Anti-GPRC5D antigen binding proteins and uses thereof

Abstract

This application relates to an EC 50 The present application relates to an isolated antigen binding protein that binds the GPCR family C group 5 member D (GPRC5D) protein at a specific binding level. The present application also relates to chimeric antigen receptors of said antigen binding protein and their use in the treatment of tumors.

Description

[Technical field]

[0001] Technical Field The present application relates to the field of biopharmaceuticals, in particular to anti-GPRC5D antigen binding proteins, chimeric antigen receptors comprising said antigen binding proteins, and their use in the treatment of tumors. [Background technology]

[0002] Background technology Multiple myeloma (MM) is the second most common malignant hematological disease and the second leading cause of cancer mortality. MM is a malignant tumor of plasma cells, and is often accompanied by multiple osteolytic lesions, renal dysfunction, bone marrow infiltration, hypercalcemia, and anemia. Currently, the mainstream treatment for MM is systemic chemotherapy, which has serious side effects and is incurable.

[0003] The G protein-coupled receptor class C group 5 member D (GPRC5D) protein is an atypical surface orphan receptor. GPRCD5, like other C5 class receptors, has a short amino terminus and is conformationally very similar to the C4 class. GPRCD5 expression in normal tissues is restricted to hair follicles, but it is specifically and highly expressed in the bone marrow of MM patients and is highly associated with plasma cell tumor burden and genetic abnormalities.

[0004] Although novel drug interventions such as protease inhibitors (PIs), immunomodulatory agents (IMiDs), and monoclonal antibodies (mAbs) have enriched the treatment of MM, there are still high relapse rates and drug resistance. Chimeric antigen receptor T cell (CAR-T) therapy has shown significant efficacy and controllable toxicity in MM, but mainstream CAR-T cell therapy targeting B cell maturation antigen (BCMA) has a target escape problem for MM patients with BCMA-negative or low BCMA expression. GPRC5D is specifically and highly expressed in plasma cells, making it an ideal therapeutic target for MM, but drug therapeutic products targeting it are not yet mature. Summary of the Invention

[0005] Summary of the Invention The present application provides an isolated antigen binding protein capable of specifically binding to G protein-coupled receptor class C member 5D (GPRC5D) protein. The present application also provides a chimeric antigen receptor comprising the antigen binding protein, and a cell comprising and / or expressing the chimeric antigen receptor, the cell having one or more of the following characteristics: (1) high proliferation ability, (2) ability to kill target cells expressing GPRC5D, (3) secreting cytokines upon stimulation by target cells, (4) having high proliferation ability upon stimulation by target cells, and (4) suppressing tumor growth.

[0006] In one aspect, the present application provides an isolated antigen binding protein capable of binding a G protein-coupled receptor class C group 5 member D (GPRC5D) protein with an EC50 value of less than 1.0 μg / mL.

[0007] In some embodiments, the antigen binding protein comprises an antibody or an antigen-binding fragment thereof.

[0008] In some embodiments, the antigen-binding fragment is a Fab, Fab', F(ab)2, Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and / or dAb.

[0009] In some embodiments, the antigen-binding fragment is a VHH.

[0010] In some embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0011] In some embodiments, the antigen binding protein is capable of binding to a protein in competition with a reference antibody, wherein the reference antibody comprises an antibody heavy chain variable region, VH, wherein the VH of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and wherein the reference antibody is selected from any of the following groups of amino acid sequences: (1) HCDR1: SEQ ID NO: 102, HCDR2: SEQ ID NO: 103, and HCDR3: SEQ ID NO: 104; (2) HCDR1: SEQ ID NO: 105, HCDR2: SEQ ID NO: 106, and HCDR3: SEQ ID NO: 107; (3) HCDR1: SEQ ID NO: 109, HCDR2: SEQ ID NO: 110, and HCDR3: SEQ ID NO: 111; (4) HCDR1: SEQ ID NO: 105, HCDR2: SEQ ID NO: 114, and HCDR3: SEQ ID NO: 115; (5) HCDR1: SEQ ID NO: 116, HCDR2: SEQ ID NO: 117, and HCDR3: SEQ ID NO: 118; (6) HCDR1: SEQ ID NO: 119, HCDR2: SEQ ID NO: 120, and HCDR3: SEQ ID NO: 121; (7) HCDR1: SEQ ID NO: 123, HCDR2: SEQ ID NO: 124, and HCDR3: SEQ ID NO: 125; (8) HCDR1: SEQ ID NO: 127, HCDR2: SEQ ID NO: 128, and HCDR3: SEQ ID NO: 129; (9) HCDR1: SEQ ID NO: 130, HCDR2: SEQ ID NO: 131, and HCDR3: SEQ ID NO: 132; (10) HCDR1: SEQ ID NO: 95, HCDR2: SEQ ID NO: 96, and HCDR3: SEQ ID NO: 97; (11) HCDR1: SEQ ID NO: 133, HCDR2: SEQ ID NO: 134, and HCDR3: SEQ ID NO: 135; (12) HCDR1: SEQ ID NO: 136, HCDR2: SEQ ID NO: 134, and HCDR3: SEQ ID NO: 137; (13) HCDR1: SEQ ID NO: 138, HCDR2: SEQ ID NO: 139, and HCDR3: SEQ ID NO: 140; (14) HCDR1: SEQ ID NO: 142, HCDR2: SEQ ID NO: 143, and HCDR3: SEQ ID NO: 144; (15) HCDR1: SEQ ID NO: 145, HCDR2: SEQ ID NO: 146, and HCDR3: SEQ ID NO: 147; (16) HCDR1: SEQ ID NO: 148, HCDR2: SEQ ID NO: 149, and HCDR3: SEQ ID NO: 150; (17) HCDR1: SEQ ID NO: 152, HCDR2: SEQ ID NO: 153, and HCDR3: SEQ ID NO: 154; (18) HCDR1: SEQ ID NO: 123, HCDR2: SEQ ID NO: 155, and HCDR3: SEQ ID NO: 156; (19) HCDR1: SEQ ID NO: 157, HCDR2: SEQ ID NO: 153, and HCDR3: SEQ ID NO: 158; (20) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 160, and HCDR3: SEQ ID NO: 161; (21) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 162, and HCDR3: SEQ ID NO: 163; (22) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 164, and HCDR3: SEQ ID NO: 165; (23) HCDR1: SEQ ID NO: 166, HCDR2: SEQ ID NO: 167, and HCDR3: SEQ ID NO: 168; (24) HCDR1: SEQ ID NO: 169, HCDR2: SEQ ID NO: 153, and HCDR3: SEQ ID NO: 170; (25) HCDR1: SEQ ID NO: 157, HCDR2: SEQ ID NO: 171, and HCDR3: SEQ ID NO: 172; (26) HCDR1: SEQ ID NO: 174, HCDR2: SEQ ID NO: 175, and HCDR3: SEQ ID NO: 176; (27) HCDR1: SEQ ID NO: 177, HCDR2: SEQ ID NO: 178, and HCDR3: SEQ ID NO: 179; (28) HCDR1: SEQ ID NO: 180, HCDR2: SEQ ID NO: 181, and HCDR3: SEQ ID NO: 182; (29) HCDR1: SEQ ID NO: 184, HCDR2: SEQ ID NO: 106, and HCDR3: SEQ ID NO: 185; (30) HCDR1: SEQ ID NO: 186, HCDR2: SEQ ID NO: 187, and HCDR3: SEQ ID NO: 188; (31) HCDR1: SEQ ID NO: 189, HCDR2: SEQ ID NO: 190, and HCDR3: SEQ ID NO: 191; (32) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 192, and HCDR3: SEQ ID NO: 179; (33) HCDR1: SEQ ID NO: 193, HCDR2: SEQ ID NO: 194, and HCDR3: SEQ ID NO: 195; and (34) HCDR1: SEQ ID NO: 196, HCDR2: SEQ ID NO: 197, and HCDR3: SEQ ID NO: 198.

[0012] In some embodiments, the antigen binding protein comprises at least one CDR derived from an antibody heavy chain variable region VH, said VH being selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:70.

[0013] In some embodiments, the antigen binding protein comprises an HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:215 or SEQ ID NO:218.

[0014] In some embodiments, the antigen binding protein comprises an HCDR3, and the HCDR3 is selected from the group consisting of SEQ ID NO:97, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:111, SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:140, SEQ ID NO:144, SEQ ID NO:147, SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:168, SEQ ID NO:170, SEQ ID SEQ ID NO:172, SEQ ID NO:176, SEQ ID NO:179, SEQ ID NO:182, SEQ ID NO:185, SEQ ID NO:188, SEQ ID NO:191, SEQ ID NO:195 and SEQ ID NO:198.

[0015] In some embodiments, the antigen binding protein comprises HCDR2, and said HCDR2 is X 1 X 2 X 3 X 4 X 5 X 6 X 7 T, wherein X 1 is I, T, X 2 is N, S or T, X 3 A, P, R, S or W, X 4 is G, R, S, T or absent, X 5 is D, G, or X 6 is G or S and X 7 is D, G, I, N, R, S, T or V.

[0016] In some embodiments, the antigen binding protein comprises an HCDR2, and the HCDR2 is selected from the group consisting of SEQ ID NO:96, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:124, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:134, SEQ ID NO:139, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:149, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:167, SEQ ID NO:171, SEQ ID NO:175, SEQ ID NO:178, SEQ ID SEQ ID NO:181, SEQ ID NO:187, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194 and SEQ ID NO:197.

[0017] In some embodiments, the antigen binding protein comprises an HCDR1, and the HCDR1 is GX 2 X 3 X 4 SX 6 X 7 X 8 wherein X comprises the amino acid sequence shown in 2 is F, G, I, L, N, R, S or Y, X 3 is I, T, X 4 is F, L, X 6 is I, L, N, R, S, T or Y, X 7 is D, N or Y and X 8 is A, D, G, I, N, R, S, T, V or Y.

[0018] In some embodiments, the antigen binding protein comprises an HCDR1, and the HCDR1 is selected from the group consisting of SEQ ID NO:95, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:109, SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:127, SEQ ID NO:130, SEQ ID NO:133, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:166, SEQ ID NO:169, SEQ ID NO:174, SEQ ID NO:177, SEQ ID NO:180, SEQ ID NO:184, SEQ ID SEQ ID NO:186, SEQ ID NO:189, SEQ ID NO:193 and SEQ ID NO:196.

[0019] In some embodiments, the antigen binding protein comprises an HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 are selected from any of the following groups of amino acid sequences: (1) HCDR1: SEQ ID NO: 102, HCDR2: SEQ ID NO: 103, and HCDR3: SEQ ID NO: 104; (2) HCDR1: SEQ ID NO: 105, HCDR2: SEQ ID NO: 106, and HCDR3: SEQ ID NO: 107; (3) HCDR1: SEQ ID NO: 109, HCDR2: SEQ ID NO: 110, and HCDR3: SEQ ID NO: 111; (4) HCDR1: SEQ ID NO: 105, HCDR2: SEQ ID NO: 114, and HCDR3: SEQ ID NO: 115; (5) HCDR1: SEQ ID NO: 116, HCDR2: SEQ ID NO: 117, and HCDR3: SEQ ID NO: 118; (6) HCDR1: SEQ ID NO: 119, HCDR2: SEQ ID NO: 120, and HCDR3: SEQ ID NO: 121; (7) HCDR1: SEQ ID NO: 123, HCDR2: SEQ ID NO: 124, and HCDR3: SEQ ID NO: 125; (8) HCDR1: SEQ ID NO: 127, HCDR2: SEQ ID NO: 128, and HCDR3: SEQ ID NO: 129; (9) HCDR1: SEQ ID NO: 130, HCDR2: SEQ ID NO: 131, and HCDR3: SEQ ID NO: 132; (10) HCDR1: SEQ ID NO: 95, HCDR2: SEQ ID NO: 96, and HCDR3: SEQ ID NO: 97; (11) HCDR1: SEQ ID NO: 133, HCDR2: SEQ ID NO: 134, and HCDR3: SEQ ID NO: 135; (12) HCDR1: SEQ ID NO: 136, HCDR2: SEQ ID NO: 134, and HCDR3: SEQ ID NO: 137; (13) HCDR1: SEQ ID NO: 138, HCDR2: SEQ ID NO: 139, and HCDR3: SEQ ID NO: 140; (14) HCDR1: SEQ ID NO: 142, HCDR2: SEQ ID NO: 143, and HCDR3: SEQ ID NO: 144; (15) HCDR1: SEQ ID NO: 145, HCDR2: SEQ ID NO: 146, and HCDR3: SEQ ID NO: 147; (16) HCDR1: SEQ ID NO: 148, HCDR2: SEQ ID NO: 149, and HCDR3: SEQ ID NO: 150; (17) HCDR1: SEQ ID NO: 152, HCDR2: SEQ ID NO: 153, and HCDR3: SEQ ID NO: 154; (18) HCDR1: SEQ ID NO: 123, HCDR2: SEQ ID NO: 155, and HCDR3: SEQ ID NO: 156; (19) HCDR1: SEQ ID NO: 157, HCDR2: SEQ ID NO: 153, and HCDR3: SEQ ID NO: 158; (20) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 160, and HCDR3: SEQ ID NO: 161; (21) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 162, and HCDR3: SEQ ID NO: 163; (22) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 164, and HCDR3: SEQ ID NO: 165; (23) HCDR1: SEQ ID NO: 166, HCDR2: SEQ ID NO: 167, and HCDR3: SEQ ID NO: 168; (24) HCDR1: SEQ ID NO: 169, HCDR2: SEQ ID NO: 153, and HCDR3: SEQ ID NO: 170; (25) HCDR1: SEQ ID NO: 157, HCDR2: SEQ ID NO: 171, and HCDR3: SEQ ID NO: 172; (26) HCDR1: SEQ ID NO: 174, HCDR2: SEQ ID NO: 175, and HCDR3: SEQ ID NO: 176; (27) HCDR1: SEQ ID NO: 177, HCDR2: SEQ ID NO: 178, and HCDR3: SEQ ID NO: 179; (28) HCDR1: SEQ ID NO: 180, HCDR2: SEQ ID NO: 181, and HCDR3: SEQ ID NO: 182; (29) HCDR1: SEQ ID NO: 184, HCDR2: SEQ ID NO: 106, and HCDR3: SEQ ID NO: 185; (30) HCDR1: SEQ ID NO: 186, HCDR2: SEQ ID NO: 187, and HCDR3: SEQ ID NO: 188; (31) HCDR1: SEQ ID NO: 189, HCDR2: SEQ ID NO: 190, and HCDR3: SEQ ID NO: 191; (32) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 192, and HCDR3: SEQ ID NO: 179; (33) HCDR1: SEQ ID NO: 193, HCDR2: SEQ ID NO: 194, and HCDR3: SEQ ID NO: 195; and (34) HCDR1: SEQ ID NO: 196, HCDR2: SEQ ID NO: 197, and HCDR3: SEQ ID NO: 198.

[0020] In some embodiments, the antigen binding protein comprises an antibody heavy chain variable region VH, wherein said VH comprises a framework region H-FR1, wherein the C-terminus of said H-FR1 and the N-terminus of said HCDR1 are linked directly or indirectly, and wherein said H-FR1 comprises the amino acid sequence shown in SEQ ID NO:98.

[0021] In some embodiments, the VH in the antigen binding protein comprises a framework region H-FR2, said H-FR2 being located between said HCDR1 and said HCDR2, and said H-FR2 comprising the amino acid sequence set forth in SEQ ID NO:216.

[0022] In some embodiments, the H-FR2 in the antigen binding protein comprises an amino acid sequence set forth in any one of SEQ ID NO:99, SEQ ID NO:108, SEQ ID NO:112, SEQ ID NO:122, SEQ ID NO:151, and SEQ ID NO:173.

[0023] In some embodiments, the VH in the antigen binding protein comprises a framework region H-FR3, said H-FR3 being located between said HCDR2 and said HCDR3, and said H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:217.

[0024] In some embodiments, the H-FR3 in the antigen binding protein comprises an amino acid sequence set forth in any one of SEQ ID NO:100, SEQ ID NO:113, SEQ ID NO:126, SEQ ID NO:141, and SEQ ID NO:183.

[0025] In some embodiments, the VH in the antigen binding protein comprises a framework region H-FR4, wherein the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and wherein the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:101.

[0026] In some embodiments, the VH in the antigen binding protein is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:70.

[0027] In some embodiments, the antigen binding protein is a VHH and the VHH is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:70.

[0028] In some embodiments, the antigen binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from an IgG.

[0029] In some embodiments, the antigen binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from human IgG.

[0030] In some embodiments, the antigen binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from human IgG1.

[0031] In another aspect, the present application provides a chimeric antigen receptor comprising a targeting moiety, wherein said targeting moiety comprises said antigen binding protein.

[0032] In some embodiments, the chimeric antigen receptor comprises a costimulatory signal region, wherein the cellular costimulatory signal region comprises an intracellular costimulatory signal region derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand for CD83, CD40, and MyD88.

[0033] In some embodiments, the costimulatory signal region in the chimeric antigen receptor is an intracellular costimulatory signal region derived from 4-1BB.

[0034] In some embodiments, the costimulatory signal region in the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:78.

[0035] In some embodiments, the chimeric antigen receptor comprises an intracellular signaling region, the intracellular signaling region comprising an intracellular signaling region derived from one or more proteins selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3epsilon, CD79a, CD79b, FcεRIgamma, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a structural domain containing at least one ITAM.

[0036] In some embodiments, the intracellular signaling region in the chimeric antigen receptor is a signaling structural domain derived from CD3ζ.

[0037] In some embodiments, the intracellular signaling region in the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:80.

[0038] In some embodiments, the chimeric antigen receptor comprises a transmembrane region, the transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0039] In some embodiments, the transmembrane region in the chimeric antigen receptor is a transmembrane region derived from CD8.

[0040] In some embodiments, the transmembrane region in the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:76.

[0041] In some embodiments, the chimeric antigen receptor comprises a hinge region between the targeting moiety and the transmembrane region, the hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0042] In some embodiments, the hinge region in the chimeric antigen receptor is a hinge region derived from CD8.

[0043] In some embodiments, the hinge region in the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:74.

[0044] In some embodiments, the chimeric antigen receptor also comprises a low density lipoprotein receptor-related protein, or a fragment thereof, wherein the low density lipoprotein receptor-related protein, or a fragment thereof, is located C-terminal to the intracellular signaling region.

[0045] In some embodiments, the low density lipoprotein receptor-related protein or fragment thereof in the chimeric antigen receptor comprises one or more selected from the group consisting of low density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.

[0046] In some embodiments, the low density lipoprotein receptor-related protein or fragment thereof in the chimeric antigen receptor is low density lipoprotein receptor-related protein 6 or a fragment thereof.

[0047] In some embodiments, the low density lipoprotein receptor-related protein or fragment thereof in the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:84.

[0048] In some embodiments, the chimeric antigen receptor comprises a signal peptide.

[0049] In some embodiments, the signal peptide in the chimeric antigen receptor is the signal peptide derived from the CD8 protein.

[0050] In some embodiments, the signal peptide in the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:72.

[0051] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence set forth in any one of SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203 and SEQ ID NO:204.

[0052] In some embodiments, the chimeric antigen receptor comprises a nucleotide sequence set forth in any one of SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, and SEQ ID NO:91.

[0053] In another aspect, the present application provides a polypeptide comprising said antigen binding protein.

[0054] In another aspect, the present application further provides an isolated nucleic acid molecule or molecules encoding said antigen binding protein and / or said chimeric antigen receptor.

[0055] In some embodiments, the nucleic acid molecule also comprises a promoter.

[0056] In some embodiments, the promoter in the nucleic acid molecule is a constitutive promoter.

[0057] In some embodiments, the promoter in the nucleic acid molecule is an EF1α promoter.

[0058] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in any one of SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, and SEQ ID NO:91.

[0059] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0060] In some embodiments, the vector is a viral vector.

[0061] In some embodiments, the vector is a lentiviral vector.

[0062] In another aspect, the present application provides a cell comprising the antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, and / or the vector.

[0063] In some embodiments, the cell is an immune effector cell.

[0064] In some embodiments, the cells comprise T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells and / or pluripotent stem cells.

[0065] In some embodiments, the cell is a T cell.

[0066] In another aspect, the present application provides a method of modulating said antigen binding protein and / or said chimeric antigen receptor comprising culturing said cells under conditions that allow expression of said antigen binding protein and / or said chimeric antigen receptor.

[0067] In another aspect, the present application provides a method of preparing an engineered immune effector cell, comprising introducing the vector into an immune effector cell.

[0068] In another aspect, the present application provides a pharmaceutical composition comprising the antigen binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector, and / or the cell, and optionally a pharma- ceutically acceptable vector.

[0069] In another aspect, the present application provides the use of the antigen binding protein, the antigen-engaging receptor, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition in the preparation of a medicament used to prevent, treat, and / or alleviate a disease or condition associated with abnormal expression of GPRC5D.

[0070] In some embodiments, in said use, said disease or condition associated with aberrant expression of GPRC5D comprises a tumor.

[0071] In some embodiments, the tumor in said use comprises a solid tumor.

[0072] In some embodiments, the tumor in said use comprises a non-solid tumor.

[0073] In some embodiments, the tumor in said use comprises a hematological tumor and / or a lymphoma.

[0074] In some embodiments, the tumor in said use comprises a myeloma.

[0075] In another aspect, the present application provides a method for preventing, treating, and / or alleviating a disease or condition associated with abnormal expression of GPRC5D, comprising administering to a subject in need thereof the antigen-binding protein, the antigen-engaging receptor, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition.

[0076] In some embodiments, the disease or condition associated with aberrant expression of GPRC5D in said method comprises a tumor.

[0077] In some embodiments, the tumor in said methods comprises a solid tumor.

[0078] In some embodiments, the tumor in said methods comprises a non-solid tumor.

[0079] In some embodiments, the tumor in the methods comprises a hematological tumor and / or a lymphoma.

[0080] In some embodiments, the tumor in said methods comprises a myeloma.

[0081] Those skilled in the art can easily ascertain other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the contents of the present application will enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention of the present application. Therefore, the accompanying drawings and the description of the present application are merely illustrative and not restrictive. [Brief description of the drawings]

[0082] Description of the drawings Particular features of the invention are set forth in the appended claims. A better understanding of the features and advantages of the invention can be obtained by reference to the exemplary embodiments described in detail herein and the accompanying drawings, the brief description of which is set forth below. 1A to 1J show the binding activity of the anti-CPRC5D antibodies described in the present application to GPRC5D on the cell surface, analyzed by flow cytometry. FIG. 2 shows the components and connection order of the CAR in the chimeric antigen receptor lentiviral expression vector and GPRC5D core plasmid described in this application. Figures 3A to 3C show the expansion fold of cultured GPRC5D CAR-T cells by activation and the CAR positivity rate of T cells after infection. Figures 4A to 4D show the killing ability of GPRC5D CAR-T cells against target cells (CHO cells / MM1S cells). Figures 5A and 5B show cytokine (IL2\IFN-γ) secretion status after killing of GPRC5D CAR-T cells in vitro. FIG. 6 shows targeted fold expansion of GPRC5D CAR-T cells. Figures 7A-7D show the cytokine secretion, tumor elimination capacity and safety of GPRC5D CAR-T cells in mice. Figures 8A to 8D and 9A to 9D show that anti-GPRC5D antibodies do not bind to cells that do not express the GPRC5D antigen: A: A549 cells, B: KERA cells, C: MCF-7 cells, D: MSC cells. 10A to 10D show the experimental results of the endocytosis effect of anti-GPRC5D. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0083] Specific embodiments In the following, embodiments of the present invention will be described using certain specific embodiments, but those familiar with this technology can easily understand other advantages and effects of the present invention from the disclosure in this application.

[0084] Term definition In the present application, the term "isolated antigen-binding protein" generally refers to a protein having antigen-binding ability that is removed from a naturally occurring state. The "isolated antigen-binding protein" includes a portion that binds an antigen and, optionally, allows the antigen-binding portion to adopt a conformation of the framework or framework portion that promotes its antigen binding. The antigen-binding protein may, for example, include a protein framework region (FR) derived from an antibody or an alternative protein framework region or an artificial framework region with grafted variable regions (CDRs) or CDR derivatives. For example, the antigen-binding protein may include an antibody or an antigen-binding fragment thereof. For example, the antigen-binding protein may bind to a GPRC5D protein. For example, the antigen-binding protein may bind to a GPRC5D protein in competition with a reference antibody. For example, the antigen-binding protein may include an antibody heavy chain variable region VH. For example, the antigen-binding protein may include at least one CDR derived from an antibody heavy chain variable region VH. For example, the VH may include HCDR3, HCDR2 and / or HCDR1. For example, the VH may comprise a framework region H-FR1, and the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1. For example, the VH may comprise a framework region H-FR2, and the H-FR2 is located between the HCDR1 and the HCDR2. For example, the VH may comprise a framework region H-FR3, and the H-FR3 is located between the HCDR2 and the HCDR3. For example, the VH may comprise a framework region H-FR4, and the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3. For example, the antigen binding protein may be a VHH. For example, the antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from IgG. For example, the antibody heavy chain constant region may be derived from human IgG. For example, the antibody heavy chain constant region may be derived from human IgG1.

[0085] The term "antibody" as used includes complete antibodies and binding fragments thereof. Usually, fragments compete with the complete antibody from which they are derived for specific binding to an antigen. Optionally, the antibody or binding fragment thereof may be chemically conjugated to other proteins or expressed in the form of a fusion protein with other proteins. For example, the antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody. For example, the binding protein of the antibody or binding fragment thereof may include GPRC5D. For example, the antibody or binding fragment thereof may have specificity for GPRC5D.

[0086] The term "antigen-binding fragment" refers to a portion of a complete antibody and to the antigen-determining variable region of the complete antibody. For example, the antigen-binding fragment may include Fab, Fab', F(ab')2, Fv fragments and single chain Fv fragments, tandem Fv fragments, VHH, and bispecific antibodies. For example, the antigen-binding fragment may be a VHH. For example, the antigen-binding fragment may bind to GPRC5D. For example, the antigen-binding fragment may have specificity for GPRC5D.

[0087] In this application, the term "VHH" generally refers to an antibody that comprises a variable antigen-binding domain of a heavy chain antibody. VHH may also be called a Nanobody (Nb) and / or a single domain antibody. For example, the VHH may bind to GPRC5D. For example, the VHH may have specificity for GPRC5D.

[0088] In the present application, the antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. The term "heavy chain constant region" refers to three structural domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The term "light chain constant region" refers to one structural domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) and regions called framework regions (FRs) interspersed with more conserved regions. Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues and factors.

[0089] In this application, the term "reference antibody" refers to an antibody that competitively binds to the same epitope as the isolated antigen-binding protein GPRC5D. The reference antibody may include a heavy chain variable region VH. For example, the reference antibody may have three CDR sequences. For example, the VH of the reference antibody may include HCDR1, HCDR2 and HCDR3. For example, the CDR sequence may be identical to the CDR sequence of the isolated antigen-binding protein.

[0090] In the present application, the term "G protein-coupled receptor class C group 5 member D (GPRC5D) protein" refers to an atypical surface orphan receptor. GPRCD5, like other C5 class receptors, is conformationally very similar to the C4 class due to its short amino terminus. GPRCD5 expression in normal tissues is restricted to hair follicles, but is also significantly highly expressed in the bone marrow of multiple myeloma patients and is highly associated with plasma cell tumor burden and genetic abnormalities. For example, GPRC5D as described in the present application may refer to GPRC5D that is specifically expressed in MM patients.

[0091] As used herein, the term "IgG" refers to a polypeptide belonging to the antibody category essentially encoded by the recognized immunoglobulin gamma gene. In humans, this category includes IgG1, IgG2, IgG3, and IgG4. In mice, this category includes IgG1, IgG2a, IgG2b, and IgG3.

[0092] In the present application, the term "chimeric antigen receptor" (CAR) generally refers to a recombinant polypeptide comprising at least an extracellular domain capable of specifically binding to an antigen or target, a transmembrane region, and an intracellular structural domain. For example, the extracellular structural domain and the transmembrane region comprise a hinge region. For example, the chimeric antigen receptor may comprise a low density lipoprotein receptor-related protein or a fragment thereof. For example, the chimeric antigen receptor may comprise a signal peptide. When the extracellular structural domain of the CAR binds to a target antigen on the surface of a target cell, the CAR clusters and transmits an activation stimulus to the cell comprising the CAR. The CAR redirects the specificity of immune effector cells, activates proliferation, cytokine production, and mediates phagocytosis and / or generation of death molecules of cells expressing the target antigen in a major histocompatibility (MHC)-independent manner. For example, the extracellular structure may comprise the antigen-binding protein. For example, the extracellular structure may specifically bind to GPRC5D.

[0093] In the present application, the term "intracellular structural domain" refers to an intracellular structural domain including any cleavage portion sufficient to transmit an activation signal. The intracellular structural domain may include an intracellular signal region and / or a costimulatory signal region. The term "intracellular signal region" refers to an intracellular region capable of generating a signal that promotes an immune effector sub-function of a CAR cell (e.g., a CART cell or a NK cell expressing a CAR). For example, the intracellular signal region may include an intracellular signal region of one or more proteins selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3epsilon, CD79a, CD79b, FcεRIgamma, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a structural domain comprising at least one ITAM. For example, the intracellular signal region may be derived from the signal transduction structural domain of CD3zeta. The term "costimulatory signal region" refers to a portion of the CAR that can transmit an effector signal in the intracellular signal region. For example, the co-stimulatory signal region can include an intracellular co-stimulatory signal region derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand for CD83, CD40, and MyD88. For example, the co-stimulatory signal region can be an intracellular co-stimulatory signal region derived from 4-1BB.

[0094] As used herein, the term "transmembrane domain" refers to a structural domain of a peptide, polypeptide or protein that is capable of spanning the cytoplasmic membrane and is capable of anchoring an extracellular structural domain to the cell membrane. For example, the transmembrane region may comprise a transmembrane domain of one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the transmembrane region may be a transmembrane region derived from CD8.

[0095] In this application, the term "hinge region" refers to a portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 structural domains, e.g., from about position 216 to about position 230 according to the EU numbering system of Kabat. A hinge region is usually a dimeric molecule consisting of two polypeptides with the same amino acid sequence. The hinge region usually contains about 25 amino acid residues and is flexible, allowing independent movement of the antigen binding regions. The hinge region can be subdivided into three structural domains: the upper, middle, and lower hinge structural domains. For example, the hinge region may comprise a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. For example, the hinge region may be derived from CD8.

[0096] In the present application, the term "low-density lipoprotein receptor-related protein" refers to a cell surface protein belonging to endocytic receptors, which is widely distributed in the body, has a large tissue variation, and its main function is to take up cholesterol into cells for cell proliferation and synthesis of steroid hormones and bile salts. For example, the low-density lipoprotein receptor-related protein may be derived from a vertebrate. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may be located at the C-terminus of the intracellular signal region. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may include one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may be low-density lipoprotein receptor-related protein 6 or a fragment thereof.

[0097] In the present application, the term "signal peptide" refers to a leader sequence at the amino-terminus (N-terminus) of a nascent CAR protein that directs the nascent protein to the endoplasmic reticulum during or after translation, leading to subsequent surface expression. For example, the signal peptide may be a signal peptide derived from the CD8 protein. For example, the signal peptide therein comprises the amino acid sequence shown in SEQ ID NO:72.

[0098] In this application, the terms "polypeptide", "peptide" and "protein" can be used interchangeably and refer to a polymer of amino acid residues. The terms can be used to refer to amino acid polymers in which one or more amino acid residues are artificial synthetic chemical mimics of their corresponding naturally occurring amino acids, and can be used to refer to naturally occurring amino acid polymers, amino acid polymers containing modified residues, and non-naturally occurring amino acid polymers. For example, the polypeptide can include the antigen-binding protein.

[0099] In this application, the term "nucleic acid molecule" includes DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. The term "promoter" generally refers to a DNA sequence that affects the expression of a selected DNA sequence in a cell by regulating the expression of the selected DNA sequence operably linked to the promoter. For example, the nucleic acid molecule may code for a corresponding antigen binding protein and / or chimeric antigen receptor. For example, the nucleic acid molecule may include a promoter. For example, the promoter may be a constitutive promoter. For example, the promoter may be an EF1α promoter.

[0100] In the present application, the term "vector" generally refers to a molecule to which one or more nucleic acid molecules of the present application can be attached. For example, the vector can be a viral vector. For example, the vector can be a lentiviral vector.

[0101] In the present application, the term "cell" refers to a cell into which a nucleic acid can be transfected, and the term "cell" includes prokaryotic cells used for propagation of plasmids, and eukaryotic cells used for nucleic acid expression and production of the encoded polypeptide. For example, the cell can include the antigen binding protein, the nucleic acid molecule, and / or the vector. For example, the cell can be an immune effector cell. The term "immune effector cell" refers to an immune cell that is typically involved in an immune response and exerts an effector function. For example, exerting the effector function can include removing foreign antigens, or promoting an immune effector response. For example, immune effector cells can include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells, and / or pluripotent stem cells. For example, immune effector cells can be T cells.

[0102] In this application, the term "pharmaceutical composition" generally refers to a chemical or biological composition suitable for administration to a mammalian individual. For example, the pharmaceutical composition may include the antigen binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector and / or the cell, and any pharma- ceutical acceptable vector. The pharmaceutical composition may be used to prevent, treat, and / or alleviate a disease or condition associated with abnormal expression of GPRC5D. For example, the disease or condition associated with abnormal expression of GPRC5D may include a tumor. For example, the tumor may include a solid tumor and / or a non-solid tumor. For example, the tumor may include a hematological tumor and / or a lymphoma. For example, the tumor may include a myeloma.

[0103] Detailed Description of the Invention Isolated antigen-binding proteins In one embodiment, the present application provides an isolated antigen binding protein capable of binding G protein-coupled receptor class C group 5 member D (GPRC5D) protein with an EC50 value of less than 1.0 μg / mL. For example, the antigen binding protein described herein may bind to GPRC5D protein with an EC50 value of less than about 0.9 μg / mL, less than about 0.8 μg / mL, less than about 0.7 μg / mL, less than about 0.6 μg / mL, less than about 0.5 μg / mL, less than about 0.4 μg / mL, less than about 0.3 μg / mL, less than about 0.2 μg / mL, less than about 0.1 μg / mL or less. For example, the antigen binding protein described herein may bind to GPRC5D protein expressed on the cell surface with an EC50 value of less than about 0.9 μg / mL. For example, binding of an antigen binding protein to a GPRC5D protein expressed on the surface of a cell described herein can be detected using flow cytometric fluorescence sorting (FACS), for example, using an iQue Screener flow cytometer.

[0104] In the present application, the antigen-binding protein may include an antibody or an antigen-binding fragment thereof. In the present application, the antigen-binding fragment may be a Fab, Fab', F(ab)2, Fv fragment, F(ab') 2In the present application, the antibody may include a monoclonal antibody, a chimeric antibody, a humanized antibody and a fully human antibody.

[0105] CDR CDRs are complementarity determining regions of an antibody, which are a part of the antibody variable region, and the amino acid residues in this region contact an antigen or an antigen epitope, thereby specifically identifying and binding to the antigen.

[0106] In the present application, the isolated antigen binding protein may comprise at least one CDR in an antibody heavy chain variable region VH, for example, the VH may be selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:70.

[0107] In the present application, the antigen binding protein may comprise an HCDR3. For example, the HCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:215: AAX 3 X 4 X 5 X 6 X 7 X8 X 9 X 10 X 11 X 12 X 13 X 14 YX 16 Y (SEQ ID NO:215), where X 3 = F, G, R, S, T or V, X 4 = A, G, L, P, R, S or T,X 5 = A, G, K, R, V or Y, X 6 = P, R, S, V or Y, X 7 = A, F, G, I, N, P, S, W or Y,X 8 = A, F, L, P, R, T or Y, X 9 = A, G, L, R, S, T or Y, X 10 = L, P, Q, S, T, W or Y, X 11 = D, G, N, R, S, T or Y,X 12 = A, D, E, G, P, R, S or Y, X 13 = A, G, L, N, R, S or Y, X 14 = E, G, L, N, R, T or V, and X 16 = D or N. For example, the sequence may be partitioned according to the IMGT rules.

[0108] In the present application, the antigen binding protein may comprise an HCDR3. For example, the HCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:218: AAX 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 YX 13 Y (SEQ ID NO:218), where X 3 = D, F, G, K, N, R, S, T or V, X 4 = D, G, I, L, R, S or T,X 5 = A, F, G, R, V or Y, X 6 = G, P, R, S, T, W or Y, X7 = A, G, L, N, R, S, W or Y, X 8 = D, F, G, L, N, R, S, T or Y, X 9 = A, D, G, I, L, P, R, S, T or Y,X 10 = G, L, N, R, S, T, W or Y, X 11 = D, G, L, N, P, R or T,X 13 = D or N.

[0109] In the present application, the antigen binding protein may comprise an HCDR3, and the HCDR3 may be selected from the group consisting of SEQ ID NO:97, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:111, SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:140, SEQ ID NO:144, SEQ ID NO:147, SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:168, SEQ ID NO:170, SEQ ID SEQ ID NO:172, SEQ ID NO:176, SEQ ID NO:179, SEQ ID NO:182, SEQ ID NO:185, SEQ ID NO:188, SEQ ID NO:191, SEQ ID NO:195 and SEQ ID NO:198.

[0110] In the present application, the antigen binding protein may comprise an HCDR2. For example, the HCDR2 of the antigen binding protein may comprise X 1 X 2 X 3 X 4 X 5 X 6 X 7 T, wherein X 1 =I or T,X2 =N, S or T,X 3 = A, P, R, S or W, X 4 = G, R, S, T or absent, X 5 =D or G,X 6 = G or S, and X 7 = D, G, I, N, R, S, T or V. For example, the sequence may be partitioned according to the IMGT rules.

[0111] In the present application, the antigen binding protein may comprise an HCDR2, and the HCDR2 may be selected from the group consisting of SEQ ID NO:96, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:124, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:134, SEQ ID NO:139, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:149, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:167, SEQ ID NO:171, SEQ ID NO:175, SEQ ID NO:178, SEQ ID SEQ ID NO:181, SEQ ID NO:187, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194 and SEQ ID NO:197.

[0112] In the present application, the antigen binding protein may comprise an HCDR1. For example, the HCDR1 of the antigen binding protein may comprise: GX 2 X 3 X 4 SX 6 X 7 X 8 wherein X is an amino acid sequence as shown in 2 = F, G, I, L, N, R, S or Y, X 3 =I or T,X 4 = F or L,X 6= I, L, N, R, S, T or Y,X 7 = D, N or Y, and X 8 = A, D, G, I, N, R, S, T, V or Y. For example, the sequence may be partitioned according to the IMGT rules.

[0113] In the present application, the antigen binding protein may comprise HCDR1, and the HCDR1 may be selected from the group consisting of SEQ ID NO:95, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:109, SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:127, SEQ ID NO:130, SEQ ID NO:133, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:166, SEQ ID NO:169, SEQ ID NO:174, SEQ ID NO:177, SEQ ID NO:180, SEQ ID NO:184, SEQ ID SEQ ID NO:186, SEQ ID NO:189, SEQ ID NO:193 and SEQ ID NO:196.

[0114] In the present application, the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, wherein HCDR1 is selected from the group consisting of SEQ ID NO:95, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:109, SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:127, SEQ ID NO:130, SEQ ID NO:133, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:142, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:166, SEQ ID NO:169, SEQ ID NO:174, SEQ ID NO:177, SEQ ID NO:180, SEQ ID NO:184, SEQ and wherein said HCDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NO:96, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:124, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:134, SEQ ID NO:139, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:149, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:189, SEQ ID NO:193 and SEQ ID NO:196, wherein said HCDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NO:96, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:124, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:134, SEQ ID NO:139, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:149, SEQ ID NO:153 and wherein the HCDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NO:97, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:111, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:175, SEQ ID NO:178, SEQ ID NO:181, SEQ ID NO:187, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, and SEQ ID NO:197.NO:118, SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:140, SEQ ID NO:144, SEQ ID NO:147, SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:176, SEQ ID NO:179, SEQ ID NO:182, SEQ ID NO:185, SEQ ID NO:188, SEQ ID NO:191, SEQ ID NO:195 and SEQ ID It comprises any one of the amino acid sequences set forth in NO:198.

[0115] In the present application, the antigen binding protein may comprise HCDR1, HCDR2, HCDR3, and the HCDR1, HCDR2, HCDR3 may comprise any of the following groups of amino acid sequences: (1) HCDR1: SEQ ID NO: 102, HCDR2: SEQ ID NO: 103, and HCDR3: SEQ ID NO: 104; (2) HCDR1: SEQ ID NO: 105, HCDR2: SEQ ID NO: 106, and HCDR3: SEQ ID NO: 107; (3) HCDR1: SEQ ID NO: 109, HCDR2: SEQ ID NO: 110, and HCDR3: SEQ ID NO: 111; (4) HCDR1: SEQ ID NO: 105, HCDR2: SEQ ID NO: 114, and HCDR3: SEQ ID NO: 115; (5) HCDR1: SEQ ID NO: 116, HCDR2: SEQ ID NO: 117, and HCDR3: SEQ ID NO: 118; (6) HCDR1: SEQ ID NO: 119, HCDR2: SEQ ID NO: 120, and HCDR3: SEQ ID NO: 121; (7) HCDR1: SEQ ID NO: 123, HCDR2: SEQ ID NO: 124, and HCDR3: SEQ ID NO: 125; (8) HCDR1: SEQ ID NO: 127, HCDR2: SEQ ID NO: 128, and HCDR3: SEQ ID NO: 129; (9) HCDR1: SEQ ID NO: 130, HCDR2: SEQ ID NO: 131, and HCDR3: SEQ ID NO: 132; (10) HCDR1: SEQ ID NO: 95, HCDR2: SEQ ID NO: 96, and HCDR3: SEQ ID NO: 97; (11) HCDR1: SEQ ID NO: 133, HCDR2: SEQ ID NO: 134, and HCDR3: SEQ ID NO: 135; (12) HCDR1: SEQ ID NO: 136, HCDR2: SEQ ID NO: 134, and HCDR3: SEQ ID NO: 137; (13) HCDR1: SEQ ID NO: 138, HCDR2: SEQ ID NO: 139, and HCDR3: SEQ ID NO: 140; (14) HCDR1: SEQ ID NO: 142, HCDR2: SEQ ID NO: 143, and HCDR3: SEQ ID NO: 144; (15) HCDR1: SEQ ID NO: 145, HCDR2: SEQ ID NO: 146, and HCDR3: SEQ ID NO: 147; (16) HCDR1: SEQ ID NO: 148, HCDR2: SEQ ID NO: 149, and HCDR3: SEQ ID NO: 150; (17) HCDR1: SEQ ID NO: 152, HCDR2: SEQ ID NO: 153, and HCDR3: SEQ ID NO: 154; (18) HCDR1: SEQ ID NO: 123, HCDR2: SEQ ID NO: 155, and HCDR3: SEQ ID NO: 156; (19) HCDR1: SEQ ID NO: 157, HCDR2: SEQ ID NO: 153, and HCDR3: SEQ ID NO: 158; (20) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 160, and HCDR3: SEQ ID NO: 161; (21) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 162, and HCDR3: SEQ ID NO: 163; (22) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 164, and HCDR3: SEQ ID NO: 165; (23) HCDR1: SEQ ID NO: 166, HCDR2: SEQ ID NO: 167, and HCDR3: SEQ ID NO: 168; (24) HCDR1: SEQ ID NO: 169, HCDR2: SEQ ID NO: 153, and HCDR3: SEQ ID NO: 170; (25) HCDR1: SEQ ID NO: 157, HCDR2: SEQ ID NO: 171, and HCDR3: SEQ ID NO: 172; (26) HCDR1: SEQ ID NO: 174, HCDR2: SEQ ID NO: 175, and HCDR3: SEQ ID NO: 176; (27) HCDR1: SEQ ID NO: 177, HCDR2: SEQ ID NO: 178, and HCDR3: SEQ ID NO: 179; (28) HCDR1: SEQ ID NO: 180, HCDR2: SEQ ID NO: 181, and HCDR3: SEQ ID NO: 182; (29) HCDR1: SEQ ID NO: 184, HCDR2: SEQ ID NO: 106, and HCDR3: SEQ ID NO: 185; (30) HCDR1: SEQ ID NO: 186, HCDR2: SEQ ID NO: 187, and HCDR3: SEQ ID NO: 188; (31) HCDR1: SEQ ID NO: 189, HCDR2: SEQ ID NO: 190, and HCDR3: SEQ ID NO: 191; (32) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 192, and HCDR3: SEQ ID NO: 179; (33) HCDR1: SEQ ID NO: 193, HCDR2: SEQ ID NO: 194, and HCDR3: SEQ ID NO: 195; and (34) HCDR1: SEQ ID NO: 196, HCDR2: SEQ ID NO: 197, and HCDR3: SEQ ID NO: 198.

[0116] FR In the present application, the antibody framework region FR refers to the portion of the antibody variable region that is present between the more divergent (i.e., hypervariable) CDRs. These framework regions are typically called frameworks 1 to 4 (FR1, FR2, FR3, and FR4), and provide a scaffold for three-dimensionally presenting the six CDRs (three from the heavy chain and three from the light chain) to form an antigen-binding surface.

[0117] In the present application, the antigen binding protein may comprise H-FR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:98.

[0118] In the present application, the antigen binding protein may comprise H-FR2, wherein the H-FR2 has the amino acid sequence shown in SEQ ID NO:216: MGWFRQAPGKGRELVAX. 17 (SEQ ID NO:216), wherein said X 17 = A, F, G, R, S or V. For example, the sequence may be partitioned according to the IMGT rules.

[0119] In the present application, the antigen binding protein may comprise an H-FR2, and the H-FR2 comprises an amino acid sequence set forth in any one of SEQ ID NO:99, SEQ ID NO:108, SEQ ID NO:112, SEQ ID NO:122, SEQ ID NO:151, and SEQ ID NO:173.

[0120] In the present application, the antigen binding protein may comprise H-FR3, and the H-FR3 has the amino acid sequence shown in SEQ ID NO:217: 1 YPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYC (SEQ ID NO:217), wherein said X 1 =H, F, S, N or Y. For example, the sequence may be partitioned according to the IMGT rules.

[0121] In the present application, the antigen binding protein may comprise an H-FR3, and the H-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NO:100, SEQ ID NO:113, SEQ ID NO:126, SEQ ID NO:141, and SEQ ID NO:183.

[0122] In the present application, the antigen binding protein may comprise an H-FR4, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:101.

[0123] In the present application, the antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1, H-FR2, H-FR3 and H-FR4 may comprise the amino acid sequences set forth in SEQ ID NO:98, SEQ ID NO:216, SEQ ID NO:217 and SEQ ID NO:101, respectively.

[0124] In the present application, the antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:98, the H-FR2 may comprise the amino acid sequence set forth in any one of SEQ ID NO:99, SEQ ID NO:108, SEQ ID NO:112, SEQ ID NO:122, SEQ ID NO:151 and SEQ ID NO:173, the H-FR3 may comprise the amino acid sequence set forth in any one of SEQ ID NO:100, SEQ ID NO:113, SEQ ID NO:126, SEQ ID NO:141 and SEQ ID NO:183, and the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:101.

[0125] In the present application, the antigen binding protein may comprise an antibody heavy chain variable region VH, and the VH may be selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:70.

[0126] In the present application, the antigen-binding fragment may be a VHH, and the VHH may be selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:70.

[0127] heavy chain constant region In the present application, the isolated antigen binding protein may comprise a heavy chain constant region. The heavy chain constant region refers to a region comprising at least three heavy chain constant structural domains, CH1, CH2, and CH3. Non-limiting, exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting, exemplary heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to one antibody isoform. For example, an antibody comprising a gamma constant region is an IgG antibody, an antibody comprising a delta constant region is an IgD antibody, and an antibody comprising an alpha constant region is an IgA antibody. An antibody comprising a mu constant region is an IgM antibody, and an antibody comprising an epsilon constant region is an IgE antibody. A particular isoform may be further subdivided into subclasses. For example, an IgG antibody is classified into IgG1 (gamma), IgG2 (delta), and IgD (alpha). 1 IgG2(γ 2 IgG3(γ 3 constant region), and IgG4 (γ 4IgA antibodies include, but are not limited to, IgA1 (α 1 IgA2 (including the constant region) and IgA2 (α 2 IgM includes, but is not limited to, IgM1 and IgM2 (including a constant region) antibodies.

[0128] In the present application, the antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from IgG. In the present application, the antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from human IgG. In the present application, the antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from human IgG1.

[0129] Chimeric Antigen Receptor In another aspect, the present application provides a chimeric antigen receptor (CAR), which can comprise a targeting moiety that binds to a GPRC5D protein, for example, the targeting moiety that binds to a GPRC5D protein can be an antigen binding protein described herein.

[0130] For example, a CAR described herein can comprise a VHH, wherein the VHH is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:70.

[0131] In the present application, the CAR comprises an extracellular targeting moiety that binds to the GPRC5D protein, and may also comprise an intracellular structural domain.

[0132] In the present application, the CAR may include an intracellular costimulatory signal region for providing a stimulatory signal. For example, the costimulatory signal region may include an intracellular costimulatory signal region of one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand for CD83, CD40, and MyD88.

[0133] For example, the costimulatory signal region can be an intracellular costimulatory signal region derived from 4-1BB. For example, the costimulatory signal region can include the amino acid sequence shown in SEQ ID NO:78.

[0134] In certain embodiments, the CAR may comprise an intracellular signaling region that may comprise a structural domain having at least one ITAM motif. The intracellular signaling structural domain may transmit an activated signal to the inside of a cell. For example, the intracellular signaling region may comprise an intracellular signaling region derived from one or more proteins selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3epsilon, CD79a, CD79b, FcεRIgamma, FcεRIbeta, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a structural domain that comprises at least one ITAM.

[0135] For example, the intracellular signaling region may be derived from the signal transduction structural domain of CD3ζ. For example, the intracellular signaling region may comprise the amino acid sequence shown in SEQ ID NO:80.

[0136] In certain embodiments, the CAR may comprise a transmembrane domain, which is a sequence in a cell surface protein that spans the cell membrane, and may comprise a hydrophobic alpha helix. The transmembrane domain may be derived from any type I transmembrane protein. The transmembrane domain may be a synthetic sequence predicted to form a hydrophobic helix. For example, the transmembrane region may comprise a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0137] For example, the transmembrane region can be a transmembrane region derived from CD8. For example, the transmembrane region can include the amino acid sequence shown in SEQ ID NO:76.

[0138] In certain embodiments, the CAR may comprise a hinge region that may be located between the extracellular targeting moiety and the transmembrane domain. For example, the hinge region may comprise one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0139] For example, the hinge region can be a hinge region derived from CD8. For example, the hinge region can comprise the amino acid sequence shown in SEQ ID NO:74.

[0140] In the present application, the N-terminus of the targeting portion of the CAR-binding GPRC5D protein may also comprise a signal peptide. For example, the signal peptide may be a signal peptide derived from CD8 protein. For example, the signal peptide may comprise the amino acid sequence shown in SEQ ID NO:72.

[0141] In the present application, the CAR may comprise a low density lipoprotein receptor-related protein or a fragment thereof. For example, the low density lipoprotein receptor-related protein or a fragment thereof may be located at the C-terminus of the CAR. For example, the low density lipoprotein receptor-related protein or a fragment thereof may comprise a low density lipoprotein receptor-related protein 1-12 or a functional fragment thereof. For example, the low density lipoprotein receptor-related protein or a fragment thereof may be a low density lipoprotein receptor-related protein 6 or a fragment thereof. For example, the low density lipoprotein receptor-related protein or a fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:84.

[0142] In the present application, the low density lipoprotein receptor-related protein or a fragment thereof in the CAR may be connected to the C-terminus of the CAR via a self-cleaving peptide (e.g., a 2A peptide such as T2A, P2A, E2A, etc.). For example, the low density lipoprotein receptor-related protein or a fragment thereof may be connected to the C-terminus of the intracellular signal region via T2A.

[0143] In the present application, the CAR may comprise, from the N-terminus to the C-terminus, a targeting moiety that binds to the GPRC5D protein (e.g., the antigen binding protein, and also the VHH described in the present application), the hinge region, the transmembrane domain, the costimulatory signal region and the intracellular signal region, in sequence. For example, the CAR can comprise, sequentially from N-terminus to C-terminus, the VHH, a hinge region derived from CD8, a transmembrane region derived from CD8, a costimulatory signal region derived from 4-1BB, and an intracellular signal region derived from CD3zeta, and the VHH is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID The amino acid sequence may include any one of SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:70.

[0144] In the present application, the CAR may include, sequentially from N-terminus to C-terminus, a targeting moiety that binds to the CPRC50D protein (e.g., the antigen binding protein, and also the VHH described herein), the hinge region, the transmembrane domain, the costimulatory signal region, the intracellular signal region, and the low density lipoprotein receptor-related protein or a fragment thereof. For example, the CAR can comprise, sequentially from N-terminus to C-terminus, the VHH, a hinge region derived from CD8, a transmembrane region derived from CD8, a costimulatory signal region derived from 4-1BB, an intracellular signal region derived from CD3zeta, and a low density lipoprotein receptor-related protein of the amino acid sequence shown in SEQ ID NO:84 or a fragment thereof, and the VHH can be selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID The amino acid sequence may include any one of SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:70.

[0145] In the present application, the CAR may comprise, in order from the N-terminus to the C-terminus, a signal peptide, a targeting moiety that binds to the GPRC5D protein (e.g., the antigen binding protein, and also the VHH described in the present application), the hinge region, the transmembrane domain, the costimulatory signal region and the intracellular signal region.

[0146] In the present application, the CAR may comprise, sequentially from N-terminus to C-terminus, a signal peptide, a targeting moiety that binds to GPRC5D protein (e.g., the antigen binding protein, and also the VHH described in the present application), the hinge region, the transmembrane domain, the costimulatory signal region, the intracellular signal region, and the low density lipoprotein receptor-related protein or a fragment thereof.

[0147] For example, the chimeric antigen receptor can include an amino acid sequence set forth in any one of SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, and SEQ ID NO:204. For example, the chimeric antigen receptor can include an amino acid sequence set forth in any one of SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:214.

[0148] For example, the chimeric antigen receptor can be encoded by a nucleotide sequence set forth in any one of SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, and SEQ ID NO:91.

[0149] nucleic acid molecule In another aspect, the present application also provides one or more nucleic acid molecules, which can be in isolated form of nucleotides, deoxynucleotides and / or ribonucleotides of any length, encoding the isolated antigen binding protein and / or chimeric antigen receptor.

[0150] For example, the nucleic acid molecule can include a promoter. For example, the promoter can be a constitutive promoter. For example, the promoter can be the EF1α promoter.

[0151] For example, the nucleic acid molecule may be any of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID The nucleic acid molecule may comprise a nucleotide sequence as set forth in any one of SEQ ID NO:65, SEQ ID NO:67, and SEQ ID NO:69. For example, the nucleic acid molecule may comprise a nucleotide sequence as set forth in any one of SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, and SEQ ID NO:91.

[0152] vector In another aspect, the present application provides a vector that may contain the nucleic acid molecule. The vector may transform, transduce or transfect a host cell, thereby expressing the genetic material elements that it carries in the host cell. For example, the vector may contain a promoter, a transcription factor, an enhancer, a replicon, a selection element and a reporter gene. For example, the vector may contain components that aid in entry into a cell. In order for the nucleic acid molecule to replicate in the vector, the 5' and 3' ends of the nucleic acid molecule may also contain long terminal repeat sequences.

[0153] For example, the vector can be a viral vector. For example, the vector can be a lentiviral vector.

[0154] cell In another aspect, the present application provides a cell comprising said isolated antigen binding protein, said chimeric antigen receptor, said nucleic acid molecule and / or said vector. The cell may comprise the progeny of a single cell. Due to spontaneous, accidental or deliberate mutations, the progeny may not necessarily be identical (morphologically or genomically of the total DNA cross-complement) to the primordial mother cell.

[0155] In some embodiments, the cells may be immune effector cells. In some embodiments, the cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells, and / or pluripotent stem cells. For example, In some embodiments, the cell may be a T cell.

[0156] Pharmaceutical Compositions In another aspect, the present application provides a pharmaceutical composition, which may comprise the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector and / or the cell, and optionally a pharma- ceutically acceptable adjuvant.

[0157] In some embodiments, the pharmaceutical compositions may also include suitable formulations of one or more (pharmaceutical effective) supports, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Acceptable components of the compositions are preferentially non-toxic to the recipient at the dosages and concentrations used. Pharmaceutical compositions of the present invention may include liquid, frozen, and lyophilized compositions.

[0158] In some embodiments, the pharma- ceutically acceptable adjuvants can include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents that are compatible with drug administration and are typically safe, non-toxic, and not biologically or otherwise undesirable.

[0159] In some embodiments, the pharmaceutical composition may include parenteral, transdermal, intracavitary, intraarterial, intrathecal and / or intranasal administration or injection directly into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the use of the pharmaceutical composition may be by different modes, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal use.

[0160] Preparation method In another aspect, the present application provides a method for preparing said isolated antigen binding protein and / or said chimeric antigen receptor, said method may comprise culturing said cell under conditions in which said antigen receptor and / or said chimeric antigen receptor is expressed.

[0161] The present application also provides a method for preparing a modified immune effector cell, comprising introducing the vector into an immune cell.

[0162] use In another aspect, the present application provides the use of said isolated antigen binding protein, said chimeric antigen receptor, said nucleic acid molecule, said vector, said cell and / or said pharmaceutical composition in the preparation of a medicament, wherein said medicament is for the prevention, alleviation and / or treatment of tumors.

[0163] In another aspect, the present application provides a method for preventing, alleviating and / or treating a tumor, the method may comprise administering to a subject the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition.

[0164] In another aspect, the present application provides the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition for use in the prevention, alleviation and / or treatment of a tumor.

[0165] In the present application, the tumor may include a solid tumor and / or a non-solid tumor.

[0166] In the present application, the tumor may include a hematological tumor and / or a lymphoma.

[0167] In the present application, the tumor may include a myeloma. In the present application, the tumor may include multiple myeloma.

[0168] In this application, the subject may include a human or a non-human animal.

[0169] In another aspect, the present application provides a polypeptide comprising said antigen binding protein.In another aspect, the present application provides an antibody-drug conjugate comprising said isolated antigen binding protein.

[0170] In another aspect, the present application provides a kit or drug administration device comprising said isolated antigen binding protein, said chimeric antigen receptor, said nucleic acid molecule, said vector, said cell and / or said pharmaceutical composition.

[0171] The following examples are not intended to be limited by theory and are only used to illustrate the antigen-binding proteins, preparation methods, uses, etc. of the present application, and are not used to limit the scope of the present application. EXAMPLES

[0172] Working Example Example 1. Screening of anti-GPRC5D antibodies using the artificial synthetic nano antibody library NanoOri_1.0 First, we compared the structure of 296 nanoantibodies without repeat sequences registered in the Protein Data Bank (PDB) database and determined three types of CDRs mutation strategies with CDR3 lengths of 14, 17, and 21 amino acids. Caplacizumab, a nanoantibody already on the market, was selected as the backbone, and the nucleotide sequence of Caplacizumab was fully genetically synthesized and cloned into the HP153 phage vector. After that, the single-stranded DNA of HP153 was extracted, and the CDRs of the nanoantibody were mutated using the Kunkel Mutagenesis method to obtain double-stranded DNA. The double-stranded DNA was electroporated into competent E. coli SS320 previously infected with M13KO7 helper phage, and the phage supernatant was collected after overnight culture, and finally, a diversity of 1.44 × 10 was obtained as a seed library. 10 We constructed a synthetic nanoantibody library, NanoOri_1.0 (Shanghai Yuaneng Origincell Technology Co., Ltd.), and performed antibody sequence screening.

[0173] The GPRC5D full-length sequence (cat:HG24447-UT) purchased from Sino Biological Company was constructed into the company's existing lentiviral vector, and lentiviral transduction was performed to prepare overexpressing cell lines of target cells CHO-GPRC5D and target cells 293-GPRC5D. The target cells CHO-GPRC5D and 293-GPRC5D were used to perform four rounds of alternating panning of the artificial synthetic nano-antibody library (Shanghai Yuaneng Origincell Technology Co., Ltd.).

[0174] One antibody was taken from the artificial synthetic nano antibody library NanoOri_1.0, and reprecipitated with PEG / NaCl. The target cells CHO-GPRC5D and 293-GPRC5D were used as an alternating screen for cell panning. 500 μL of phage and 500 μL of 10% FBS / PBS buffer were mixed to a final volume of 1 mL, placed in a 2 mL low-adsorption EP tube, confined at 4 °C for 1 h, and gently rotated or periodically shaken. CHO-GPRC5D cells were collected by centrifugation at 140 g for 10 min and washed once with 10 mL of PBS. After counting and the viable cell count reaching 95% or more, the CHO-GPRC5D cells were washed three times with 5% FBS / PBS buffer. Then, the cell count was adjusted to 1 × 10 7 The cells were resuspended in 1 mL of 5% FBS / PBS buffer until the phage antibody library was fully absorbed. During this time, the cells were kept on ice. The cells were centrifuged at 140 g for 2 min at 4°C, resuspended in 1 mL of the trapped phage antibody library, rotated gently or swirled periodically at 4°C or room temperature for 2 h, the supernatant was removed, and the collected cells were resuspended in 1 mL of 5% FBS / PBS buffer, washed twice, and placed in a new 2 mL low-adsorption EP tube after the final suspension to avoid elution of phages nonspecifically adsorbed to the EP tube.

[0175] The cells were resuspended in 1 mL of 100 nM Gly-HCl (pH 2.2), incubated at room temperature for 10 min, centrifuged at 140 g for 2 min at 4°C, and the supernatant was transferred to a new EP tube and 25 μL of 2 M Tris neutralizing solution was added. Approximately 1 mL of the neutralizing solution was further neutralized with approximately 500 μL of 7.5% FBS / PBS buffer.

[0176] Three adsorbed CHO cells were centrifuged in the same manner as CHO-GPRC5D cells. The eluted and neutralized phages were resuspended in CHO cells. They were then gently rotated or periodically shaken for 30 min at 4°C. They were then centrifuged at 140g for 2 min at 4°C, and the supernatant was used for the next adsorption step. This procedure was repeated three times. The final phage supernatant was used to infect TG1 cells.

[0177] A second round of cell panning was performed in the same manner, with the number of target cells CHO-GPRC5D increased to 5 × 10 6 The third and fourth rounds of cell panning were performed in the same manner, with the target cells changed to 293-GPRC5D and the cell number increased to 1 × 10 6 The number of washes was increased to 10.

[0178] After four rounds of panning, the output phages were found to be significantly enriched specifically for the target cells. A cell-based monophage ELISA was used to identify the phage antibody clones obtained by panning: CHO-GPRC5D target cells were cultured at 3 × 10 4 The plate was coated with cells / well and left at 4℃ overnight. Next, non-specific binding sites were blocked with 5% skim milk powder. After thorough washing, the monoclonal phage supernatant (pre-mixed with skim milk powder) was taken and placed in a 96-well plate and incubated for 2h. After thorough washing, Anti-M13-HRP was added and reacted for 45min. After thorough washing, TMB was added to develop color, and after acting at room temperature for 5-10min, the reaction was terminated with sulfuric acid, and the OD value of each well was measured at 450nm.

[0179] Thirty-four phage antibody clones (i.e., the isolated GPRC5D antibodies described in this application) that specifically bind to human GPRC5D were obtained by ELISA and were sequenced to identify single-chain V HThe H gene sequences were obtained, and the 10 phage antibody clones were named 3B5, 3E7, 4A2, 4B3, 3E9, 3F5, 3G4, 4A4, 4E12, 4F10, 505A1, 505A3, 505A11, 505B12, 505D2, 505F1, 505H3, 505H8, 505H10, 506A5, 506A8, 506B6, 506B12, 506C2, 506C5, 506C8, 506C11, 506D2, 506D8, 506E1, 506F5, 506G1, 506H2 and 506H9, respectively. The CDR sequences of the 34 clones are shown in Table 1, and the amino acid sequences of the variable regions are SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:9 As shown in SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:70.

[0180] [Table 1]

[0181] Example 2. Anti-GPRC5D V H Construction of HhFc fusion antibody and its eukaryotic expression and purification Phage antibody clones 3B5, 3E7, 4A2, 4B3, 3E9, 3F5, 3G4, 4A4, 4E12, 4F10, 505A1, 505A3, 505A11, 505B12, 505D2, 505F1, 505H3, 505H8, 505H10, 506A5, 506A8, 506B6, 506B12, 506C2, 506C5, 506C8, 506C11, 506D2, 506D8, 506E1, 506F5, 506G1, 506H2 and 506H9, and positive control antibody ET150-5 The scFv (patent CN107428829A) was redesigned and constructed into a eukaryotic expression vector containing Fc (IgG1): phage antibody clones and V of 3B5, 3E7, 4A2, 4B3, 3E9, 3F5, 3G4, 4A4, 4E12, 4F10, 505A1, 505A3, 505A11, 505B12, 505D2, 505F1, 505H3, 505H8, 505H10, 506A5, 506A8, 506B6, 506B12, 506C2, 506C5, 506C8, 506C11, 506D2, 506D8, 506E1, 506F5, 506G1, 506H2 and 506H9. H Primers were designed to PCR amplify H, and the PCR product was recombination cloned into pcDNA3.4-hFc vector that was double digested with SfiI and NotI restriction endonucleases. After correctly sequencing, the plasmids were transfected into 293F cells to transiently express and purified by Protein A affinity column to obtain 3B5, 3E7, 4A2, 4B3, 3E9, 3F5, 3G4, 4A4, 4E12, 4F10, 505A1, 505A3, 505A11, 505B12, 505D2, 505F1, 505H3, 505H8, 505H10, 506A5, 506A8, 506B6, 506B12, 506C2, 506C5, 506C8, 506C11, 506D2, 506D8, 506E1, 506F5, 506G1, 506H2 and 506H9 antibodies. The SEC purity of the antibody portion is shown in Table 2.

[0182] [Table 2]

[0183] Example 3. Detection of binding activity of anti-GPRC5D antibody to cell surface GPRC5D The cell surface target antigen (GPRC5D) binding activity of the antibodies (i.e., 3B5, 3E7, 4A2, 4B3, 3E9, 3F5, 3G4, 4A4, 4E12, 4F10, 505A1, 505A3, 505A11, 505B12, 505D2, 505F1, 505H3, 505H8, 505H10, 506A5, 506A8, 506B6, 506B12, 506C2, 506C5, 506C8, 506C11, 506D2, 506D8, 506E1, 506F5, 506G1, 506H2 and 506H9 antibodies obtained in Example 2) was assayed using PBS containing 0.1% BSA as a buffer solution, using iQue Detection was performed by flow cytometry fluorescence sorting (FACS) using a Screener flow cytometer (purchased from IntelliCyt). 6 Target cells (i.e., MM1S myeloma cells) were prepared at 100 μg / mL, and 30 μL was added to each well of a 96-well sharp bottom plate (corning 3894). The detection antibody concentration was adjusted to 10 μg / mL with buffer, and the antibody was diluted at a 3-fold ratio to form eight concentration gradients. 30 μL / well of the prepared antibody was added to the spread target cells and mixed evenly. Incubated in a refrigerator at 4°C for 1 h. 150 μL of buffer was added to each well, centrifuged at 300 g for 5 min, the supernatant was discarded, and the cells were shaken to loosen, and washed once. Fluorescent secondary antibody (ab98593) was prepared at a ratio of 1:200 using buffer, and 30 μl / well was added to the cells and mixed evenly, and incubated in a refrigerator at 4°C for 30 min. 150 μL of buffer was added to each well, centrifuged at 300 g for 5 min, the supernatant was discarded, and the cells were shaken to loosen. The wells were washed once. 35 μL of buffer solution was added to each well, mixed uniformly, and then detected by flow cytometer. The analysis results of affinity binding test by flow cytometry are shown in Table 3 and Figure 1, and the anti-GPRC5D VHH of the present application can bind to GPRC5D on the cell surface, and the binding ability is equal to or higher than that of the positive control ET150-5 scFv.

[0184] [Table 3]

[0185] Example 4. Construction of chimeric antigen receptor lentiviral expression vector The CAR lentivirus core empty plasmid (self-constructed by Shanghai Yuaneng Origincell Technology Co., Ltd. and containing the ori2 novel element, hereinafter referred to as the CAR lentivirus empty vector) was double-digested using SphI and NotI endonucleases (purchased from NEB) to produce a linearized fragment of 8992 bp, which was recovered by gel cutting and then mixed with the VHH fragments of 3B5, 3E7, 4A2, 4B3, 3E9, 3F5, 3G4, 4A4, 4E12 and 4F10 obtained in Example 1 at a molar ratio of 1:3. Homologous recombination was performed to transform DH5α competent cells. Single colonies were picked and identified by sequencing. The elements and connection order of each part of CAR in the CAR lentiviral empty vector and GPRC5D core plasmid (3B5VHH-CAR-ori2, 3E7-CAR-ori2, 4A2-CAR-ori2, 4B3-CAR-ori2, 3E9-CAR-ori2, 3F5-CAR-ori2, 3G4-CAR-ori2, 4A4-CAR-ori2, 4E12-CAR-ori2 and 4F10-CAR-ori2) are shown in Figure 2, and the amino acid sequences are shown in order as SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203 and SEQ ID NO: 204.

[0186] Example 5. Lentivirus packaging and viral titration As an example, the lentiviral vector system used to construct the present invention belongs to the third generation, and the system is composed of three plasmids: a packaging plasmid psPAX2 encoding Gag-Pol protein and Rev protein, a PMD2.G plasmid encoding the envelope protein VSV-G, and a core plasmid, that is, each CAR lentiviral plasmid containing a VHH sequence in the above Example 4 (i.e., 3B5VHH-CAR-ori2, 3E7-CAR-ori2, 4A2-CAR-ori2, 4B3-CAR-ori2, 3E9-CAR-ori2, 3F5-CAR-ori2, 3G4-CAR-ori2, 4A4-CAR-ori2, 4E12-CAR-ori2, and 4F10-CAR-ori2). The expression of the CAR gene in each CAR lentiviral plasmid is controlled by the elongation factor-1α (EF-1α) promoter.

[0187] 5.1 The lentivirus packaging process is as follows: (1) Add approximately 2.5 × 10 293T cells (usually cells within 20 generations) in 5 mL of 293T cell complete medium (high-glucose DMEM containing 10% FBS). 6 After mixing thoroughly, inoculate the cells into a 6 cm dish (ensure that the cell confluence is 80-90% after 20-24 hours) and incubate at 37℃, 5% CO 2 The cells were cultured overnight in an incubator. (2) After about 20-24 hours, 300 μL of Opti-MEM medium was taken, and a total of 6.3 μg of the three plasmids (added in a mass ratio of CAR lentivirus core plasmid:psPAX2:PMD2.G=4:3:2) was added, mixed well, and then 18.9 μL of FuGENEHD transfection reagent (Promega, E2311) was added, mixed carefully and thoroughly, and then allowed to stand at room temperature for 15-20 minutes. Next, the mixture was carefully added dropwise to a 6 cm dish, the dish was carefully shaken, and then incubated at 37°C, 5% CO 2 The cells were cultured overnight in an incubator. (3) Approximately 20-24 h after transfection, the supernatant from the 6 cm dish was carefully discarded, and 5 mL of complete 293T cell medium was slowly added. (4) Approximately 48 hours after transfection, the supernatant (containing dead cells and debris) from the 6 cm dish was collected and centrifuged at 3,000 g for 5 minutes. The supernatant virus solution after removing the cell debris was transferred to a new centrifuge tube, aliquoted, and stored at -80°C.

[0188] 5.2 Lentivirus titration was performed as follows. (1) The virus solution prepared above was quickly thawed. (2) 293T cells in good growth state (usually cells within 20 generations) were taken, the upper layer waste liquid was discarded, and the cells were washed with PBS, and then digested with 0.25% pancreatic enzyme (GIBICO) at 37°C for about 3 min. After the cells were completely digested, a specified volume of 293T cell complete medium was added to terminate the reaction. Samples were taken and counted, and the cell density was confirmed to be 2.0 × 10 5 The cells were then inoculated into 6-well plates at an inoculation volume of 2.5 mL / well, with polybrene added to a final concentration of 10 μg / mL. (3) Next, the virus solution was added to each of the 6-well plates. Typically, three sample gradients of 2 μL, 5 μL, and 10 μL were prepared for each virus solution, and 1-2 blank wells to which no virus solution was added were prepared as negative controls for flow cytometry measurements. (4) Cell culture plates are incubated at 37°C and 5% CO 2 The cells were cultured statically in an incubator at 37 °C. (5) After 48 hours of static culture, the upper layer of the waste liquid was discarded, the cells were washed with PBS, and then digested with 0.25% pancreatic enzyme (GIBICO) at 37°C for about 3 minutes. After the cells were completely digested, a specified volume of 293T cell complete medium was added to terminate the reaction. Samples were taken and counted, yielding about 5 × 10 5 Cells were placed into a 1.5 mL sterile centrifuge tube and marked. (6) The solution was adjusted to 1.2-1.5 mL using NBS solution (PBS solution containing 1% newborn calf serum), centrifuged at 4°C and 500 g for 5 minutes, and the supernatant was discarded. (7) 1 mL of NBS was added to each tube, the tubes were gently mixed by infusing, and the tubes were centrifuged at 4°C and 500 g for 5 minutes, and the supernatant was thoroughly discarded. (8) 50 μL of NBS and 0.8 μg of Biotin-Goat anti-human F(ab)2 antibody (109-066-006) were added to each tube, gently mixed with a pipette (no primary antibody was added to the negative control), and incubated at 4°C for 60 min. (9) After the incubation was completed, 1 mL of NBS was directly added to resuspend the cells, followed by centrifugation at 4°C and 500 g for 5 minutes, and the supernatant was discarded. (10) 50 μL of NBS and APC streptavidin (554067) fluorescent secondary antibody (no protein was added to the negative control) were added to each tube and incubated at 4° C. for 30 minutes. (11) Step (9) was repeated. (12) Each tube contained 200 μL of NBS-resuspended cells. (13) The percentage of fluorescent expression was detected using a BD FACS CantoII flow cytometer, and the data were processed.

[0189] Results are usable only if the positive-negative control positive rate for each test tube is between 5%-20%.

[0190] Lentiviral titer (IM / mL) = number of cells plated × (test tube positivity rate – control tube positivity rate) / volume of virus inoculated (mL).

[0191] As a result, the titer range of each CAR virus containing the above VHH was 1-10 × 10 6 IM / mL.

[0192] Example 6. T cell infection and expansion of BCMA CAR-T cells in vitro The method for preparing CAR-T cells containing the GPRC5D antibody sequence and the positive control sequence 109 scFv (Patent CN107428829A) is as follows: (1) Blood samples collected by the apheresis device were isolated using Ficoll density gradient centrifugation to obtain human peripheral blood mononuclear cells (PBMCs) (apheresis blood was provided by volunteers from the Ark Program of Shanghai Yuaneng Origincell Technology Co., Ltd.). (2) PBMCs were sorted by CD3 positive magnetic beads to obtain CD3+ T cells with purity >90%. For T cell sorting method, refer to the product instruction manual (MACS, DS130-050-101). (3) Resuspend the CD3+ T cells using T cell complete medium (X-VIVO 15 (Lonza) + 5% FBS + cytokines) and add 3x the cell volume of already washed CD3 / CD28 Dynabeads (Gibco, 40203D) and supplement with T cell complete medium to achieve a cell density of 1.0-1.2 × 10 6 Adjust the cell count to 100 cells / mL and incubate the cells at 37°C, 5% CO. 2 The cells were activated and cultured in an incubator (recording is at D0). (4) Lentiviral transduction is performed 20-24 h after T cell activation. (5) T cells activated for 20-24 h were collected and centrifuged at 500 g for 5 min. The cells were then resuspended in a given volume of complete T cell medium, sampled and counted, and the virus solutions whose titers were measured in Example 5 were added at a ratio such that the virus reinfection index (MOI) was 2-5. Polybrene was then added to a final concentration of 5 μg / mL, and the complete T cell medium was supplemented to adjust the cell density to 0.6-1.0 × 10 6 The cells were then incubated at 37°C, 5% CO 2 The cells were cultured in an incubator, and T cells not infected with the lentivirus were collected separately as a negative control group. (6) Approximately 20-24 h after viral transduction, T cells from each group were collected and centrifuged at 500 g for 5 min. The cells were then resuspended in a given volume of T cell complete medium. After sampling and counting, the cells were supplemented with T cell complete medium to obtain a cell density of 0.5-0.7 × 10 6 Adjusted to cells / mL. (7) After that, count the CAR-T cells as per 1-2, replenish the T cell complete medium according to the actual situation, and adjust the cell density to 0.5-1.0 × 10 6 Adjusted to cells / mL. (8) Typically, Dynabeads were removed on day 7 of culture, and the entire culture cycle took approximately 12 days.

[0193] The experimental results are shown in Figures 3A-3D. The total amplification fold of GPRC5D CAR-T cells activated and cultured for 12 days was 100-600 times, and the CAR positive rate after infection was 75%-95%, which could be used for cytological functional experiments.

[0194] Example 7. Evaluation of in vitro target killing activity of GPRC5D CAR-T cells To accurately detect the killing ability of CAR-T cells against target cells, the GPRC5D gene was introduced into CHO cells via lentivirus, and GPRC5D-positive cells, i.e., CHO-GPRC5D cells, were selected using a flow sorter. The target cells were CHO-GPRC5D cells, and the negative cells were CHO-negative cells. The effector cells cultured until day 9 were incubated with the target cells for 20 h at effector to target ratios of 1:3, 1:1, and 3:1, respectively, and the viability of the target cells (i.e., the killing ability of CAR-T cells) was determined by the LDH method.

[0195] The cell killing toxicity can be calculated using the following formula:

[0196] TIFF2024534847000004.tif15170

[0197] As shown in Figure 4A-C, when compared with blank effector T cells, anti-GPRC5D CAR-T cells and the 109 positive control showed significant killing effects against target cells in a gradient-dependent manner, whereas no killing effects against non-target cells were observed.

[0198] In another method to detect the killing effect, MM1S-lucifurase was used as target cells, and the killing effect was determined by detecting the luciferase level of the remaining target cells after co-incubation with effector CAR-T cells for 6 h. The detection results at an effector to target ratio of 1:1 are shown in Figure 4D, and the results show that the present CAR-T cells have a significant killing effect compared to T cells.

[0199] Example 8. Cytokine secretion of GPRC5D CAR-T cells after in vitro killing Referring to Example 7, a target cell killing experiment was carried out in a 96-well plate, the effector cell to target cell ratio was 1:1, and the number of effector cells in each 96-well was 2×10 4 Target and effector cells were added sequentially to each group, with two replicate wells for each effector-to-target ratio, and two separate effector cell wells for each group to detect background factor secretion by effector cells.

[0200] Approximately 24 h after target cell killing, the 96-well plate was centrifuged and the supernatant from each well was collected (supernatants should be stored in a -80°C refrigerator if not tested immediately).

[0201] Effector cells were centrifuged to remove the supernatant and then cultured at 3 × 10 5 The CAR-T cells were diluted to 100 μL / mL and 100 μL was set aside from each well. They were mixed with non-target cells (i.e., CAR-T cells that were not cultured with tumor cells) and MM1S cells in a round-bottom 96-well plate at an effector-to-target ratio of 1:1, and after 24 h of co-incubation, the supernatant was aspirated and the IL-2 and IFN-γ contents in the supernatant were detected by R&D Cytokine Kit.

[0202] As shown in Figure 5, without stimulation with MM1S cells, each CAR-T cell group secreted small amounts of IL-2 and IFN-γ, but when stimulated with MM1S cells, the present application produced more IFN-γ secretion during target cell killing compared to the positive control 109-ori2 selected in the present application.

[0203] Example 9. Targeted proliferation ability of GPRC5D CAR-T cells In this example, untreated MM1S cells were selected as target cells to detect the target proliferation ability of CAR-T cells.

[0204] This is usually performed after culturing CAR-T cells for about 9-12 days, but the CAR positivity rate of each group was detected in advance, and blank effector T cells were used to adjust the CAR positivity rate of each group so that it was consistent (X-VIVO 15 medium, which does not contain any additives, was selected as the medium).

[0205] CAR-T cells and MM1S cells were given the first target stimulation at an effector-to-target ratio of 1:1, co-incubated for 4-5 days, after which all cells were harvested, sampled, counted, and given the second target stimulation at a ratio of 1:3 (total CAR-T cells:new MM1S cells), co-incubated for another 4-5 days, and then given the third target stimulation (the ratio between the two cells was the same as the second target stimulation). During the third target stimulation, the appropriate amount of X-VIVO 15 medium was replenished every 1-2 days depending on the cell growth status.

[0206] As shown in Figure 6, after 2-3 rounds of target stimulation of GPRC5D CAR-T cells, the fold expansion of our CAR-T was comparable to the positive control, whereas the T cell co-incubated wells and MM1S target cell wells alone showed little expansion.

[0207] Example 10. GPRC5D CAR-T Mouse In Vivo Study We further verified the tumor-eliminating and tumor-suppressing effects of GPRC5D CAR-T cells in vivo using NSG mice. Specifically, we divided the mice into three groups: a T cell group, a positive control group, and a GPRC5D CAR-T cell group, and performed the test using five mice in each group. First, NSG mice were treated with 1 × 10 7 MM1S cells were subcutaneously inoculated at a dose of 150 cells / mouse, and approximately 2 weeks later (when tumor size was 50-150 mm 3CAR-T (or T) cells cultured for 10 to 12 days were injected into the tail vein of three groups, and the dose of CAR-T cells was 2 × 10 6 (The total number of inoculated cells for each mouse was standardized to T cells.) After inoculation of CAR-T (or T) cells, cytokine secretion was detected from the peripheral blood of the mice on the 7th and 14th days after inoculation of CAR-T (or T) cells, and the tumor size and body weight of the mice were measured three times a week and observed for three weeks.

[0208] Regarding cytokine secretion, as shown in Figures 7B and 7C, the GPRC5D CAR-T cell group produced more IFN-γ from within the mouse body and for a longer duration (IFN-γ detected on day 14 after CAR-T administration was significantly higher than that in the control group), but the secretion of other cytokines such as IL-2 was very low, and the difference between the groups was very small.

[0209] In terms of the tumor disappearance rate, as shown in Figure 7E, GPRC5D CAR-T cells were basically consistent with the positive control group, and showed a very significant tumor disappearance effect compared with the T cell group.

[0210] As shown in Figure 7D, after inoculation of CAR-T (or T) cells into NSG mice, the mouse weight was measured every 2 to 3 days, the mouse weight curve was plotted, and the mouse hair color, excrement, food intake, body movement, and mouse death status were observed and recorded. The mouse weights of the GPRC5D CAR-T cell and positive control groups showed a stable growth trend over time, which was consistent with the weight curve trend of the T group mice. During the test period, the mouse hair color of the GPRC5D CAR-T cell and positive control groups was bright, and the behavior, food intake, urine and feces colors were all normal, confirming that the GPRC5D CAR-T cells have good safety in the mouse body.

[0211] Example 11. Specificity test of GPRC5D antibody A non-specific binding test of the antibodies (i.e., the 3B5, 3E7, 4A2, 4B3, 3E9, 3F5, 3G4, 4A4 and 4E12 antibodies obtained in Example 2) to the cell surface target antigen (GPRC5D) was carried out by flow cytometry fluorescence sorting (FACS) using PBS containing 0.1% BSA as a buffer solution, using an iQue Screener flow cytometer (purchased from IntelliCyt). That is, target cells (i.e., cells that do not express the GPRC5D antigen, such as A549 cells, KERA cells, MCF-7 cells, and MSC cells) were diluted to 1x10 6 The concentration of each antibody was set to 1:1 cells / mL and added to a 96-well sharp bottom plate (corning 3894) at 30 μL / well. A detection antibody was prepared at a concentration of 20 μg / mL using buffer, and the antibody was diluted 3-fold to form eight concentration gradients. The prepared antibodies at different concentrations were added to the plated target cells at 30 μL / well, mixed well, and incubated in a refrigerator at 4 °C for 1 h. 150 μL of buffer was added to each well, centrifuged at 300 g for 5 min, the supernatant was discarded, the cells were shaken to loosen, and the washing was repeated once. A fluorescent secondary antibody (ab98593) was prepared at a ratio of 1:200 using buffer, added to the cells at 30 μL / well, mixed well, and incubated in a refrigerator at 4 °C for 30 min. 150 μL of buffer was added to each well, centrifuged at 300 g for 5 min, the supernatant was discarded, the cells were shaken to loosen, and the washing was repeated once. 35 μL of buffer solution was added to each well, mixed thoroughly, and then detected using a flow cytometer.

[0212] The results of affinity binding experiments by flow cytometry are shown in Figures 8A to 8D and Figures 9A to 9D, and indicate that the anti-GPRC5D antibodies 3B5, 3E7, 4A2, 4B3, 3E9, 3F5, 3G4, 4A4 and 4E12 do not bind nonspecifically to the above-mentioned cells, suggesting that the anti-GPRC5D antibodies have strong specificity.

[0213] Example 12. Endocytosis effect experiment of GPRC5D antibody The wild-type cell line MM.1S cells and NCIH929-GPRC5D cells overexpressing the GPRC5D target antigen were selected, and the specific operation procedures were as follows: 5x10 4 The target cells were coated on a 96-well V-bottom plate at 30 μL / well, for a total of two plates (plate A and plate B). The antibody dilution concentration started at 20 μg / mL, and diluted in a 3-fold ratio gradient, setting up a total of seven concentration gradients, and added to the 96-well V-bottom plate coated with the target cells at 30 μL / well, mixed well, incubated for 1 h, and washed once with PBS containing 0.1% BSA. Based on the antibody dilution concentration, the antibody was diluted 1:200 and added with 30 μL / well of Anti-Human IgG-Fc (Dylight650) secondary antibody, incubated at 4 °C for 30 min, washed once with PBS containing 0.1% BSA, and then added 50 μL of complete medium (RPMI1640 + 10% FBS). Plate A and plate B were incubated in an incubator at 4 °C and 37 °C for 2 h, respectively, and washed once with PBS containing 0.1% BSA. 30 μL of fluorescence quenching buffer was added to each well and left at 4°C for 10 min, after which PBS containing 0.1% BSA was added, the cells were washed once, and the cells were resuspended in 30 μL / well of PBS containing 0.1% BSA and detected using an intellicyt iQue3 flow cytometer.

[0214] The results are shown in Figures 10A-10D and demonstrate that the GPRC5D antibodies provided herein have varying degrees of endocytosis efficacy at 37°C.

[0215] The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the embodiments described herein will be apparent to those skilled in the art and remain within the scope of the appended claims and their equivalents.

Claims

1. An isolated antigen-binding protein capable of binding to a G protein-coupled receptor class C group 5 member D (GPRC5D) protein. wherein the antigen binding protein comprises HCDR1, HCDR2 and HCDR3; said HCDR1 comprises an amino acid sequence represented by GX2X3X4SX6X7X8, wherein X2 is F, G, I, L, N, R, S or Y, X3 is I or T, X4 is F or L, X6 is I, L, N, R, S, T or Y, X7 is D, N or Y, and X8 is A, D, G, I, N, R, S, T, V or Y; said HCDR2 comprises the amino acid sequence set forth in X1, X2, X3, X4, X5, X6, X7, T, wherein Xi is I or T, X2 is N, S or T, X3 is A, P, R, S or W, X4 is G, R, S, T or absent, X5 is D or G, X6 is G or S, and X7 is D, G, I, N, R, S, T or V; The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:215 or SEQ ID NO:

218.

2. The antigen-binding protein of claim 1 , comprising an antibody or an antigen-binding fragment thereof.

3. The antigen-binding fragments include Fab, Fab', F(ab), 2 , Fv fragment, F(ab') 2 3. The antigen binding protein of claim 2, comprising a ., scFv, di-scFv, VHH and / or dAb.

4. The antigen-binding protein of claim 1, comprising HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise any pair of amino acid sequences selected from the group consisting of: (1) HCDR1: SEQ ID NO: 102, HCDR2: SEQ ID NO: 103, and HCDR3: SEQ ID NO: 104; (2) HCDR1: SEQ ID NO: 105, HCDR2: SEQ ID NO: 106, and HCDR3: SEQ ID NO: 107; (3) HCDR1: SEQ ID NO: 109, HCDR2: SEQ ID NO: 110, and HCDR3: SEQ ID NO: 111; (4) HCDR1: SEQ ID NO: 105, HCDR2: SEQ ID NO: 114, and HCDR3: SEQ ID NO: 115; (5) HCDR1: SEQ ID NO: 116, HCDR2: SEQ ID NO: 117, and HCDR3: SEQ ID NO: 118; (6) HCDR1: SEQ ID NO: 119, HCDR2: SEQ ID (7) HCDR1: SEQ ID NO: 123, HCDR2: SEQ ID NO: 124, and HCDR3: SEQ ID NO: 125; (8) HCDR1: SEQ ID NO: 127, HCDR2: SEQ ID NO: 128, and HCDR3: SEQ ID NO: 129; (9) HCDR1: SEQ ID NO: 130, HCDR2: SEQ ID NO: 131, and HCDR3: SEQ ID NO: 132; (10) HCDR1: SEQ ID NO: 95, HCDR2: SEQ ID NO: 96, and HCDR3: SEQ ID NO: 97; (11) HCDR1: SEQ ID NO: 133, HCDR2: SEQ ID NO: 134, and HCDR3: SEQ ID NO: 135; (12) HCDR1: SEQ ID NO: 136, HCDR2: SEQ ID NO: 134, and HCDR3: SEQ ID NO: 137; (13) HCDR1: SEQ ID NO: 138, HCDR2: SEQ ID NO: 139, and HCDR3: SEQ ID NO: 140; (14) HCDR1: SEQ ID NO: 142, HCDR2: SEQ ID NO: 143, and HCDR3: SEQ ID NO: 144; (15) HCDR1: SEQ ID NO: 145, HCDR2: SEQ ID NO: 146, and HCDR3: SEQ ID NO: 147; (16) HCDR1: SEQ ID NO: 148, HCDR2: SEQ ID NO: 149, and HCDR3: SEQ ID NO: 150; (17) HCDR1: SEQ ID NO: 152, HCDR2: SEQ ID NO: 153, and HCDR3: SEQ ID NO: 154; (18) HCDR1: SEQ ID NO: 123, HCDR2: SEQ ID NO: 155, and HCDR3: SEQ ID NO: 156; (19) HCDR1: SEQ ID NO: 157, HCDR2: SEQ ID NO: 153, and HCDR3: SEQ ID NO: 158; (20) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 160, and HCDR3: SEQ ID NO: 161; (21) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 162, and HCDR3: SEQ ID NO: 163; (22) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 164, and HCDR3: SEQ ID NO: 165; (23) HCDR1: SEQ ID NO: 166, HCDR2: SEQ ID NO: 167, and HCDR3: SEQ ID NO: 168; (24) HCDR1: SEQ ID NO: 169, HCDR2: SEQ ID NO: 153, and HCDR3: SEQ ID NO: 170; (25) HCDR1: SEQ ID NO: 157, HCDR2: SEQ ID NO: 171, and HCDR3: SEQ ID NO: 172; (26) HCDR1: SEQ ID NO: 174, HCDR2: SEQ ID NO: 175, and HCDR3: SEQ ID NO: 176; (27) HCDR1: SEQ ID NO: 177, HCDR2: SEQ ID NO: 178, and HCDR3: SEQ ID NO: 179; (28) HCDR1: SEQ ID NO: 180, HCDR2: SEQ ID NO: 181, and HCDR3: SEQ ID NO: 182; (29) HCDR1: SEQ ID NO: 184, HCDR2: SEQ ID NO: 106, and HCDR3: SEQ ID NO: 185; (30) HCDR1: SEQ ID NO: 186, HCDR2: SEQ ID NO: 187, and HCDR3: SEQ ID NO: 188; (31) HCDR1: SEQ ID NO: 189, HCDR2: SEQ ID NO: 190, and HCDR3: SEQ ID NO: 191; (32) HCDR1: SEQ ID NO: 159, HCDR2: SEQ ID NO: 192, and HCDR3: SEQ ID NO: 179; (33) HCDR1: SEQ ID NO: 193, HCDR2: SEQ ID NO: 194, and HCDR3: SEQ ID NO: 195; and (34) HCDR1: SEQ ID NO: 196, HCDR2: SEQ ID NO: 197, and HCDR3: SEQ ID NO:

198.

5. and the VHH is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID The antigen binding protein of claim 1, comprising an amino acid sequence as set forth in any one of SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:

70.

6. 2. The antigen binding protein of claim 1, comprising an antibody heavy chain constant region, wherein the antibody heavy chain constant region is derived from IgG.

7. 13. A chimeric antigen receptor comprising a targeting moiety, the targeting moiety comprising the antigen binding protein of claim 1.

8. comprising a costimulatory signal region, an intracellular signal region, a transmembrane region and / or a hinge region; Where: the costimulatory signal region comprises an intracellular costimulatory signal region derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand for CD83, CD40, and MyD88; the intracellular signal region comprises an intracellular signal region derived from one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma-associated herpesvirus (HSKV), DAP10, DAP-12, and a domain containing at least one ITAM domain; the transmembrane region comprises a transmembrane region derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM; the hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT; The chimeric antigen receptor of claim 7.

9. The costimulatory signal region is an intracellular costimulatory signal region derived from 4-1BB and comprises the amino acid sequence shown in SEQ ID NO: 78; the intracellular signal region is an intracellular signal region derived from CD3ζ and comprises the amino acid sequence shown in SEQ ID NO: 80; the transmembrane domain is a transmembrane domain derived from CD8 and comprises the amino acid sequence shown in SEQ ID NO: 76; and / or The hinge region is a transmembrane region derived from CD8 and comprises the amino acid sequence shown in SEQ ID NO:

74. The chimeric antigen receptor of claim 8.

10. The chimeric antigen receptor of claim 7, further comprising a low density lipoprotein receptor-related protein or a fragment thereof.

11. The chimeric antigen receptor of claim 10, wherein the low density lipoprotein receptor-related protein or a fragment thereof is low density lipoprotein receptor-related protein 6 or a fragment thereof.

12. The chimeric antigen receptor of claim 11, wherein the low density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:

84.

13. The chimeric antigen receptor of claim 8, comprising an amino acid sequence as set forth in any one of SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203 and SEQ ID NO:

204.

14. 13. An isolated nucleic acid molecule or molecules encoding the antigen binding protein of claim 1.

15. An isolated nucleic acid molecule or molecules encoding the chimeric antigen receptor described in claim 7.

16. A vector comprising the nucleic acid molecule of claim 15.

17. A cell comprising the chimeric antigen receptor of claim 7.

18. 18. The cell of claim 17, which is an immune effector cell.

19. 18. The cell of claim 17, comprising a T cell, a B cell, a natural killer cell (NK cell), a macrophage, a NKT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte, a peripheral blood mononuclear cell, an embryonic stem cell, a lymphoid progenitor cell and / or a pluripotent stem cell.

20. A method for preparing a modified immune effector cell, comprising introducing into an immune effector cell the vector described in claim 16.

21. 20. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1 to 6, a chimeric antigen receptor according to any one of claims 7 to 13, a nucleic acid molecule according to claim 14 or 15, a vector according to claim 16, and / or a cell according to any one of claims 17 to 19.

22. 22. A pharmaceutical composition according to claim 21 for use in the treatment, prevention and / or alleviation of a disease or condition associated with the abnormal expression of GPRC5D.

23. The pharmaceutical composition of claim 22, wherein the disease or condition associated with the abnormal expression of GPRC5D comprises a tumor, the tumor comprising a solid or non-solid tumor, including a hematological tumor, lymphoma and / or myeloma.

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