cells showed an increase in the pro-inflammatory cytokines INF-γ, TNF-α, or IL-2. The increase in each of the pro-inflammatory cytokines was associated with the increase in GPRC5D expression. The cell lines were co-cultured with GPRC5D CAR-T cells. Co-culture with MM.1S showed the highest Although co-culture with KMS11 cells also resulted in high INF-γ and TNF-α expression, co-culture with KMS11 cells resulted in the highest INF-γ and TNF-α expression. The K562 cell line was used as a negative control and non-GPR Basal background cytokine levels were demonstrated with C5D-expressing cell lines.

[0300] 24 hours after transient transfection with CAR-expressing mRNA, primary pan T cells were Autologous peripheral blood mononuclear cells (PBMCs) were co-cultured with multiple myeloma cell line H929 (1:1: After 16 hours of co-culture, the supernatants were collected. All three populations (CAR-T, H929 Cytokine profiling by MSD of supernatants from co-cultures containing PBMCs (autologous PBMCs) The analysis was performed on control co-cultures without CAR-T cells, PBMCs, or H929 alone. The results were compared with those of each cultured cell population (Figure 4). As in Figure 3, the results were compared with those of the mock and CAR-T cells. A, CAR-T B, CAR-T C, or isotype CAR-T cells were administered to H929 and When H929 cells were co-cultured with PBMCs, TNF-α was increased. Increased IL-α and IL-2 were observed in CAR-T A and CAR-T B cells. When RT-CC cells were co-cultured with H929 and PBMCs, limited or undetectable (li IFN-γ release was observed at a limited-to-no detectable level. TC cells showed a decrease in cytokine release, consistent with the decrease in T cell activation markers in Figure 2. CAR-T, H929, and PBMC cells were cultured alone as negative controls. , indicated background cytokine levels.

[0301] Example 4: Targeted cytotoxicity of GPRC5D-expressing tumor cell lines: high / low / non-expressing GPRC5 D target cell line The cytotoxic potential of CAR-T cells against GPRC5D-expressing myeloma cells was assessed using flow cytometry. Primary pan-T cells (transiently transfected with one of the three CARs previously described) were used to assess the efficacy of CAR-mediated immunization. (expressed in the IL-11 region) at the indicated effector:target (E:T) ratios on fluorescently labeled myeloma cells. The cells were co-cultured with the following strains: MM1R, H929, and K562 for 8 hours. At this point, the co-cultures were viable. The killing rate was calculated relative to the number of viable targets cultured without CAR-T cells. The absolute ratio of viable (viable dye-negative) target (CTV-positive) cells remaining in the co-culture (Figure 5). CAR A, CAR B, CAR C, isotype CAR, and Mo ckT cells were cultured in the GPRC5D+ cell lines MM1R and H929 or GPRC5D-K562 The cells were co-cultured for 8 hours. Various effector-to-target ratios were analyzed, and CAR A, CAR B, and It was shown that CAR C cells can induce cytotoxicity in MM1R cells. Increased toxicity correlated with a higher effector-to-target ratio. The k control was included as a control and showed approximately 20-30% cytotoxicity. CAR A and CAR B cells induced cytotoxicity of H929 cells. These data show that the cells exhibited cytotoxic activity similar to that of the isotype and mock cells. The specificity of CAR A cells and CAR B cells for MM1R and H929 cells was investigated. However, specificity for K562 cells (GPRC5D) is not shown.

[0302] Example 5: Targeted cytotoxicity of GPRC5D-expressing tumor cell lines: T cells derived from multiple myeloma patients cell The cytotoxic potential of CAR-T cells against GPRC5D-expressing myeloma cells was assessed using flow cytometry. The T cells of primary multiple myeloma patients (the two CARs mentioned above) were evaluated by immunocytochemistry. Transiently expressing one of the 5'-terminal nucleotides, the effector:target (E:T) ratios indicated, were transfected with fluorescently labeled 5'-terminal nucleotides. The identified myeloma cell line MM.1S or the control GPRC5D-negative cell line KG-1 for 48 hours At this point, the co-cultures were stained with a viability dye. The cytotoxicity rate was calculated as 0.05, which indicates cell death. Viability was assessed as the percentage of fluorescently labeled cells that stained positive for the viability dye (Figure 6). Primary multiple tumors transduced with either GC5B81_LH or GC5B483_LH Both T cells from multiple myeloma patients showed increased amounts of effector cells compared to mock T cells. GPRC5D-KG-1 cells were treated with GPRC5D+MM.1S cells and induced cytotoxicity. When co-cultured with cell lines GC5B81_LH or GC5B483_LH GPRC5D There was no observable cytotoxicity with CAR-T cells. These data are in agreement with the GPRC Killing of GPRC5D+ cells by multiple myeloma T cells transduced with 5D CAR These results indicate that GPRC5D cells can be induced to die, but not to die.

[0303] Example 6: Antigen-stimulated proliferative response of α-GPRC5D CAR-T cells The proliferation potential of α-GPRC5D CAR-T cells was assessed by flow cytometry. Fluorescently labeled pan-T cells (α-GPRC5D CAR or isotype CAR control) were transiently cultured. The α-glucanase (e.g., α-glucanase ... as the absolute number of viable (viable dye-negative) CAR-T cells with a fluorescently labeled (CTV) Proliferation in response to H929 was measured 4 days after α-CD3 / CD28 bead stimulation. The CAR-T proliferation was compared with that of CAR-Ts cultured alone (no stimulation control). (Figure 7) Mock, isotype CAR, CAR-T A, CAR-T B, and CAR- TC cells were stimulated with GPRC5D+H929 cells, CD3 / CD28 beads, or without stimulation. After 4 days, CD4 and CD8 populations were analyzed for total viability and GPRC5D This reflects the proliferation ability of cells in response to antigen. Used as a control, all cell populations showed an increase in CD4 events compared to no stimulation. Only CAR-TA and CAR-T B cells showed significant improvement compared to isotype and mock. The CD8 population showed robust increases in numbers in response to H929 cells. Similar results were observed in the CD8 population. However, CAR-TC showed no proliferative response in response to H929 cells. Collectively, these data suggest that CAR-T A and CAR-T B cells , showing proliferation in response to GPRC5D+H929 cells.

[0304] Example 7: Expression of GPRC5D-CAR in healthy donor T cells Using the Miltenyi Biotec T cell TRANSACT system, six Primary human pan-T cells were activated and expanded. 24 hours after activation, T cells were treated with GPR Nucleotide sequence encoding C5D GC5B680-N68S-LH CAR (sequence Cells were transduced with lentivirus containing IgG (number 90). Cells were grown for 12 days and transduced on days 6 and 1. On day 0, the cells were visualized by flow cytometry using a commercially available rabbit anti-human H+L detection antibody. CAR expression was measured. The results are shown in Figure 8. Data are the mean ± SD from six healthy donors. The gray bar just to the left of the black bar (GCB6800-N68S-LH) represents SD. The shaded bar (Mock) indicates a bar obtained using a commercial antibody in non-transduced (Mock) cells. The black bars represent the background CAR detection. CAR-T cells transduced with N68S-LH are shown.

[0305] Example 8: Expression of CD4, CD8, and memory markers in GPRC5D CAR-T cells current Pan T cells from six healthy donors were treated with GPRC5D GC5B680-N68S-L H CAR encoding the nucleotide sequence (GPRC5D CAR; SEQ ID NO: 90) Mock (non-transduced) cells and transduced cells [surface Cells bearing CAR (CAR-expressing or CAR + ) and cells without surface CAR (CAR Non-expressing or CAR - ) to form a "T cell pool" containing surface CD4 (CD4 + ) The proportion of cells with surface CD8 (CD8 + The data were evaluated for the proportion of cells with The data in Figure 9C show that, as expected, GPRC5D CAR , CD4 + and CD8 + Figure 9A-B shows that the signal was detected in both T cells and erythrocytes. 9C also showed that the CD4:CD8 ratio was significantly higher than that of CAR + cells, CAR -cells and mock cells These results indicate that the cellular responses were similar across all cell populations evaluated.

[0306] The T cell subtypes of the CAR-T population were characterized using two surface markers, CD45RA and CD62. Using L, four memory cell populations were identified: effector memory and RA. + T cells (T EMRA+ ), effector memory cells T EM , naive T cells and memory stem cells T N / SCM , and central memory T cells (T CM ) were distinguished. The data are shown in Figures 9D-9F. is a flow cytogram representing a single donor sample, using two surface markers. We highlight the gating strategies used to identify four distinct memory populations. Figures 9E and 9F show that GPRC5D GC5B680-N68S-LH is N / SCM and T CM It was shown that the expression of IL-1 in IL-1 cells was predominant in IL-1 cells, which may contribute to high proliferation, survival, and It is found to have therapeutic efficacy.

[0307] Example 9: Assay of GPRC5D CAR-T cytotoxicity at 6 hours and 24 hours GPRC5D GC5B680-N68S-LH trait from one of six healthy donors Transduced T cells or non-transduced (Mock) T cells were treated at various E:T ratios: (i) Lucif GPRC5D containing the ferrase transgene + (ii) cultures of MM.1S tumor cells; or GPRC5D containing a luciferase transgene - It was added to cultures of K562 tumor cells. Cellular damage was assessed at each E:T ratio after 6 or 24 hours of incubation. Data are shown in Figure 10A for the 6 hour time point and in Figure 10B for the 24 hour time point. The relative lysis rate was measured after incubation in the presence of CAR-T for (i) 6 or 24 hours. The luciferase signal in the irradiated tumor cells was measured at the same time points after incubation. (ii) Divide the luciferase signal in the tumor cells by the calculated value and multiply by 100; and (iii) Multiply by 100. The formula was calculated by subtracting the number from the CAR-T tumor luminescence. / mean tumor-only emission) × 100. The dotted line highlights zero lysis. Negative lysis is , indicating cell growth.

[0308] Example 10: Assay of GPRC5D CAR-T cytotoxicity at 48 hours GPRC5D GC5B680-N68S-LH transduced and non-transduced (Mock ) T cells were prepared from healthy donors. (i) GPRC5D GC5B680-N68S Either (ii) LH-transduced T cells or (iii) transduced (Mock) T cells were treated with various E :T ratio, GPRC5D + H929, GPRC5D + MM.1S, GPRC5D + MM .1R, GPRC5D + MOLP-2, GPRC5D + EJM and GPRC5D - K56 2. Cells labeled with carboxyfluorescein succinimidyl ester (CFSE) After 48 hours of incubation, cells were stained with a live / dead dye to identify dead cells. Cells and CFSE + The percentage of cells (% cytotoxicity) was assessed. The cytotoxicity rate was calculated using a logarithmic A four-parameter nonlinear regression curve fit was plotted against the transformed x-axis (E:T ratio). Data are shown as mean ± SD in Figures 11A-11F. kNegative control T cells express all GPRC5D genes except EJM. + Some cells in cell lines Induced injury, but to a lesser extent than in GPRC5D GC5B680-N68S-LH cells Without being bound by theory, cell injury The reduced toxicity likely results from an allogeneic response. The cytotoxicity of the α-glucan varied depending on the cell line.

[0309] Example 11: Assay of GPRC5D CAR-T activation at 48 hours GPRC5D GC5B680-N68S-LH transduced and non-transduced (Mock ) T cells were prepared from healthy donors. GPRC5D GC5B680-N68S-LH Transduced or non-transduced (Mock) T cells were transduced with GPRC5D at an E:T ratio of 3:1. + H929, GPRC5D + MM.1S, GPRC5D + MOLP-2, GPRC5D + E JM, and GPRC5D - Carboxyfluorescein succinate labeled in K562 cells After 48 hours of incubation, cells were incubated with CFSE. The cells were stained with live / dead dye, anti-CD25, anti-CD3, and anti-CAR visual markers. Cells were assessed for surface CD25 expression at 1000 rpm. Data are presented in Figure 12 as mean ± SD. As shown in Figure 12, less than 13% of background surface CD25 expression was observed. , observed in mock cells (i.e., no activation). In contrast, GC5B680-N 68S-LH CAR-T cells were shown to be highly resistant to leukemia, as indicated by elevated CD25 expression levels. GPRC5D + Activated in the presence of cell lines (i) Mock and (ii) GC5B6 Both 80-N68S-LH CAR-T cells alone and incubation with K562 cells Low levels of surface CD25 expression were observed following incubation with either the IgG4 or IgG4 receptor agonist alone.

[0310] Example 12: Assay of GPRC5D CAR-T multifunctionality GC5B680-N68S-LH transduced (CAR-T) cells were treated with PMA / ionomycin (as a positive control), GPRC5D + Cell lines H929 (H929), GPRC5D - K 562 (K562) cells (CFSE-labeled), or alone for 18 hours. The cells were incubated at an E:T ratio of 1:2, in which case the cells were either H929 or K562. The cells were then incubated in the presence of live / dead dye, anti-CD3, and anti-idiotype, followed by anti-IFN-γ, anti-IL-2, and anti-T After fixation, permeabilization, and intracellular staining with NF-α, the stained cells were then flow-transferred. Cytometric analysis was performed. Percentages of individual cytokine levels were calculated using Boolean (hierarchical) Following analysis of the flow cytogram using gating, the percentages were calculated as follows: The results are shown in Tables 3 to 6 below. Tables 3 to 6 also show the multifunctional cytokine release in each group. The levels are highlighted in grey and the average sum is displayed.

[0311] [Table 3]

[0312] [Table 4]

[0313] [Table 5]

[0314] [Table 6]

[0315] Example 13: GPRC5D CAR-T proliferation assay GC5B680-N68S-LH cells were prepared using cells from a single donor (i) GC5B680-N68S-LH transduced cells and (ii) Mock cells were cultured in G PRC5D + Incubation was performed in the presence or absence of cells (MM.1S and H929). Mock (untransduced) cells were used as a negative control. By assaying the degree of dilution of ACE Violet (CTV) dye concentration, After incubation for 1 h, the extent of cell proliferation was measured. The amount is shown on the X-axis of the graph in Figure 13. The CAR was isolated using an anti-idiotype antibody. + The cells were stained. The degree of staining was , represented by the Y-axis of the graph in FIG.

[0316] As shown in Figure 13, mock- and CTV-labeled CAR T cells alone or in combination with GPR C5D - As seen at low to zero dilutions of CTV dye in the presence of K562 cells The agonist CD3 / CD28 beads (positive pair) showed little or no cell proliferation. As expected, the IL-16 / IL-16 T cells (which served as a control) resulted in the expansion of all T cell populations evaluated. CAR-T cells were transfected with GPRC5D + In combination with target cells (H929 or MM.1S), When incubated, proliferation was primarily driven by cells expressing GC5B680-N68S-LH on their surface. Transduced cells (CAR + ) (as shown in the graph in Figure 13). The proliferation of GC5B680-N68S-LH cells was enhanced by GPRC5D + Enhanced in the presence of target cells The data shown in Figure 13 suggest that the cellular markers of fibroblasts prepared from other donors may be involved in the cellular markers of fibroblasts. This represents a similar observation made using cells.

[0317] Example 14: Assay of GPRC5D CAR-T proliferation GC5B680-N68S-LH (CAR-T) cells were treated with the positive control staurosporine, GPRC5D + H929 cells, GPRC5D - K562 cells or alone for 24 hours The E:T ratio was 1:2, and GC5B680-N68S-LH (CAR-T) The cells were then used for incubation with H929 or K562 cells. , live / dead dye, anti-CD3, anti-idiotype, and anti-cleaved caspase 3 / 7 dye. The stained cells were analyzed by flow cytometry. The data are shown in the graph in Figure 14. As shown in Figure 14, staurosporine is expressed as CAR + and CAR - T cells In both groups, cleaved caspase 3 / 7 staining was induced. Starting after 2 hours, there were 50-65% positive cells. + T cell populations and CARs - Negative control incubations (K562 and CART alone) and H showed a significant difference in caspase 3 / 7 levels between incubation in the presence of 929 and did.

[0318] Example 15: Established MM.1S human MM xenografts in NSG-B2M mice For GC5B680-N68S-LH, GC5B680-N68S-HL, GP5B 83_N24T_N31S-LH and GP5B83_N24T_N31S-HL are valid Sexuality assay GC5B680-N68S-LH (SEQ ID NO: 86), GC5B680-N68S-HL (SEQ ID NO: 85), GP5B83_N24T_N31S-LH (SEQ ID NO: 84), and G P5B83_N24T_N31S-HL (SEQ ID NO: 83) transduced T cells (CAR-T cells The antitumor efficacy of the MM.1S human multiple myeloma (MM) tumor cells was evaluated in established subcutaneous (sc) MM.1S human multiple myeloma (MM) tumor cells. In a seed transplantation model, female NSG B2m (alternatively, NOD-scid Il2rg nul l B2m null , or NOD-scid gamma B2m) . (i) GC5B680-N68S-LH, (ii) GC5B680-N68S-HL, (iii) GP5B83_N24T_N31S-LH, or (iv) GP5B83_N2 4T_N31S-HL CAR-T cells, 1x10 6 CAR + cells or 5x10 6 of CAR + The cells were administered intravenously (iv) once on day 13. Three groups of xenograft-bearing mice were treated with PBS; ck-transduced mice, and (iii) GPRC5D-transduced mice. Delta tumor growth inhibition in three groups of mice bearing SC (subcutaneous) MM.1S xenografts The percentage (%ΔTGI) was calculated on day 26 after tumor implantation using the following formula: ([(TVc -TVc0)-(TVt-TVt0)] / (TVc-TVc0))×100, where “T "Vc" is the mean tumor burden in a given control group, and "TVc0" is the mean initial tumor burden in a given control group. "TVt" is the mean tumor burden of the treatment group, and "TVt0" is the mean initial tumor burden of the treatment group. The amount of tumor.

[0319] Statistical significance was calculated using linear mixed-effects analysis in R, with treatment and time as fixed effects. , animals were calculated as a random effect. If individual longitudinal response trajectories were not linear, pairwise A number transformation (base 10) was performed. Information obtained from this statistical model was used to calculate the PBS vs. Paired treatment comparisons were performed with control or mock-transduced groups. P values ​​<0.05 were considered statistically significant. was considered significant.

[0320] As outlined in Table 7 and Figure 15, all four GPRC5D directional CAR-T structures Constructs were 1 x 10 compared to PBS and MockCAR-T controls. 6 and 5 x 10 6 C Both AR+ T cell dose levels induced statistically significant %ΔTGI. Complete tumor regression (with no palpable tumor) was assessed 47 days after tumor implantation.

[0321] [Table 7] PBS and MockCAR-T vs. unless noted as not significant (ns) p<0.05 vs. control. CR = Complete Response

[0322] Example 16 Against disseminated H929 IV human MM xenografts in NSG mice GC5B680-N68S-LH, GC5B680-N68S-HL, GP5B83_N Effectiveness of 24T_N31S-LH and GP5B83_N24T_N31S-HL GC5B680-N68S-LH (SEQ ID NO: 86; SEQ ID NO: 90) GC5B680-N68S-HL (encoded by SEQ ID NO: 85; SEQ ID NO: 89) nucleotide), GP5B83_N24T_N31S-LH (SEQ ID NO: No. 84; encoded by the nucleotide sequence of SEQ ID NO: 88), and GP5B83_N2 4T_N31S-HL (SEQ ID NO: 83; encoded by the nucleotide sequence of SEQ ID NO: 87) ) Efficacy of transduced T cells was assessed using female NSG™ (alternatively, NOD scid gamma) or NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ) In mice, the disseminated (iv) H929 human MM model was evaluated. GC5B680-N68S-LH, GC5B680-N68S-HL, GP5B83_N 24T_N31S-LH or GP5B83_N24T_N31S-HL expression (CAR + ) T cells were administered at 1 × 10 6 CAR + cells or 5x10 6 CAR + Thin The dose was administered intravenously in a single dose.

[0323] Survival assessment was performed and survival rates at various study days were plotted against study day. Hind limb paralysis or other clinical signs of excessive disseminated tumor burden were used as surrogate endpoints for death. Median survival was determined using Kaplan-Meier survival analysis. ILS rate (%ILS) is defined as the difference in median survival between the treatment and control groups, and ILS = ([M The mean saturation of the control group was calculated as [St-MSc] / MSc) × 100, where "MSc" is the mean saturation of the control group. "MSt" is the median survival time for a given treatment group. Surrogate endpoints were not achieved due to symptoms (ulcerative tumors, weight loss, etc.) or deaths unrelated to treatment. Animals that failed to reach the endpoint were excluded from the survival assessment. Survival rates were calculated using Kaplan-Meier method. The data were graphed using the Earle curve and analyzed using GraphPad Prism software (Bar The results were evaluated by the log-rank (Mantel-Cox) test using the NIH Standards Track [Page 11] RFC 2111 Statistical Model for Clinical Trials (SDT) August 2011 (Version 7.0).

[0324] In control mice treated with PBS, 52 days after tumor implantation (CAR + (up to 43 days after injection) The median survival time reached 50% within 4 days after tumor implantation in MockCAR-T cell-treated mice. By day 4 (35 days after CAR-T injection), median survival reached 50%. 1x10 6 GC5B680-N68S-LH CAR administered at a dose of + Cell-treated mice In this study, median survival reached 50% after 111 days (102 days after CAR-T injection). Each one is 1x10 6 GC5B680-N68S-HL C administered at a dose of 200 cells AR + Mice treated with the cells survived on day 63.5 (54.5 days after CAR-T injection). Median survival reached 50%. 1x10 6 GP5B83_N administered at a dose of 100 cells 24T_N31S-LH CAR + In mice treated with CAR cells, the The median survival time reached 50% at 84.5 days after T injection. 6 at cell dose Administered GP5B83_N24T_N31S-HL CAR + Cell-treated mice In the study, median survival reached 50% after 111 days (102 days after CAR-T injection). Increased life span (ILS) was assessed via Kaplan-Meier curves and log-rank curves. Evaluation was performed by Telcox test.

[0325] As discussed below and outlined in Table 8 and Figure 16, all four GPRC5D directional CAR-T constructs were compared to PBS and MockCAR-T controls at 1x10 6 Administration of The dose level induced a statistically significant %ILS. Animals that survived until day 111 were classified as CR. I considered it.

[0326] [Table 8] PBS and MockCAR-T vs. unless noted as not significant (ns) p<0.05 vs. control. CR = Complete Response

[0327] Progressive tumor disease burden in the PBS-treated control group starting 52 days after tumor implantation No relevant adverse general symptoms were observed, but the Mock transduction started on day 48 after tumor implantation. General signs of graft-versus-host disease were observed in the GPRC5D CAR-T treated group. It was.

[0328] The teachings of all patents, published patents, and references cited herein are hereby incorporated by reference for all purposes. No. 60 / 699,493, filed on Oct. 1, 2003, which is incorporated by reference in its entirety for the purposes of this application.

[0329] Although exemplary embodiments have been particularly shown and described, those skilled in the art will recognize that the same may be varied in many ways without departing from the spirit and scope of the appended claims. Variations in form and detail may be made without departing from the scope of the embodiments encompassed by the scope of the present invention. Understand that changes may be made.

[0330] Sequence Listing Light chain sequence 1 (GC5B483) (SEQ ID NO: 1) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIK Heavy chain sequence 1 (GC5B483) (SEQ ID NO: 2) EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSFGGRHKALFDYWGQGTLVTVSS Light chain sequence 2 (GC5B81) (SEQ ID NO: 3) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK Heavy chain sequence 2 (GC5B81) (SEQ ID NO: 4) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESRWRGYKLDYWGQGTLVTVSS Light chain sequence 3 (GC5B596) (SEQ ID NO: 5) DIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIK Heavy chain sequence 3 (GC5B596) (SEQ ID NO: 6) QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSS Linker polypeptide sequence (SEQ ID NO: 7) GTEGKSSGSGSESKST scFv sequence 1 (GC5B483) (SEQ ID NO: 8) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIKGTEGKSSGSGSESKST EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSFGGRHKALFDYWGQGTLVTVSS scFv sequence 2 (GC5B81) (SEQ ID NO: 9) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKGTEGKSSGSGSESK STQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESRWRGYKLDYWGQGTLVTVSS scFv sequence 3 (GC5B596) (SEQ ID NO: 10) DIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIKGTEGKSSGSGSES KSTQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSS Signal sequence (SEQ ID NO: 11) MAWVWTLLFLMAAAQSIQA CD137 sequence (SEQ ID NO: 12) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3z sequence (SEQ ID NO: 13) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Intracellular signaling domain (SEQ ID NO: 14) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD8a-TM sequence (SEQ ID NO: 15) IYIWAPLAGTCGVLLLSLVITLYC CD8a hinge sequence (SEQ ID NO: 16) TSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD Extracellular binding domain (GC5B483) (SEQ ID NO: 17) MAWVWTLLFLMAAAQSIQAEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIKGTEGKS SGSGSESKSTEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSFGGRHKALFDYWGQGTLVTVSS Extracellular binding domain (GC5B81) (SEQ ID NO: 18) MAWVWTLLFLLMAAAQSIQADIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKGTEGK SSGSGSESKSTQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESRWRGYKLDYWGQGTLVTVSS Extracellular binding domain (GC5B596) (SEQ ID NO: 19) MAWVWTLLFLLMAAAQSIQADIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIKGTEG KSSGSGSESKSTQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSSTTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSS Extracellular binding domain (GC5B483) (SEQ ID NO: 20) MAWVWTLLFLMAAAQSIQAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSFGGRHKALFDYW GQGTLVTVSSGTEGKSSGSGSESKSTEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIK Extracellular binding domain (GC5B81) (SEQ ID NO: 21) MAWVWTLLFLMAAAQSIQAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESRWRGYKLDYWGQ GTLVTVSSGTEGKSSGSGSESKSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK Extracellular binding domain (GC5B596) (SEQ ID NO: 22) MAWVWTLLFLMAAAQSIQAQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQG TLVTVSSGTEGKSSGSGSESKSTDIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIK scFv sequence 4 (GC5B483) (SEQ ID NO: 24) EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSFGGRHKALFDYWGQGTLVTVSS GTEGKSSGSGSESKSTEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIK scFv sequence 5 (GC5B81) (SEQ ID NO: 25) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESRWRGYKLDYWGQGTLVTVSSG TEGKSSGSGSESKSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK scFv sequence 6 (GC5B596) (SEQ ID NO: 26) QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSSGT EGKSSGSGSESKSTDIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIK pDR000074490 GC5B483-LH-CAR sequence (SEQ ID NO: 27) MAWVWTLLFLMAAAQSIQAEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTK VEIKGTEGKSSGSGSESKSTEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSF GGRHKALFDYWGQGTLVTVSSTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR pDR000074489 GC5B81-LH-CAR sequence (SEQ ID NO: 28) MAWVWTLLFLLMAAAQSIQADIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGT KVEIKGTEGKSSGSGSESKSTQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARES RWRGYKLDYWGQGTLVTVSSTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR pDR000074488 GC5B596-LH-CAR sequence (SEQ ID NO: 29) MAWVWTLLFLMAAAQSIQADIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQG TKLEIKGTEGKSSGSGSESKSTQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARV ALRVALDYWGQGTLVTVSSTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR pDR000074483 GC5B483-HL-CAR sequence (SEQ ID NO: 30) MAWVWTLLFLMAAAQSIQAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSFG GRHKALFDYWGQGTLVTVSSGTEGKSSGSGSESKSTEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVY YCQQRSNWPLTFGQGTKVEIKTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 30 pDR000074482 GC5B81-HL-CAR sequence (SEQ ID NO: 31) MAWVWTLLFLLMAAAQSIQAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESRW RGYKLDYWGQGTLVTVSSGTEGKSSGSGSESKSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQSYSTPLTFGQGTKVEIKTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR pDR000074481 GC5B596-HL-CAR sequence (SEQ ID NO: 32) MAWVWTLLFLMAAAQSIQAQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVAL RVALDYWGQGTLVTVSSGTEGKSSGSGSESKSTDIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYC QQYNRYPYTFGQGTKLEIKTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR DNA sequence of pDR000074490 GC5B483-LH-CDS (SEQ ID NO: 33) DNA sequence of pDR000074489 GC5B81-LH-CDS (SEQ ID NO: 34) DNA sequence of pDR000074488 GC5B596-LH-CDS (SEQ ID NO: 35) DNA sequence of pDR000074483 GC5B483-HL-CDS (SEQ ID NO: 36) DNA sequence of pDR000074482 GC5B81-HL-CDS (SEQ ID NO: 37) DNA sequence of pDR000074481 GC5B596-HL-CDS (SEQ ID NO: 38) HCDR1 (GC5B81) (SEQ ID NO: 39) GGTFSSY HCDR2 (GC5B81) (SEQ ID NO: 40) ESRWRGYKLDY HCDR3 (GC5B81) (SEQ ID NO: 41) IPIFGT HCDR1 (GC5B483) (SEQ ID NO: 42) GYSFTSY HCDR2 (GC5B483) (SEQ ID NO: 43) YPGKSD HCDR3 (GC5B483) (SEQ ID NO: 44) VYSFGGRHKALFDY HCDR1 (GC5B596) (SEQ ID NO: 45) GYSFTGY HCDR2 (GC5B596) (SEQ ID NO: 46) NPYNSD HCDR3 (GC5B596) (SEQ ID NO: 47) VALRVALDY LCDR1 (GC5B81) (SEQ ID NO: 48) RASQSISSYLN LCDR2 (GC5B81) (SEQ ID NO: 49) AASSLQS LCDR3 (GC5B81) (SEQ ID NO: 50) QQSYSTPLT LCDR1 (GC5B483) (SEQ ID NO: 51) RASQSVSSYLA LCDR2 (GC5B483) (SEQ ID NO: 52) DASNRAT LCDR3 (GC5B483) (SEQ ID NO: 53) QQRSNWPLT LCDR1 (GC5B596) (SEQ ID NO: 54) KASQNVATHVG LCDR2 (GC5B596) (SEQ ID NO: 55) SASYRYS LCDR3 (GC5B596) (SEQ ID NO: 56) QQYNRYPYT Human GPRC5D polypeptide (SEQ ID NO: 57) MYKDCIESTGDYFLLCDAEGPWGIILESLAILGIVVTILLLLAFLFLMRKIQDCSQWNVLPTQLLFLLSVLGLFGLAFAFIIELNQQTAPVRYFLFGVLFALCFSCLLAHASNLVKLVRGCVSFSWTTILCIAIGCSLLQIIIATEYVTLIMTRGMMFVNMTPCQLNVDFVV LLVYVLFLMALTFFVSKATFCGPCENWKQHGRLIFITVLFSIIIWVVWISMLLRGNPQFQRQPQWDDPVVCIALVTNAWVFLLLYIVPELCILYRSCRQECPLQGNACPVTAYQHSFQVENQELSRARDSDGAEEDVALTSYGTPIQPQTVDPTQECFIPQAKLSPQQDAGGV HCDR1 (GP5B83) (SEQ ID NO: 58) GGSLSSSSY HCDR2 (GP5B83) (SEQ ID NO: 59) YYSGN HCDR3 (GP5B83) (SEQ ID NO: 60) HVGYSYGRRFWYFDL LCDR1 (GP5B83) (SEQ ID NO: 61) RASQSVSSYLA LCDR2 (GP5B83) (SEQ ID NO: 62) DASNRAT LCDR3 (GP5B83) (SEQ ID NO: 63) QQRSNWPPT Heavy chain sequence (GP5B83) (SEQ ID NO: 64) QLQLQESGPGLVKPSETLSLTTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFWYFDLWGRGTLVTVSS Light chain sequence (GP5B83) (SEQ ID NO: 65) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK HCDR1 (GC5B680) (SEQ ID NO: 66) GFSLTNIRM HCDR2 (GC5B680) (SEQ ID NO: 67) FSNDE HCDR3 (GC5B680) (SEQ ID NO: 68) MRLPYGMDV LCDR1 (GC5B680) (SEQ ID NO: 69) RSSQSLVHSDGNTYLS LCDR2 (GC5B680) (SEQ ID NO: 70) KISNRFF LCDR3 (GC5B680) (SEQ ID NO: 71) MQATQFPHT Heavy chain sequence (GC5B680) (SEQ ID NO: 72) QVTLKESGPVLVKPTETLTLTCTVSGFSLTNIRMSVSWIRQPPGKALEWLAHIFSNDEKSYSSSLKSRLTISRDTSKSQVVLTLTNVDPVDTATYYCARMRLPYGMDVWGQGTTVTVSS Light chain sequence (GC5B680) (SEQ ID NO: 73) DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFFGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPHTFGQGTKLEIK Linker polypeptide sequence (SEQ ID NO: 74) GGSEGKSSGSGSESKSTGGS scFv sequence (GP5B83-HL) (SEQ ID NO: 75) QLQLQESGPGLVKPSETLSLTTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFWYFDLWGRGTLVTVSSG GSEGKSSGSGSESKSTGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK scFv sequence (GP5B83-LH) (SEQ ID NO: 76) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKGGSEGKSSGSGSESKSTGG SQLQLQESGPGLVKPSETLSLTTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFWYFDLWGRGTLVTVSS scFv sequence (GC5B680-HL) (SEQ ID NO: 77) QVTLKESGPVLVKPTETLTLTCTVSGFSLTNIRMSVSWIRQPPGKALEWLAHIFSNDEKSYSSSLKSRLTISRDTSKSQVVLTLTNVDPVDTATYYCARMRLPYGMDVWGQGTTVTVSSGGSEGK SSGSGSESKSTGGSDIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFFGVPDRFSGSGAGTDFTLKISRVEAEEDVGVYYCMQATQFPHTFGQGTKLEIK scFv sequence (GC5B680-LH) (SEQ ID NO: 78) DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFFGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPHTFGQGTKLEIKGGSEGKSSGSGSE SKSTGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTNIRMSVSWIRQPPGKALEWLAHIFSNDEKSYSSSLKSRLTISRDTSKSQVVLTLTNVDPVDTATYYCARMRLPYGMDVWGQGTTVTVSS Extracellular binding domain (GP5B83-HL) (SEQ ID NO: 79) MAWVWTLLFLLMAAAQSIQAQLQLQESGPGLVKPSETLSLTTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFWYFDLWG RGTLVTVSSGGSEGKSSGSGSESKSTGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK Extracellular binding domain (GP5B83-LH) (SEQ ID NO: 80) MAWVWTLLFLMAAAQSIQAEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYYCQQRSNWPPTFGQGTKVEIKGGSEGKSSG SGSESKSTGGSQLQLQESGPGLVKPSETLSLTTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFWYFDLWGRGTLVTVSS Extracellular binding domain (GC5B680-HL) (SEQ ID NO: 81) MAWVWTLLFLLMAAAQSIQAQVTLKESGPVLVKPTETLTLTCTVSGFSLTNIRMSVSWIRQPPGKALEWLAHIFSNDEKSYSSSLKSRLTISRDTSKSQVVLTLTNVDPVDTATYYCARMRLPYGMDVWGQGTTVT VSSGGSEGKSSGSGSESKSTGGSDIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFFGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPHTFGQGTKLEIK Extracellular binding domain (GC5B680-LH) (SEQ ID NO: 82) MAWVWTLLFLMAAAQSIQADIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFFGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPHTFGQGTKLEIKGGSE GKSSGSGSESKSTGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTNIRMSVSWIRQPPGKALEWLAHIFSNDEKSYSSSLKSRLTISRDTSKSQVVLTLTNVDPVDTATYYCARMRLPYGMDVWGQGTTVTVSS GP5B83-HL-CAR sequence (SEQ ID NO: 83) QLQLQESGPGLVKPSETLSLTCTVSGGSLSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYCARHVGYSYGRRFWYFDLWGRGTLVTVSSGGSEGKSSGSGSESKSTGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYCQQRS NWPPTFGQGTKVEIKTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR GP5B83-LH-CAR sequence(sequence number84) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKGGSEGKSSGSGSESKSTGGSQLQLQESGPGLVKPSETLSLTCTVSGGSLSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYCARHVGYSYGRRFW YFDLWGRGTLVTVSSTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR GC5B680-HL-CAR sequence (SEQ ID NO: 85) QVTLKESGPVLVKPTETLTLTCTVSGFSLTNIRMSVSWIRQPPGKALEWLAHIFSNDEKSYSSSLKSRLTISRDTSKSQVVLTLTNVDPVDTATYYCARMRLPYGMDVWGQGTTVTVS SGGSEGKSSGSGSESKSTGGSDIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFFGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYYCMQATQ FPHTFGQGTKLEIKTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR GC5B680-LH-CAR sequence (SEQ ID NO: 86) DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFFGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPHTFGQGTKLEIKGGSEGK SSGSGSESKSTGGSQVTLKESGPVLVKPTETLTLTCTVSGFSLTNIRMSVSWIRQPPGKALEWLAHIFSNDEKSYSSSLKSRLTISRDTSKSQVVLTLTNVDPVDTATYYCARMRLPYG MDVWGQGTTVTVSSTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR DNA sequence of pDR000084454 GP5B83-HL sequence (SEQ ID NO: 87) DNA sequence of GP5B83-LH sequence (SEQ ID NO: 88) DNA sequence of GC5B680-HL sequence (SEQ ID NO: 89) DNA sequence of GC5B680-LH sequence (SEQ ID NO: 90) Linker sequence (SEQ ID NO: 91) GGSEGKSSGSGSESKSTGGS Linker sequence (SEQ ID NO: 92) GGGSGGGS Linker sequence (SEQ ID NO: 93) GGGSGGGSGGGS Linker sequence (SEQ ID NO: 94) GGGSGGGSGGGSGGGS Linker sequence (SEQ ID NO: 95) GGGSGGGSGGGSGGGSGGGS Linker sequence (SEQ ID NO: 96) GGGGSGGGGSGGGGS Linker sequence (SEQ ID NO: 97) GGGGSGGGGSGGGGSGGGGS Linker sequence (SEQ ID NO: 98) GGGGSGGGGSGGGGSGGGGSGGGGS Linker sequence (SEQ ID NO: 99) GSTSGSGKPGSGEGSTKG Linker sequence (SEQ ID NO: 100) IRPRAIGGSKPRVA Linker sequence (SEQ ID NO: 101) GKGGSGKGGSGKGGS Linker sequence (SEQ ID NO: 102) GGKGSGGKGSGGKGS Linker sequence (SEQ ID NO: 103) GGGKSGGGKSGGGKS Linker sequence (SEQ ID NO: 104) GKGKSGKGKSGKGKS Linker sequence (SEQ ID NO: 105) GGGKSGGKGSGKGGS Linker sequence (SEQ ID NO: 106) GKPGSGKPGSGKPGS Linker sequence (SEQ ID NO: 107) GKPGSGKPGSGKPGSGKPGS Linker sequence (SEQ ID NO: 108) GKGKSGKGKSGKGKSGKGKS Linker sequence (SEQ ID NO: 109) STAGDTHLGGEDFD Linker sequence (SEQ ID NO: 110) GEGGSGEGGSGEGGS Linker sequence (SEQ ID NO: 111) GGEGSGGEGSGGEGS Linker sequence (SEQ ID NO: 112) GEGESGEGESGEGES Linker sequence (SEQ ID NO: 113) GGGESGGEGSGEGGS Linker sequence (SEQ ID NO: 114) GEGESGEGESGEGESGEGES Linker sequence (SEQ ID NO: 115) GSTSGSGKPGSGEGSTKG Linker sequence (SEQ ID NO: 116) PRGASKSGSASQTGSAPGS Linker sequence (SEQ ID NO: 117) GTAAAGAGAAGGAAAGAAG Linker sequence (SEQ ID NO: 118) GTSGSSGSGSGGSGSGGGG Linker sequence (SEQ ID NO: 119) GKPGSGKPGSGKPGSGKPGS Linker sequence (SEQ ID NO: 120) GSGS Linker sequence (SEQ ID NO: 121) APAPAPAPAP Linker sequence (SEQ ID NO: 122) APAPAPAPAPAPAPAPAPAP Linker sequence (SEQ ID NO: 123) AEAAAKEAAAKEAAAAKEAAAAKEAAAAKAAA Hinge sequence (SEQ ID NO: 124) EPKSCDKTHTCPPCP Hinge sequence (SEQ ID NO: 125) ERKCCVECPPCP Hinge sequence (SEQ ID NO: 126) ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP Hinge sequence (SEQ ID NO: 127) ESKYGPPCPSCP The inventions described in the original claims of this application are listed below. [Invention 1] A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, the extracellular antigen-binding domain 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; 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; 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; 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 heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 45, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 46, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 47 Including, the extracellular antigen-binding domain binds to an anti-G protein receptor family C group 5 member D (GPRC5D) antigen; The chimeric antigen receptor (CAR). [Invention 2] the extracellular antigen-binding domain comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 66, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 67, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 68, further comprising 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; the extracellular antigen-binding domain comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 58, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 59, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 60, further comprising 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; the extracellular antigen-binding domain comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 39, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 40, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 41, 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; the extracellular antigen-binding domain comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 42, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 43, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 44, further comprising 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 the extracellular antigen-binding domain comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 45, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 46, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 47, further comprising 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; The CAR according to Invention 1. [Invention 3] The CAR according to Invention 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 NOs: 73, 65, 1, 3, and 5, or a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 72, 64, 2, 4, and 6, or a combination of an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 65, 1, 3, and 5 and an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 72, 64, 2, 4, and 6. [Invention 4] the extracellular antigen-binding domain 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; 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; 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; 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 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; A CAR according to any one of Inventions 1 to 3. [Invention 5] 5. The CAR according to any one of Inventions 1 to 4, wherein the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv). [Invention 6] The CAR according to Invention 5, wherein the scFv comprises a linker polypeptide between the light chain variable region and the heavy chain variable region. [Invention 7] The CAR of invention 6, wherein the linker polypeptide comprises the amino acid sequence of SEQ ID NO: 7. [Invention 8] The CAR according to any one of Inventions 5 to 7, wherein the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 78, 77, 75, 76, 8, 9, 10, 24, 25, and 26. [Invention 9] 9. The CAR according to any one of Inventions 1 to 8, wherein the extracellular antigen-binding domain comprises a signal polypeptide. [Invention 10] The CAR of invention 9, wherein the signal polypeptide comprises the amino acid sequence of SEQ ID NO: 11. [Invention 11] 11. The CAR according to any one of inventions 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 zeta chain (CD3z) component, a cluster of differentiation (CD27) component, a cluster of differentiation superfamily member component, and combinations thereof. [Invention 12] 12. The CAR of claim 11, wherein the CD137 component comprises the amino acid sequence of SEQ ID NO: 12. [Invention 13] 12. The CAR of claim 11, wherein the CD3z component comprises the amino acid sequence of SEQ ID NO: 13. [Invention 14] The CAR of invention 11, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 14. [Invention 15] 15. The CAR according to any one of inventions 1 to 14, wherein the transmembrane domain comprises a CD8a transmembrane region (CD8A-TM) polypeptide. [Invention 16] The CAR of invention 15, wherein the CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO: 15. [Invention 17] 17. The CAR according to any one of Inventions 1 to 16, further comprising a hinge region linking the transmembrane domain to the extracellular antigen-binding domain. [Invention 18] 18. The CAR according to Invention 17, wherein the hinge region is a CD8a hinge region. [Invention 19] 19. The CAR of invention 18, wherein the CD8a hinge region comprises the amino acid sequence of SEQ ID NO: 16. [Invention 20] 20. The CAR according to any one of Inventions 1 to 19, wherein the extracellular antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 82, 81, 80, 79, 17, 18, 19, 20, 21, and 22. [Invention 21] 21. The CAR according to any one of Inventions 1 to 20, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 86, 85, 83, 84, 27, 28, 29, 30, 31, and 32. [Invention 22] An isolated lymphocyte expressing a CAR according to any one of inventions 1 to 21. [Invention 23] 23. The isolated lymphocyte according to claim 22, wherein the lymphocyte is a T lymphocyte. [Invention 24] 24. The isolated lymphocyte according to claim 23, wherein the T lymphocyte is a naive T cell. [Invention 25] 24. The isolated lymphocyte according to claim 23, wherein the T lymphocyte is a memory stem T cell. [Invention 26] 24. The isolated lymphocyte according to claim 23, wherein the T lymphocyte is a central memory T cell. [Invention 27] 27. The isolated lymphocyte according to any one of inventions 23 to 26, wherein the T lymphocyte is CD4+. [Invention 28] 27. The isolated lymphocyte according to any one of inventions 23 to 26, wherein the T lymphocyte is CD8+. [Invention 29] 27. The isolated lymphocyte according to any one of inventions 23 to 26, wherein the T lymphocyte is CD4+ and CD8+. [Invention 30] An isolated nucleic acid molecule encoding a CAR according to any one of inventions 1 to 29. [Invention 31] 31. The isolated nucleic acid molecule of claim 30, wherein the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 90, 89, 87, 88, 33, 34, 35, 36, 37, and 38. [Invention 32] 31. The isolated nucleic acid molecule of claim 30, 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 NOs: 90, 89, 87, 88, 33, 34, 35, 36, 37, and 38. [Invention 33] A vector comprising the nucleic acid molecule according to any one of Inventions 30 to 32. [Invention 34] A cell that expresses the nucleic acid molecule according to any one of inventions 30 to 32. [Invention 35] 30. A pharmaceutical composition comprising an effective amount of lymphocytes according to any one of inventions 22 to 29. [Invention 36] 30. A pharmaceutical composition comprising an effective amount of lymphocytes according to any one of inventions 22 to 29 and a pharmaceutically acceptable excipient. [Invention 37] A CAR according to any one of Inventions 1 to 21, or a pharmaceutical composition according to Invention 35 or 36, for use in therapy. [Invention 38] A CAR according to any one of Inventions 1 to 21, or a pharmaceutical composition according to Invention 35 or 36, for use in a method for treating a subject having cancer. [Invention 39] 1. A method of treating a subject having cancer, comprising: administering a therapeutically effective amount of the lymphocytes according to any one of Inventions 22 to 29 to a subject in need of treatment, whereby the lymphocytes induce the death of cancer cells in the subject. The method. [Invention 40] 39. The method of Invention 39, or the CAR or the pharmaceutical composition for use in the method of Invention 38, 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 myelogenous leukemia (CML), smoldering multiple myeloma (SMM), multiple myeloma (MM), acute myeloid leukemia (AML), and combinations thereof. [Invention 41] The method of Invention 39 or 40, or the CAR or the pharmaceutical composition for use in the method of Invention 38 or 40, wherein the cancer is multiple myeloma. [Invention 42] 1. A method for targeted killing of cancer cells, comprising: contacting the cancer cells with the lymphocytes according to any one of Inventions 22 to 29, thereby causing the lymphocytes to induce death of the cancer cells. The method. [Invention 43] 43. The method of invention 42, 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 non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), smoldering multiple myeloma (SMM), multiple myeloma (MM), acute myeloid leukemia (AML), and combinations thereof. [Invention 44] 43. The method of claim 42, wherein the cancer cells are multiple myeloma cells. [Invention 45] 1. A method for detecting the presence of cancer in a subject, comprising: (a) contacting a cell sample obtained from the subject with a CAR according to invention 1 to form a CAR-cell complex; and (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the subject. The method.

Claims

[Claim 1] A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, The extracellular antigen-binding domain comprises: a heavy chain variable region comprising 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, and a light chain variable region comprising a light chain complementarity determining region 1 (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, or a heavy chain variable region comprising 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, and a light chain variable region comprising a light chain complementarity determining region 1 (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; the extracellular antigen-binding domain binds to a G protein receptor family C group 5 member D (GPRC5D) antigen; The chimeric antigen receptor (CAR).

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