Chimeric antigen receptors targeting GPRC5D and uses thereof

Abstract

The present application relates to a chimeric antigen receptor, comprising a first antigen-binding domain targeting G protein-coupled receptor class C group 5 member D (GPRC5D) protein and a second antigen-binding domain targeting GPRC5D protein. The present application further provides a cell comprising said chimeric antigen receptor and uses thereof.

Description

[Technical field]

[0001] The present application relates to the biomedicine field, in particular to a novel anti-GPRC5D chimeric antigen receptor, cells containing said chimeric antigen receptor, and their use in the treatment of tumors. [Background technology]

[0002] Multiple myeloma (MM) is the second most common malignant hematological disease and ranks second in cancer mortality. MM is a malignant tumor of plasma cells, often accompanied by multiple osteolytic lesions, impaired renal function, bone marrow invasion, hypercalcemia, and anemia. The current main treatment for MM is systemic chemotherapy, which has severe side effects and cannot be completely cured.

[0003] G protein-coupled receptor class C group 5 member D (GPRC5D) protein is an atypical surface orphan receptor. GPRCD5, like other C5 family receptors, is structurally very similar to the C4 family due to a short amino terminus. Its expression in normal tissues is restricted to hair follicles, but it is also specifically and highly expressed in bone marrow of MM patients and is highly correlated with plasma cell neoplasm burden and gene mutations.

[0004] Although the treatment of MM has expanded with the intervention of new drugs such as protease inhibitors (PIs), immunomodulatory drugs (IMiDs) and monoclonal antibodies (mAbs), the relapse rate is still very high and drug resistance is occurring. Meanwhile, chimeric antigen receptor T cell (CAR-T) treatment has brought about remarkable therapeutic effects and controllable toxicity in MM, among which the mainstream CAR-T cell therapy targeting B cell maturation antigen (BCMA) has a problem of target escape in MM patients with BCMA-negative or low BCMA expression. GPRC5D is specifically 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] [Contents of the invention] The present application provides a chimeric antigen receptor comprising two antigen-binding domains, both of which can target GPRC5D. The present application further provides a cell comprising the chimeric antigen receptor. The cell has one or more of the following properties: (1) strong proliferation ability, (2) ability to kill target cells expressing GPRC5D, (3) secretion of cytokines under stimulation of target cells, (4) relatively strong proliferation ability under stimulation of target cells, (4) suppression of tumor growth, and (5) favorable safety.

[0006] According to one aspect, the present application provides a chimeric antigen receptor comprising a first antigen-binding domain that targets G protein-coupled receptor class C group 5 member D (GPRC5D) protein and a second antigen-binding domain that targets GPRC5D protein.

[0007] In some embodiments, the first antigen-binding domain and / or the second antigen-binding domain of the chimeric antigen receptor comprises an antibody or an antigen-binding fragment thereof.

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

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

[0010] In some embodiments, the first antigen binding domain of the chimeric antigen receptor comprises at least one CDR of an antibody heavy chain variable region VH, wherein the VH comprises the amino acid sequence set forth in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 30.

[0011] In some embodiments, the first antigen binding domain of the chimeric antigen receptor comprises an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:33.

[0012] In some embodiments, the first antigen binding domain of the chimeric antigen receptor comprises an HCDR3 comprising the amino acid sequence set forth in any one of SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:22, and SEQ ID NO:27.

[0013] In some embodiments, the first antigen-binding domain of the chimeric antigen receptor comprises an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (IX1X2X3X4GX5T, where X1 is N or T, X2 is S or W, X3 is G or S, X4 is D or G, and X5 is N, S, or T).

[0014] In some embodiments, the first antigen binding domain of the chimeric antigen receptor comprises an HCDR2 comprising the amino acid sequence set forth in any one of SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21, and SEQ ID NO:26.

[0015] In some embodiments, the first antigen binding domain of the chimeric antigen receptor comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:35.

[0016] In some embodiments, the first antigen binding domain of the chimeric antigen receptor comprises an HCDR1 comprising the amino acid sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:20, and SEQ ID NO:25.

[0017] In some embodiments, the first antigen-binding domain of the chimeric antigen receptor comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (IX1X2X3X4GX5T, where X1 is N or T, X2 is S or W, X3 is G or S, X4 is D or G, and X5 is N, S, or T), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33.

[0018] In some embodiments, the first antigen binding domain of the chimeric antigen receptor comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2, HCDR3 are (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 8, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 10; (2) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 17, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 18; (3) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 21, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 22; and (4) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27; The present invention includes any set of amino acid sequences selected from the group consisting of:

[0019] In some embodiments, the first antigen-binding domain of the chimeric antigen receptor comprises an H-FR1 comprising the amino acid sequence set forth in SEQ ID NO:11, and the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1.

[0020] In some embodiments, the first antigen-binding domain of the chimeric antigen receptor comprises an H-FR2 comprising the amino acid sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 28, and SEQ ID NO: 23, wherein the H-FR2 is located between the HCDR1 and the HCDR2.

[0021] In some embodiments, the first antigen-binding domain of the chimeric antigen receptor comprises an H-FR3 comprising the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 29, and the H-FR3 is positioned between the HCDR2 and the HCDR3.

[0022] In some embodiments, the first antigen-binding domain of the chimeric antigen receptor comprises an H-FR4 comprising the amino acid sequence set forth in SEQ ID NO: 14, wherein the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3.

[0023] In some embodiments, the first antigen binding domain of the chimeric antigen receptor comprises a VH comprising the amino acid sequence set forth in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 30.

[0024] In some embodiments, the first antigen-binding domain of the chimeric antigen receptor is a VHH comprising the amino acid sequence set forth in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 30.

[0025] In some embodiments, the second antigen binding domain of the chimeric antigen receptor comprises at least one CDR of an antibody heavy chain variable region VH, wherein the VH comprises the amino acid sequence set forth in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 30.

[0026] In some embodiments, the second antigen binding domain of the chimeric antigen receptor comprises an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33.

[0027] In some embodiments, the second antigen binding domain of the chimeric antigen receptor comprises an HCDR3 comprising the amino acid sequence set forth in any one of SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:22, and SEQ ID NO:27.

[0028] In some embodiments, the second antigen binding domain of the chimeric antigen receptor comprises an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (IX1X2X3X4GX5T, where X1 is N or T, X2 is S or W, X3 is G or S, X4 is D or G, and X5 is N, S, or T).

[0029] In some embodiments, the second antigen binding domain of the chimeric antigen receptor comprises an HCDR2 comprising the amino acid sequence set forth in any one of SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21, and SEQ ID NO:26.

[0030] In some embodiments, the second antigen binding domain of the chimeric antigen receptor comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:35.

[0031] In some embodiments, the second antigen binding domain of the chimeric antigen receptor comprises an HCDR1 comprising the amino acid sequence set forth in any one of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:20, and SEQ ID NO:25.

[0032] In some embodiments, the second antigen-binding domain of the chimeric antigen receptor comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (IX1X2X3X4GX5T, where X1 is N or T, X2 is S or W, X3 is G or S, X4 is D or G, and X5 is N, S, or T), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33.

[0033] In some embodiments, the second antigen binding domain of the chimeric antigen receptor comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2, HCDR3 are (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 8, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 10; (2) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 17, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 18; (3) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 21, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 22; and (4) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27; The present invention includes any set of amino acid sequences selected from the group consisting of:

[0034] In some embodiments, the second antigen-binding domain of the chimeric antigen receptor comprises an H-FR1 comprising the amino acid sequence set forth in SEQ ID NO:11, and the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1.

[0035] In some embodiments, the second antigen-binding domain of the chimeric antigen receptor comprises an H-FR2 comprising the amino acid sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 28, and SEQ ID NO: 23, wherein the H-FR2 is located between the HCDR1 and the HCDR2.

[0036] In some embodiments, the second antigen-binding domain of the chimeric antigen receptor comprises an H-FR3 comprising the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 29, and the H-FR3 is located between the HCDR2 and the HCDR3.

[0037] In some embodiments, the second antigen-binding domain of the chimeric antigen receptor comprises an H-FR4 comprising the amino acid sequence set forth in SEQ ID NO: 14, wherein the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3.

[0038] In some embodiments, the second antigen binding domain of the chimeric antigen receptor comprises a VH comprising the amino acid sequence set forth in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 30.

[0039] In some embodiments, the second antigen binding domain of the chimeric antigen receptor is a VHH comprising the amino acid sequence set forth in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 30.

[0040] In some embodiments, the first antigen binding domain and the second antigen binding domain of the chimeric antigen receptor comprise HCDR1, HCDR2 and HCDR3, respectively, and the sequences of HCDR1, HCDR2 and HCDR3 of the first antigen binding domain and the second antigen binding domain are (1) a first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; (2) a first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; (3) a first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; (4) a first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; (5) a first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; (6) a first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; (7) a first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; (8) a first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; (9) a first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; (10) a first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; (11) A first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; (12) A first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; (13) A first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; and a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10. (14) A first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; (15) a first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; and (16) A first antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; a second antigen-binding domain, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; The amino acid sequence is any set of amino acid sequences selected from the group consisting of:

[0041] In some embodiments, the first antigen binding domain and the second antigen binding domain of the chimeric antigen receptor target the same epitope of GPRC5D.

[0042] In some embodiments, the first antigen-binding domain and the second antigen-binding domain of the chimeric antigen receptor comprise the same or different amino acid sequences.

[0043] In some embodiments, the first antigen binding domain and the second antigen binding domain of the chimeric antigen receptor are directly or indirectly linked.

[0044] In some embodiments, the first antigen binding domain and the second antigen binding domain of the chimeric antigen receptor are linked via a linker.

[0045] In some embodiments, the linker comprises an amino acid sequence of (GGGGS) n , where n is any positive integer from 1 to 10.

[0046] In some embodiments, the linker comprises an amino acid sequence of (EAAAK) n , where n is any positive integer from 1 to 10.

[0047] In some embodiments, the chimeric antigen receptor comprises a costimulatory signal region, wherein 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.

[0048] In some embodiments, the costimulatory signal region of the chimeric antigen receptor is an intracellular costimulatory signal region derived from 4-1BB.

[0049] In some embodiments, the costimulatory signal region of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:4.

[0050] In some embodiments, the chimeric antigen receptor comprises an intracellular signaling domain, the intracellular signaling domain 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 domain containing at least one ITAM.

[0051] In some embodiments, the intracellular signaling domain of the chimeric antigen receptor is a signaling domain derived from CD3ζ.

[0052] In some embodiments, the intracellular signaling domain of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:5.

[0053] 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.

[0054] In some embodiments, the transmembrane region of the chimeric antigen receptor is a transmembrane region derived from CD8.

[0055] In some embodiments, the transmembrane region of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:3.

[0056] In some embodiments, the chimeric antigen receptor further comprises a hinge 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.

[0057] In some embodiments, the hinge region of the chimeric antigen receptor is a hinge region derived from CD8.

[0058] In some embodiments, the hinge region of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:2.

[0059] In some embodiments, the chimeric antigen receptor further comprises a low density lipoprotein receptor-related protein, or a fragment thereof.

[0060] In some embodiments, the low density lipoprotein receptor-related protein or fragment thereof is located C-terminal to the intracellular signaling region.

[0061] In some embodiments, the low density lipoprotein receptor-related protein or a fragment thereof comprises one or more proteins selected from the group consisting of low density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof.

[0062] In some embodiments, the low density lipoprotein receptor-related protein or fragment thereof is low density lipoprotein receptor-related protein 5 and / or 6 or a fragment thereof.

[0063] In some embodiments, the low density lipoprotein receptor-related protein or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:7.

[0064] In some embodiments, the chimeric antigen receptor further comprises a signal peptide.

[0065] In some embodiments, the signal peptide is derived from the signal peptide of the CD8 protein.

[0066] In some embodiments, the signal peptide of the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO:1.

[0067] According to another aspect, the present application further provides one or more isolated nucleic acid molecules encoding said chimeric antigen receptor.

[0068] In some embodiments, the nucleic acid molecule further comprises a promoter.

[0069] In some embodiments, the promoter is a constitutive promoter.

[0070] In some embodiments, the promoter is an EF1α promoter.

[0071] In another aspect, the present application further provides a carrier comprising the nucleic acid molecule.

[0072] In some embodiments, the carrier is a viral carrier.

[0073] In some embodiments, the carrier is a lentiviral carrier.

[0074] According to another aspect, the present application further provides a cell comprising said chimeric antigen receptor, said nucleic acid molecule, and / or said carrier.

[0075] In some embodiments, the cell is an immune effector cell.

[0076] 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.

[0077] In some embodiments, the cell is a T cell.

[0078] According to another aspect, the present application further provides a method of producing the chimeric antigen receptor, comprising culturing the cell under conditions allowing expression of the chimeric antigen receptor.

[0079] According to another aspect, the present application further provides a method of producing a modified immune effector cell, the method comprising introducing said carrier into an immune effector cell.

[0080] According to another aspect, the present application further provides a pharmaceutical composition comprising said chimeric antigen receptor, said nucleic acid molecule, said carrier, and / or said cell, and optionally a pharma- ceutically acceptable carrier.

[0081] According to another aspect, the present application further provides a use of said chimeric antigen receptor, said nucleic acid molecule, said carrier, said cell, and / or said pharmaceutical composition in the manufacture of a medicament for preventing, treating and / or ameliorating a disease and / or condition.

[0082] In some embodiments, the disease and / or condition comprises a disease and / or condition associated with aberrant expression of GPRC5D.

[0083] In some embodiments, the disease and / or condition associated with aberrant expression of GPRC5D comprises a tumor.

[0084] In some embodiments, the tumor comprises a solid tumor.

[0085] In some embodiments, the tumor comprises a non-solid tumor.

[0086] In some embodiments, the tumor comprises a non-solid tumor.

[0087] In some embodiments, the tumor comprises a hematological tumor and / or lymphoma.

[0088] In some embodiments, the tumor comprises a myeloma.

[0089] In some embodiments, the myeloma comprises refractory / relapsed multiple myeloma.

[0090] According to another aspect, the present application further provides a method for preventing, treating and / or alleviating a disease and / or condition, comprising administering the cells and / or the pharmaceutical composition to a subject in need thereof.

[0091] In some embodiments, the disease and / or condition comprises a disease and / or condition associated with aberrant expression of GPRC5D.

[0092] In some embodiments, the disease and / or condition associated with aberrant expression of GPRC5D comprises a tumor.

[0093] In some embodiments, the tumor comprises a solid tumor.

[0094] In some embodiments, the tumor comprises a non-solid tumor.

[0095] In some embodiments, the tumor comprises a non-solid tumor.

[0096] In some embodiments, the tumor comprises a hematological tumor and / or lymphoma.

[0097] In some embodiments, the tumor comprises a myeloma.

[0098] In some embodiments, the myeloma comprises refractory / relapsed multiple myeloma.

[0099] Those skilled in the art can easily recognize other aspects and advantages of the present application from the following detailed description. The following detailed description merely illustrates and describes exemplary embodiments of the present application. As will be recognized by those skilled in the art, given the content of the present application, those skilled in the art can modify the specific embodiments disclosed without departing from the spirit and scope of the invention related to the present application. Correspondingly, the drawings and description in the present application are merely illustrative and not limiting. [Brief description of the drawings]

[0100] Particular features of the invention related to this application are set forth in the appended claims. A better understanding of the features and advantages of the invention related to this application can be obtained by reference to the following detailed description of exemplary embodiments and drawings, the brief description of which is as follows: [Figure 1] FIG. 1 shows the components and linkage order of each part of the chimeric antigen receptor described in the present application. [Figure 2A] Shows the positive rate of T cells after double binding domain GPRC5D double VHH CAR lentivirus infection. [Figure 2B] Shows the positive rate of T cells after single VHH CAR lentivirus infection. [Diagram 3] Shown is the in vitro expansion curve of single VHH CAR-T cells. [Figure 4A] Shows CD107a expression of dual binding domain GPRC5D double VHH CAR-T cells. [Figure 4B] Shows CD107a expression on single VHH CAR-T cells. [Figure 5A] Shows specific killing of dual binding domain GPRC5D double VHH CAR-T cells. [Figure 5B] Shows specific killing of single VHH CAR-T cells. [Figure 6A] Dual binding domain GPRC5D double VHH CAR-T cytokine secretion levels. [Figure 6B] Single VHH CAR-T cytokine secretion levels are shown. [Figure 7A] Shows fold target proliferation of dual binding domain GPRC5D double VHH CAR-T cells. [Figure 7B] Target fold expansion of single VHH CAR-T cells is shown. [Figure 8] CAR-T cell memory phenotype flow cytometry chart. [Figure 9] Phenotype of CD8+ CAR-T cells. [Figure 10] Shows expression of activation markers in CD3+ CAR-T cells. [Figure 11] Shows the expression of exhaustion markers in CD3+ CAR-T cells. [Figure 12] Confocal images of the CAR-T cell immune synapse are shown. [Figure 13] Quantitative analysis of the distance between the MTOC and synapses. [Figure 14] 1 shows a flowchart of an in vivo tumor suppression experiment in NSG mice. [Figure 15A-15D] FIG. 13 shows the in vivo tumor suppression effect in subcutaneous tumor model NSG mice. [Figure 16] FIG. 13 shows the in vivo tumor suppression effect in orthotopic transplant model NSG mice. [Figure 17] 4 shows a chart of antibody affinity detection. [Figure 18] 1 shows a flow chart of antibody binding activity. [Figure 19] 1 shows a flow chart of competitive binding between G5VHH1 and G5VHH2. [Figure 20]Figure 2 shows the cross-binding activity of dual binding domain VHH-hFc antibodies with target antigens of different species. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0101] Hereinafter, embodiments of the present invention will be described with reference to specific examples, and those skilled in the art will easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0102] Definition of Terms The term "antibody" as used includes complete antibodies and binding fragments thereof. Generally, fragments compete with the complete antibody from which they are derived for specific binding to an antigen. Optionally, the antibody or binding fragment thereof can 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, or 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 be specific for GPRC5D.

[0103] 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, 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 be specific for GPRC5D.

[0104] In this application, "VHH" or "V H The term "VHH" is used interchangeably and generally refers to an antibody comprising a variable antigen-binding domain of a heavy chain antibody. VHHs are also called Nanobodies (Nbs) and / or single domain antibodies. For example, the VHHs can bind to GPRC5D. For example, the VHHs may be specific for GPRC5D.

[0105] 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 consists of a heavy chain variable region (VH) and a heavy chain constant region. The term "heavy chain constant region" consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The term "light chain constant region" consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors.

[0106] In this application, the term "G protein-coupled receptor class C group 5 member D (GPRC5D) protein" is an atypical surface orphan receptor. GPRCD5, like other C5 family receptors, is structurally very similar to the C4 family due to its short amino terminus. Its expression in normal tissues is limited to hair follicles, but it is also prominently expressed in the bone marrow of MM patients and is highly correlated with plasma cell tumor burden and gene mutations. For example, GPRC5D in this application may refer to GPRC5D that is specifically expressed in the body of MM patients.

[0107] In the present application, the term "chimeric antigen receptor" (CAR) generally refers to a recombinant polypeptide comprising at least an extracellular domain that specifically binds to an antigen or target, a transmembrane region, and an intracellular domain. For example, the chimeric antigen receptor may further comprise a low density lipoprotein receptor-related protein or a fragment thereof. For example, the chimeric antigen receptor may comprise a signal peptide. Binding of the extracellular domain of the CAR to a target antigen on the surface of a target cell triggers CAR clustering and transmits an activation stimulus to the CAR-containing cell. CARs can redirect the specificity of immune effector cells and trigger proliferation, cytokine production, phagocytosis and / or production of molecules that can mediate cell death expressing the target antigen in a major histocompatibility (MHC)-independent manner. For example, the extracellular structure can specifically bind to GPRC5D.

[0108] In the present application, the term "intracellular domain" is meant to include any truncated portion of the intracellular domain sufficient to transmit an activation signal. The intracellular domain may include an intracellular signaling region and / or a costimulatory signaling region. The term "intracellular signaling region" refers to an intracellular region capable of generating a signal that promotes immune effector function of a CAR-containing cell (e.g., a CART cell or an NK cell expressing a CAR). For example, the intracellular signaling region may include an intracellular signaling 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 domain containing at least one ITAM. For example, the intracellular signaling region may be a signaling domain derived from CD3zeta. The term "co-stimulatory signal region" refers to a portion of the CAR of the intracellular signal region that can transmit an effector signal. For example, the co-stimulatory signal region may 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 may be an intracellular co-stimulatory signal region derived from 4-1BB.

[0109] As used herein, the term "transmembrane domain" refers to a domain of a peptide, polypeptide or protein that is capable of spanning the cytoplasmic membrane. These domains can be used to anchor the extracellular 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.

[0110] In the present application, the term "hinge region" refers to a portion of an antibody heavy chain polypeptide that links the CH1 domain and the CH2 domain, for example, from about position 216 to about position 230 according to the EU numbering system of Kabat. A hinge region is generally a dimeric molecule consisting of two polypeptides having the same amino acid sequence. A hinge region generally contains about 25 amino acid residues and is flexible, allowing independent movement of the antigen-binding region. The hinge region can be subdivided into three domains: the upper, middle, and lower hinge 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.

[0111] In the present application, the term "low-density lipoprotein receptor-related protein" refers to a cell surface protein belonging to an endocytosis receptor that is widely distributed in the body, has a very large tissue variability, and has a main function of taking up cholesterol into cells for cell proliferation and synthesis of steroid hormones and bile salts. For example, the low-density lipoprotein receptor-related protein can be derived from any vertebrate. For example, the low-density lipoprotein receptor-related protein or a fragment thereof is 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 species selected from the group consisting of low-density lipoprotein receptor-related proteins 1 to 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. For example, the low-density lipoprotein receptor-related protein or a fragment thereof may be low-density lipoprotein receptor-related protein 5 or a fragment thereof.

[0112] In the present application, the term "signal peptide" refers to a leader sequence at the amino-terminus (N-terminus) of the nascent CAR protein that directs the nascent protein to the endoplasmic reticulum during or after translation and is subsequently surface expressed. For example, the signal peptide is derived from the signal peptide of the CD8 protein.

[0113] 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 a selected DNA sequence that is operably linked to the promoter. For example, the nucleic acid molecule can encode a corresponding antigen binding protein and / or the chimeric antigen receptor. 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.

[0114] In the present application, the term "carrier" generally refers to a molecule to which one or more nucleic acid molecules of the present application can be attached. For example, the carrier may be a viral carrier. For example, the carrier may be a lentiviral carrier.

[0115] In the present application, the term "cell" refers to a cell into which a nucleic acid is transfected, and the term "cell" includes prokaryotic cells used for propagation of plasmids, and eukaryotic cells used for expression of nucleic acids and production of encoded polypeptides. For example, the cell may comprise the chimeric antigen receptor, the nucleic acid molecule, and / or the carrier. For example, the cell may be an immune effector cell. The term "immune effector cell" generally refers to an immune cell that participates in an immune response and performs an effector function. For example, performing the effector function may include removing foreign antigens or promoting an immune effector response. For example, the immune effector cell may comprise 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. For example, the immune effector cell may be a T cell.

[0116] 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 comprise the antigen binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the carrier and / or the cell, and an optional pharma- ceutically acceptable carrier. The pharmaceutical composition may be used for the prevention, treatment and / or alleviation of a disease or condition associated with aberrant expression of GPRC5D. For example, the disease or condition associated with aberrant expression of GPRC5D may comprise a tumor. For example, the tumor may comprise a solid tumor and / or a non-solid tumor. For example, the tumor may comprise a hematological tumor and / or a lymphoma. For example, the tumor may comprise a myeloma.

[0117] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT According to one aspect, the present application provides a chimeric antigen receptor comprising a first antigen-binding domain that targets G protein-coupled receptor class C group 5 member D (GPRC5D) protein and a second antigen-binding domain that targets GPRC5D protein.

[0118] In some embodiments, the first antigen-binding domain and / or the second antigen-binding domain of the chimeric antigen receptor may comprise an antibody or an antigen-binding fragment thereof. For example, the antigen-binding fragment comprises a Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and / or dAb. For example, the antigen-binding fragment comprises a VHH.

[0119] In the present application, the CDRs can be split by the IMGT splitting method.

[0120] First antigen-binding domain In the present application, the first antigen-binding domain of the chimeric antigen receptor can target the GPRC5D protein.

[0121] In the present application, the first antigen-binding domain of the chimeric antigen receptor may comprise at least one CDR of an antibody heavy chain variable region VH, and the VH may comprise an amino acid sequence shown in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 30.

[0122] In the present application, the first antigen-binding domain of the chimeric antigen receptor may comprise an HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 33. For example, the first antigen-binding domain of the chimeric antigen receptor comprises an HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO: 27.

[0123] In the present application, the first antigen-binding domain of the chimeric antigen receptor may comprise an HCDR2 that may comprise the amino acid sequence shown in SEQ ID NO: 34 (IX1X2X3X4GX5T, where X1 is N or T, X2 is S or W, X3 is G or S, X4 is D or G, and X5 is N, S or T). For example, the first antigen-binding domain of the chimeric antigen receptor may comprise an HCDR2 that may comprise the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 21, and SEQ ID NO: 26.

[0124] In the present application, the first antigen-binding domain of the chimeric antigen receptor may comprise an HCDR1 that may comprise the amino acid sequence shown in SEQ ID NO: 35. For example, the first antigen-binding domain of the chimeric antigen receptor may comprise an HCDR1 that may comprise the amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 20, and SEQ ID NO: 25.

[0125] In the present application, the first antigen-binding domain of the chimeric antigen receptor may comprise an HCDR1 which may comprise the amino acid sequence shown in SEQ ID NO: 35, an HCDR2 which may comprise the amino acid sequence shown in SEQ ID NO: 34 (IX1X2X3X4GX5T, where X1 is N or T, X2 is S or W, X3 is G or S, X4 is D or G, and X5 is N, S, or T), and an HCDR3 which may comprise the amino acid sequence shown in SEQ ID NO: 33.

[0126] In the present application, the first antigen-binding domain of the chimeric antigen receptor may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 8, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 10; (2) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 17, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 18; (3) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 21, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 22; and (4) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27; The present invention includes any set of amino acid sequences selected from the group consisting of:

[0127] In the present application, the first antigen-binding domain of the chimeric antigen receptor may comprise an H-FR1 which may comprise the amino acid sequence shown in SEQ ID NO: 11, and the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1.

[0128] In the present application, the first antigen-binding domain of the chimeric antigen receptor may comprise an H-FR2 which may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 12, SEQ ID NO: 28, and SEQ ID NO: 23, and the H-FR2 is located between the HCDR1 and the HCDR2.

[0129] In the present application, the first antigen-binding domain of the chimeric antigen receptor may comprise an H-FR3 which may comprise the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 29, and the H-FR3 is located between the HCDR2 and the HCDR3.

[0130] In the present application, the first antigen-binding domain of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO: 14 and may comprise an H-FR4, wherein the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3.

[0131] In the present application, the first antigen-binding domain of the chimeric antigen receptor may comprise a VH that may comprise the amino acid sequence shown in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 30.

[0132] In the present application, the first antigen-binding domain of the chimeric antigen receptor may be a VHH that may comprise the amino acid sequence shown in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 30.

[0133] Second antigen-binding domain In the present application, the second antigen binding domain of the chimeric antigen receptor can target the GPRC5D protein.

[0134] In the present application, the second antigen-binding domain of the chimeric antigen receptor may comprise at least one CDR of an antibody heavy chain variable region VH, and the VH may comprise an amino acid sequence shown in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 30.

[0135] In the present application, the second antigen-binding domain of the chimeric antigen receptor may comprise an HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 33. For example, the second antigen-binding domain of the chimeric antigen receptor comprises an HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO: 27.

[0136] In the present application, the second antigen-binding domain of the chimeric antigen receptor may comprise an HCDR2 that may comprise the amino acid sequence shown in SEQ ID NO: 34 (IX1X2X3X4GX5T, where X1 is N or T, X2 is S or W, X3 is G or S, X4 is D or G, and X5 is N, S or T). For example, the second antigen-binding domain of the chimeric antigen receptor may comprise an HCDR2 that may comprise the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 21, and SEQ ID NO: 26.

[0137] In the present application, the second antigen-binding domain of the chimeric antigen receptor may comprise an HCDR1 that may comprise the amino acid sequence shown in SEQ ID NO: 35. For example, the second antigen-binding domain of the chimeric antigen receptor may comprise an HCDR1 that may comprise the amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 20, and SEQ ID NO: 25.

[0138] In the present application, the second antigen-binding domain of the chimeric antigen receptor may comprise an HCDR1 which may comprise the amino acid sequence shown in SEQ ID NO: 35, an HCDR2 which may comprise the amino acid sequence shown in SEQ ID NO: 34 (IX1X2X3X4GX5T, where X1 is N or T, X2 is S or W, X3 is G or S, X4 is D or G, and X5 is N, S, or T), and an HCDR3 which may comprise the amino acid sequence shown in SEQ ID NO: 33.

[0139] In the present application, the second antigen-binding domain of the chimeric antigen receptor may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2, HCDR3 are (5) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 8, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 10; (6) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 17, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 18; (7) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 21, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 22; and (8) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27; The present invention includes any set of amino acid sequences selected from the group consisting of:

[0140] In the present application, the second antigen-binding domain of the chimeric antigen receptor may comprise an H-FR1 which may comprise the amino acid sequence shown in SEQ ID NO: 11, and the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1.

[0141] In the present application, the second antigen-binding domain of the chimeric antigen receptor may comprise an H-FR2 which may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 12, SEQ ID NO: 28, and SEQ ID NO: 23, and the H-FR2 is located between the HCDR1 and the HCDR2.

[0142] In the present application, the second antigen-binding domain of the chimeric antigen receptor may comprise an H-FR3 which may comprise the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 29, and the H-FR3 is located between the HCDR2 and the HCDR3.

[0143] In the present application, the second antigen-binding domain of the chimeric antigen receptor may comprise an H-FR4 which may comprise the amino acid sequence shown in SEQ ID NO: 14, and the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3.

[0144] In the present application, the second antigen-binding domain of the chimeric antigen receptor may comprise a VH that may comprise the amino acid sequence shown in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 30.

[0145] In the present application, the second antigen-binding domain of the chimeric antigen receptor may be a VHH that may comprise the amino acid sequence shown in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 30.

[0146] Chimeric Antigen Receptor In the present application, the first antigen-binding domain and the second antigen-binding domain of the chimeric antigen receptor may comprise HCDR1, HCDR2 and HCDR3, respectively.

[0147] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10.

[0148] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18.

[0149] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22.

[0150] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27.

[0151] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10.

[0152] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18.

[0153] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22.

[0154] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27.

[0155] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0156] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18.

[0157] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22.

[0158] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27.

[0159] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0160] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18.

[0161] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22.

[0162] For example, in the first antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27; and in the second antigen-binding domain, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27.

[0163] In the present application, the first antigen-binding domain and the second antigen-binding domain of the chimeric antigen receptor can target the same epitope of GPRC5D. In the present application, the first antigen-binding domain and the second antigen-binding domain of the chimeric antigen receptor can target different epitopes of GPRC5D.

[0164] In the present application, the first antigen-binding domain and the second antigen-binding domain of the chimeric antigen receptor may comprise the same or different amino acid sequences.

[0165] In the present application, the first antigen-binding domain and the second antigen-binding domain of the chimeric antigen receptor may be directly or indirectly linked, for example, the first antigen-binding domain and the second antigen-binding domain of the chimeric antigen receptor may be linked via a linker.

[0166] In the present application, the linker may comprise an amino acid sequence of (GGGGS)n, where n is any positive integer from 1 to 10. For example, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0167] In the present application, the linker may comprise an amino acid sequence of (EAAAK)n, where n is any positive integer from 1 to 10. For example, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0168] In the present application, the chimeric antigen receptor may include a costimulatory signal region, and the costimulatory signal region includes 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. For example, the costimulatory signal region of the chimeric antigen receptor may be an intracellular costimulatory signal region derived from 4-1BB. For example, the costimulatory signal region of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:4.

[0169] In the present application, the chimeric antigen receptor may include an intracellular signaling domain, and the intracellular signaling domain includes an intracellular signal 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 domain containing at least one ITAM. For example, the intracellular signaling domain of the chimeric antigen receptor may be a signaling domain derived from CD3zeta. For example, the intracellular signaling domain of the chimeric antigen receptor may include the amino acid sequence shown in SEQ ID NO:5.

[0170] In the present application, the chimeric antigen receptor may include a transmembrane region, and the transmembrane region may include 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. For example, the transmembrane region of the chimeric antigen receptor may be derived from CD8. For example, the transmembrane region of the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:3.

[0171] In the present application, the chimeric antigen receptor may further comprise a hinge region, and 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. For example, the hinge region of the chimeric antigen receptor may be a hinge region derived from CD8. For example, the hinge region of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:2.

[0172] In the present application, the chimeric antigen receptor may further 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 is located at the C-terminus of the intracellular signal region. For example, the low density lipoprotein receptor-related protein or a fragment thereof comprises one or more species selected from the group consisting of low density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof. For example, the low density lipoprotein receptor-related protein or a fragment thereof is low density lipoprotein receptor-related protein 5 and / or 6 or a fragment thereof. For example, the low density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 7.

[0173] In the present application, the chimeric antigen receptor may further comprise a signal peptide. For example, the signal peptide is derived from the signal peptide of CD8 protein. For example, the signal peptide of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:1.

[0174] In the present application, the low density lipoprotein receptor-related protein or a fragment thereof in the chimeric antigen receptor can be linked to the C-terminus of the CAR by 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 can be linked to the C-terminus of the intracellular signal region by T2A.

[0175] In the present application, the chimeric antigen receptor may comprise, in order from the N-terminus to the C-terminus, an antigen-binding domain that binds to a GPRC5D protein, the hinge region, the transmembrane domain, the costimulatory signal region, and the intracellular signal region. For example, the chimeric antigen receptor may comprise, in order from the N-terminus to the C-terminus, the antigen-binding domain, 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 CD3ζ.

[0176] In the present application, the chimeric antigen receptor may comprise, in order from the N-terminus to the C-terminus, an antigen-binding domain that binds to a GPRC5D protein, 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 chimeric antigen receptor may comprise, in order from the N-terminus to the C-terminus, the antigen-binding domain, 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 CD3ζ, and the low-density lipoprotein receptor-related protein or a fragment thereof.

[0177] For example, the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO:31 or SEQ ID NO:32.

[0178] In the present application, the chimeric antigen receptor may comprise the structure shown in the table below.

[0179] TIFF2025504803000001.tif130170, where A represents (GGGGS)n and B represents (EAAAK)n, where n is any positive integer from 1 to 10.

[0180] For example, HCDR1 of 3B5 comprises the amino acid sequence shown in SEQ ID NO:8, HCDR2 of 3B5 comprises the amino acid sequence shown in SEQ ID NO:9, HCDR3 of 3B5 comprises the amino acid sequence shown in SEQ ID NO:10, and VHH of 3B5 comprises the amino acid sequence shown in SEQ ID NO:15.

[0181] For example, HCDR1 of 3E7 comprises the amino acid sequence shown in SEQ ID NO: 16, HCDR2 of 3E7 comprises the amino acid sequence shown in SEQ ID NO: 17, HCDR3 of 3E7 comprises the amino acid sequence shown in SEQ ID NO: 18, and VHH of 3E7 comprises the amino acid sequence shown in SEQ ID NO: 19.

[0182] For example, HCDR1 of 4A2 comprises the amino acid sequence shown in SEQ ID NO:20, HCDR2 of 4A2 comprises the amino acid sequence shown in SEQ ID NO:21, HCDR3 of 4A2 comprises the amino acid sequence shown in SEQ ID NO:22, and VHH of 4A2 comprises the amino acid sequence shown in SEQ ID NO:24.

[0183] For example, HCDR1 of 4B3 comprises the amino acid sequence shown in SEQ ID NO: 25, HCDR2 of 4B3 comprises the amino acid sequence shown in SEQ ID NO: 26, HCDR3 of 4B3 comprises the amino acid sequence shown in SEQ ID NO: 27, and VHH of 4B3 comprises the amino acid sequence shown in SEQ ID NO: 30.

[0184] For example, the CD8α signal peptide may comprise the amino acid sequence shown in SEQ ID NO:1.

[0185] For example, the CD8α hinge region may comprise the amino acid sequence shown in SEQ ID NO:2.

[0186] For example, the CD8α transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO:3.

[0187] For example, the 4-1BB costimulatory signal may comprise the amino acid sequence shown in SEQ ID NO:4.

[0188] For example, the CD3 zeta intracellular signal region may comprise the amino acid sequence shown in SEQ ID NO:5.

[0189] For example, the Ori may comprise the amino acid sequence shown in SEQ ID NO:7.

[0190] nucleic acid molecule According to another aspect, the present application further provides one or more nucleic acid molecules, which may be isolated nucleotides, deoxynucleotides and / or ribonucleotides of any length, capable of encoding said chimeric antigen receptor.

[0191] For example, the nucleic acid molecule may comprise a promoter. For example, the promoter may be a constitutive promoter. For example, the promoter may be the EF1α promoter.

[0192] Carrier In another aspect, the present application further provides a carrier that may contain the nucleic acid molecule. The carrier is capable of transforming, transducing or transfecting a host cell to express the elements of its genetic material in the host cell. For example, the carrier may contain promoters, transcripts, enhancers, replicons, selection elements and reporter genes. For example, the carrier may contain components that aid in cell entry. The 5' and 3' ends of the nucleic acid molecule may further contain long terminal repeats to allow the nucleic acid molecule to replicate in the carrier.

[0193] For example, the carrier may be a viral carrier. For example, the carrier may be a lentiviral carrier.

[0194] cell In another aspect, the present application further provides a cell that may comprise said chimeric antigen receptor, said nucleic acid molecule and / or said carrier. The cell may comprise the progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be identical (in morphology of total DNA complement or genome) to the original parent cell.

[0195] 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, the cells may be T cells.

[0196] Pharmaceutical Compositions According to another aspect, the present application further provides a pharmaceutical composition comprising said chimeric antigen receptor, said nucleic acid molecule, said carrier and / or said cell, and optionally a pharma- ceutically acceptable adjuvant.

[0197] In some embodiments, the pharmaceutical composition may further comprise a suitable formulation of one or more (pharmaceutical effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. Acceptable components of the composition are preferably non-toxic to recipients at the dosages and concentrations used. Pharmaceutical compositions of the present invention may include liquids, frozen compositions and lyophilized compositions.

[0198] In some embodiments, the pharma- ceutically acceptable adjuvant may include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents that are compatible with the administration of the drug, generally safe, non-toxic, and biologically or otherwise undesirable.

[0199] In some embodiments, the pharmaceutical composition may include parenteral, gastrointestinal, transdermal, intraluminal, intraarterial, intrathecal, and / or intranasal administration, or direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, administration of the pharmaceutical composition may be by a variety of methods, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.

[0200] Manufacturing method According to another aspect, the present application further provides a method of producing said chimeric antigen receptor, which may comprise culturing said cell under conditions allowing expression of said chimeric antigen receptor.

[0201] The present application further provides a method of producing a modified immune effector cell, the method may comprise introducing the carrier into an immune cell.

[0202] use In another aspect, the present application further provides the use of said chimeric antigen receptor, said nucleic acid molecule, said carrier, said cell and / or said pharmaceutical composition in the manufacture of a medicament that can be used for the prevention, alleviation and / or treatment of a tumor.

[0203] According to another aspect, the present application further provides a method for preventing, alleviating and / or treating a tumor, which may comprise administering said chimeric antigen receptor, said nucleic acid molecule, said carrier, said cell and / or said pharmaceutical composition to a subject.

[0204] In another aspect, the present application further provides said chimeric antigen receptor, said nucleic acid molecule, said carrier, said cell and / or said pharmaceutical composition for use in the prevention, alleviation and / or treatment of a tumor.

[0205] In the present application, the tumor may include a tumor with aberrant expression of GPRC5D.

[0206] In the present application, the tumor may include a solid tumor and / or a non-solid tumor.

[0207] In the present application, the tumor may include a hematological tumor and / or a lymphoma.

[0208] In the present application, the tumor may include a myeloma. In the present application, the myeloma may include a refractory / relapsed multiple myeloma.

[0209] In this application, the subject may include a human or a non-human animal.

[0210] According to another aspect, the present application provides a reagent kit or an administration device comprising said chimeric antigen receptor, said nucleic acid molecule, said carrier, said cell and / or said pharmaceutical composition.

[0211] Without being limited by any theory, the following examples are merely intended to illustrate each technical solution of the present invention, and do not limit the scope of the present invention.

[0212] Working Example Example 1: Screening of anti-GPRC5D antibodies using the artificial synthetic nano antibody library NanoOri_1.0 296 nanoantibodies without repeat sequences and their structures in the Protein Data Bank (PDB) were aligned to determine the CDRs mutation policy, where the CDR3 lengths are 14, 17 and 21 amino acids. The commercially available nanoantibody Caplacizumab was selected as the backbone, and the complete gene synthesis of the nucleotide sequence of Caplacizumab was performed, which was then cloned into the HP153 phage vector, after which the single-stranded DNA of HP153 was extracted, and the CDRs of the nanoantibody were mutated by the Kunkel Mutagenesis method to obtain double-stranded DNA. The double-stranded DNA was electroporated into competent E. coli SS320 pre-infected with M13KO7 helper phage, and the phage supernatant was collected after overnight culture, resulting in a final diversity of 1.44 × 10 10We constructed an artificial synthetic nano antibody library NanoOri_1.0 (Yamato Scientific Biotechnology (Shanghai) Co., Ltd.) and used it as a seed bank to screen antibody sequences.

[0213] The GPRC5D full-length sequence (cat:HG24447-UT) purchased from Sino Biological was constructed into the company's existing lentiviral vector, and lentiviral transduction was performed to produce overexpressing cell lines of the target cells CHO-GPRC5D and HEK293-GPRC5D. The target cells CHO-GPRC5D and HEK293-GPRC5D were used to alternately pan the artificial synthetic nano antibody library (Yuanqi Biological Technology (Shanghai) Co., Ltd.), for a total of four rounds of panning.

[0214] One tube was taken from the artificial synthetic nano antibody library NanoOri_1.0, and PEG / NaCl was added, and the tube was reprecipitated for use. The target cells CHO-GPRC5D and HEK293-GPRC5D were selected for the alternate screening of cell panning. 500 μL of phage and 500 μL of 10% FBS / PBS buffer were taken and mixed until the final volume was 1 mL, and then placed in a 2 mL low-adsorption EP tube and blocked for 1 hour at 4 °C by gentle rotation or regular homogenous mixing. CHO-GPRC5D cells were obtained by centrifugation at 140 g for 10 min and washed once with 10 mL of PBS. Counting was performed, and when the viable cell count reached 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 so that each cell was 100%. The cells were placed on ice during this time. CHO-GPRC5D cells were centrifuged at 140 g for 2 min at 4°C, resuspended in 1 mL of blocked phage antibody library, and bound for 2 hours at 4°C or room temperature by gentle rotation or regular homogenous mixing, the supernatant was removed, and the obtained cells were resuspended in 1 mL of 5% FBS / PBS buffer, washed twice, and finally the cells were resuspended and replaced with a new 2 mL low-adsorption EP tube to avoid elution of phages nonspecifically adsorbed to the EP tube.

[0215] The cells were resuspended in 1 mL of 100 nM Gly-HCl (pH 2.2), incubated at room temperature for 10 min, and then centrifuged at 140 g for 2 min at 4°C. 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.

[0216] Three portions of the same adsorbed CHO cells were centrifuged, and the processing method was the same as that of CHO-GPRC5D cells. The neutralized phages were resuspended in CHO cells after elution. They were bound for 30 min at 4 °C with gentle rotation or regular homogenous mixing. They were centrifuged at 140 g for 2 min at 4 °C, and the supernatant was taken and used for the next adsorption process, which was carried out three times in total. The last phage supernatant was used to infect TG1 cells. The number of target CHO-GPRC5D cells was increased to 5 × 10 6 The second round of cell panning was performed using the same method, with three washes. The target cells were replaced with HEK293-GPRC5D, and the cell number was 1 × 10 6 The third and fourth rounds of cell panning were performed using the same procedure, with the number of washes increased to 10.

[0217] After four rounds of panning, significant specific enrichment of exported phages on target cells was observed. Cell-based mono phage ELISA was used to identify phage antibody clones obtained by panning. 3 × 10 4CHO-GPRC5D target cells were coated with 1000 cells / well and placed at 4°C overnight. Then, non-specific binding sites were blocked with 5% skim milk powder, and after thorough washing, the monoclonal phage supernatant (mixed with skim milk powder) was added to the 96-well plate, incubated for 2 hours, thoroughly washed, Anti-M13-HRP was added, reacted for 45 min, thoroughly washed, and TMB was added to develop color, reacted at room temperature for 5-10 min, then the reaction was stopped with 2M sulfuric acid, and the OD value of each well was measured at 450 nm.

[0218] After panning, 34 phage antibody clones capable of specifically binding to human GPRC5D were identified by ELISA and, after sequencing, were identified as single-chain V H The H gene sequence was obtained.

[0219] For example, 3B5 V H H(V H H, the amino acid sequence of which is shown in SEQ ID NO: 15), 3E7 V H H(V H H is shown in SEQ ID NO: 19), 4A2 V H H(V H H is shown in SEQ ID NO: 24) and 4B3 V H H(V H The antibody sequence (amino acid sequence of H is shown in SEQ ID NO: 30) was selected, and the cell binding activity of the antibody to the cell surface target antigen (GPRC5D) was detected by flow cytometry fluorescence sorting technique (FACS) using an iQue Screener flow cytometer (purchased from IntelliCyt) in PBS containing 0.1% BSA as a buffer solution.

[0220] Use buffer to obtain a concentration of 1 x 10 6Target cells (i.e., MM1S myeloma cells) were prepared at 100 cells / mL and plated in a conical-bottom 96-well plate (30 μL / well). 3894), and prepare a detection antibody concentration of 10μg / mL using buffer, dilute the antibody at a 3-fold ratio to form an 8-concentration gradient, add the prepared antibodies of different concentrations to the seeded target cells at 30μL / well and mix evenly, incubate in a refrigerator at 4℃ for 1 hour, add buffer at 150μL / well, centrifuge at 300g for 5 minutes, discard the supernatant, shake to loosen the cells, repeat washing once, prepare fluorescent secondary antibody (ab98593) at a mixing ratio of 1:200 using buffer, add 30μL / well to the cells and mix evenly, incubate in a refrigerator at 4℃ for 30 minutes, add buffer at 150μL / well, centrifuge at 300g for 5 minutes, discard the supernatant, shake to loosen the cells, repeat washing once, add buffer at 35μL / well and mix evenly, and then detect by flow cytometer. The binding activity of anti-GPRC5D antibodies to cell surface GPRC5D protein is shown in Table 1. Here, ET150-5scFv is a positive control antibody whose sequence is shown in patent application CN107428829A, and 3B5V H The amino acid sequence of H is shown in SEQ ID NO: 15, H The amino acid sequence of H is shown in SEQ ID NO: 19, H The amino acid sequence of H is shown in SEQ ID NO: 24, H The amino acid sequence of the anti-GPRC5D VH is shown in SEQ ID NO: 30. H Both H were able to bind to GPRC5D on the cell surface, and the binding ability was comparable to and even higher than that of the positive control ET150-5 scFv.

[0221] TIFF2025504803000002.tif56170

[0222] Example 2 Construction of CAR lentivirus expression carrier In order to obtain an anti-GPRC5D CAR-T cell product with stronger affinity and better antitumor effect, the inventors have developed 3B5V, which has a relatively excellent target cell killing activity, a relatively high target proliferation ability, and a relatively low tonic signal level in in vitro functional validation. H H, 3E7V H H, 4A2V H H and 4B3V H Four antibody sequences, named H, were linked to the CAR structure via different peptide linkers to form the double-binding domain GPRC5D double V H H CAR was constructed. Exemplary double binding domain double V H The H CAR construct is shown in Table 1. The steps for constructing the construct are as follows: Specifically, two Vs are linked by (GGGGS)n or (EAAAK)n (wherein n may be a positive integer). H The H sequence was fused to the CAR lentivirus core empty plasmid (self-constructed by Yuanqi Biotechnology (Shanghai) Co., Ltd. and containing Orinov elements, hereinafter abbreviated as CAR lentivirus empty carrier) using SphI and NotI endonucleases (purchased from NEB) to generate a 8992 bp linearized fragment, and the double VH sequence obtained by fusion was then fused to the CAR lentivirus core empty plasmid (self-constructed by Yuanqi Biotechnology (Shanghai) Co., Ltd. and containing Orinov elements, hereinafter abbreviated as CAR lentivirus empty carrier) using SphI and NotI endonucleases (purchased from NEB). H The double V binding domain was transformed into DH5α competent cells after homologous recombination. Single colonies were selected and identified by sequencing. H The linking order of the H CAR structure is detailed in Table 2.

[0223] Double V H For 32 different combinations of H CAR structures, we verified the in vitro biological functions of CAR-T, including in vitro proliferation activity, target killing ability, and cytokine secretion level, and one of them, double V H A combination of H CAR structures was exemplarily selected and named OriCAR-017 (i.e., oriCAR-DG508M), and then a single domain G5V H H1 CAR (However, the target part G5VH The amino acid sequence of H1 is shown in SEQ ID NO:19 (3E7)) and G5V H H2 CAR (However, the target part G5V H H2 (amino acid sequence shown in SEQ ID NO: 30 (4B3)), and positive control ET150-8 (amino acid sequence shown in Patent No. US10633426B2) were used to carry out CAR-T in vitro biological function experiments, and the elements and linking order of each part of each CAR lentiviral carrier were shown in detail in FIG. 1. However, the amino acid sequence of the CD8α signal peptide was shown in SEQ ID NO: 1, the amino acid sequence of the CD8α hinge region was shown in SEQ ID NO: 2, the amino acid sequence of the CD8α transmembrane region was shown in SEQ ID NO: 3, the amino acid sequence of the 4-1BB costimulatory signal was shown in SEQ ID NO: 4, the amino acid sequence of the CD3ζ intracellular signaling region was shown in SEQ ID NO: 5, and the amino acid sequence of the Ori new element was shown in SEQ ID NO: 7. An exemplary selected sequence for linker A was (GGGGS)5, and an exemplary selected sequence for linker B was (EAAAK)3.

[0224] TIFF2025504803000003.tif138170

[0225] A: (GGGGS)n, where n is an integer of 0 or more, and the amino acid sequence of GGGGS is shown in SEQ ID NO:39. B: (EAAAK)n, where n is an integer of 0 or more, and the amino acid sequence of EAAAK is shown in SEQ ID NO:40.

[0226] Example 3 Packaging of lentivirus and measurement of viral titer By way of example, the lentiviral carrier system used in the construction of the present invention belongs to the third generation, and the system comprises the following VSV packaging plasmids: pRH1, which encodes the Gag-Pol proteins; pVH3, which encodes the Rev protein; pMH2, which encodes the envelope protein VSV-G; and a core plasmid. HIt was composed of a total of four plasmids, each of which was the CAR lentiviral plasmid in Example 2 above, containing the H sequence. Expression of the CAR gene in each CAR lentiviral plasmid was regulated by the elongation factor-1α (EF-1α) promoter.

[0227] 3.1 Lentivirus packaging: (1) Approximately 2.5 x 10 6 293T cells were placed in 5 mL of 293T cell complete medium (high glucose DMEM containing 10% FBS) and mixed evenly, then inoculated into a 6 cm culture dish (ensuring that the cell confluence after 20-24 h is 80-90%) and cultured overnight in a 37°C, 5% CO incubator. (2) After about 20 to 24 hours, take 300uL of Opti-MEM medium, add a total of 6.9ug of three types of plasmids (pRH1:pMH2:pVH3:CAR lentivirus core plasmid = 1:1:2:2 by mass ratio), mix evenly, add 20.7uL of FuGENEHD transfection reagent (Promega, E2311), mix evenly carefully, and leave at room temperature for 15 to 20 minutes. Then, carefully drop the mixture into a 6cm culture dish, shake the culture dish carefully, and culture overnight in a 37℃, 5% CO2 incubator. (3) After approximately 20 to 24 h of transfection, carefully discard the upper liquid in the 6 cm culture dish, and then slowly add 5 mL of 293T cell complete medium. (4) After approximately 48 hours of transfection, the upper layer liquid (containing dead cells and debris) in the 6 cm culture dish was collected and centrifuged at 3000 g for 5 minutes. The upper layer virus liquid from which the cell debris had been removed was then transferred to a new centrifuge tube, aliquoted, and stored at -80°C.

[0228] 3.2 Detection of lentivirus titer: (1) The virus solution prepared above was quickly thawed. (2) Take 293T cells in good growth condition (generally cultured cells within 20 generations), discard the upper layer waste liquid, wash the cells with PBS, and then digest them with 0.25% trypsin (GIBCO) at 37°C for about 3 min. After the cells are completely digested, a certain volume of 293T cell complete medium is added to stop the reaction, and samples are taken and counted. Then, the cell density is adjusted to 2.0 × 10 5 / mL and polybrene was added to a final concentration of 10 ug / mL, and the cells were seeded into 6-well plates at an inoculum volume of 2.5 mL / well. (3) Next, add the above virus solutions to a 6-well plate, and generally prepare three sample loading gradients of 2uL, 5uL, and 10uL of each virus solution. In addition, 1-2 blank wells without virus solution should be added as negative controls for flow cytometry. (4) The cell culture plate was subjected to static culture in a 37°C, 5% CO2 incubator. (5) After the cells were statically cultured for 48 h, the upper layer of the waste liquid was discarded, the cells were washed with PBS, and then digested with 0.25% trypsin (GIBICO) at 37°C for about 3 minutes. After the cells were completely digested, a certain volume of 293T cell complete medium was added to stop the reaction, and samples were taken and counted, and then about 5 × 10 5 Cells were placed into a 1.5 ml sterile centrifuge tube and marked. (6) The solution was supplemented to 1.2-1.5 mL with NBS solution (PBS solution containing 1% newborn bovine 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, which was then gently mixed uniformly by blowing, centrifuged at 500 g for 5 minutes at 4°C, 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, mixed evenly by gently blowing with a pipette (no primary antibody was added to the negative control), and incubated at 4°C for 60 minutes. (9) After incubation was completed, 1 ml of NBS was directly added to resuspend the cells, followed by centrifugation at 500 g for 5 minutes at 4° C., and the supernatant was discarded. (10) 50 μL of NBS and 0.5 μg of APC streptavidin (554067) fluorescent secondary antibody (no protein was added as a negative control) were added to each tube and incubated at 4° C. for 30 minutes. (11) Step (9) was repeated. (12) 200 μL of NBS was added to each tube to resuspend the cells. (13) The percentage of fluorescent expression was detected on a BD FACS CantoII flow cytometer, and the data were processed using FlowJo-V10 software.

[0229] Results are only available if the positive rate of each test tube - the positive rate of the negative control - is between 5% and 20%. The formula for calculating the viral titer is as follows:

[0230]

number

[0231] Detection of the above V H The titer range of each CAR virus, including H, was 1–10 × 10 6 The mean mean RT-PCR level was 1.0 μg / mL.

[0232] Example 4 Infection of T cells and in vitro expansion of GPRC5D CAR-T cells Each combination of double-binding domains GPRC5D double V H H CAR antibody sequence and single V H The method for producing CAR-T cells with the HGPRC5D antibody sequence is as follows: (1) Human peripheral blood mononuclear cells (PBMCs) were isolated from blood samples collected using an apheresis device by Ficoll density gradient centrifugation (the apheresis device was provided by the Shanghai Primordial Cell Ark Project volunteers). (2) PBMCs were sorted using CD3-positive magnetic beads and were purified to obtain CD3 + T cells were obtained. The specific method for selecting T cells was described in the product's instruction manual (MACS, DS130-050-101). (3) T cell complete medium (X-VIVO 15 (Lonza) + 5% FBS + cytokines) + Resuspend the T cells and add another 3x ratio of previously washed CD3 / CD28 Dynabeads (Gibco, 40203D), then supplement with T cell complete medium until the cell density is 1.0–1.2 × 10 6 The concentration was adjusted to cells / mL and the mixture was placed in a 37°C, 5% CO2 incubator for activation culture. (4) Typically, T cells were activated for 20–24 h and then transduced with lentivirus. (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 certain volume of complete T cell medium, sampled and counted, and the virus liquid whose titer had already been measured in Example 3 was added at a viral multiplicity of infection (MOI) of 3. Polybrene was then added to a final concentration of 5 μg / mL, and the complete T cell medium was further supplemented to obtain a cell density of 0.6-1.0 × 10 6 The solution was adjusted to 1 / mL, and the cells were further cultured in an incubator at 37°C with 5% CO2. Separate T cells were taken and not infected with the lentivirus to serve as a negative control. (6) After transduction of the virus for about 20–24 h, the T cells of each group were collected and centrifuged at 500 g for 5 min. The cells were then resuspended in a certain volume of T cell complete medium, sampled and counted, and supplemented with T cell complete medium until the cell density was 0.5–0.7 × 10 6 Adjusted to cells / mL. (7) After that, count the CAR-T cells every 1-2 days, replenish with T cell complete medium according to the actual situation, and keep the cell density at 0.5-1.0 × 10 6 Adjusted to cells / mL. (8) Generally, Dynabeads were removed on the 5th day of culture, and the total culture period was about 12 days.

[0233] The experimental results are shown in Figures 2A-2B. After activation and culture amplification, the double-binding domain GPRC5D double VH The positive rate of T cell CAR after H CAR lentivirus infection ranged between 55% and 75%, with a single V H The positive rate of HGPRC5D CAR was between 80% and 90%, the CAR gene was stably expressed on T cells, and there was no significant difference between the groups. As shown in Figure 3, after in vitro CD3 / CD28 stimulation, CAR-T cells proliferated stably, especially from the 9th day of co-culture, and G5V H H1 CART and OriCAR-017 cells were G5V H It showed superior proliferation potential to H2 CAR-T and positive control ET150-8 CAR-T cells, with cumulative amplification folds of 1000-fold and 200-fold, respectively, and could be used for cytological functional experiments.

[0234] Example 5 Measurement of the expression level of degranulation molecule CD107a in GPRC5D CAR-T cells One of the important pathways for CAR-T cell cytotoxicity is the release of perforin and granzyme, and the degranulation effector of CAR-T cells is also an important expression of CAR T cell activation, so the expression level of the cell degranulation function molecule CD107a was an important indicator for evaluating the CAR T killing effector. To detect the activation ability of CAR-T against target cells, the inventors detected the expression content of CD107a in CD3 positive cells in the co-incubation well, and judged the activation and killing ability of CAR-T according to the content.

[0235] As shown in Figure 4A-4B, the different double-binding domains GPRC5D double V HH After CAR-T cells and target cells were co-cultured for 4 hours at an effector-to-target ratio of 1:1, the expression levels of CD107a by CAR-T cells in each group were not uniform, and all were higher than the expression levels of CD107a in the Mock T control cells. However, the positive rate of CD107a expression in OriCAR-017 was about 28%, which was relatively higher than that of the two single domain antibody CAR cell groups and the positive control ET150-8 group. The experiment showed that OriCAR-017 had obvious degranulation effector on specific target cells, suggesting that it had good activation and effector killing potential.

[0236] Example 6 Evaluation of in vitro effector killing activity of GPRC5D CAR-T cells In order to accurately detect the killing ability of CAR-T cells against target cells, the inventors introduced the GPRC5D gene into CHO cells by lentivirus, and then selected GPRC5D-positive cells, i.e., CHO-GPRC5D cells, by a flow sorter. The target cells were CHO-GPRC5D cells, and the negative cells were CHO-negative cells. The effector cells cultured on the 9th day were incubated with the target cells for 20 hours at an effector to target ratio of 3:1, and the survival status of the target cells (i.e., the killing ability of CAR-T cells) was determined by the LDH method.

[0237] The cell killing toxicity was calculated according to the following formula:

[0238]

number

[0239] As shown in Figures 5A-5B, the blank effector cells were compared with T cells, and the results showed that the double binding domain GPRC5D double V H H CAR-T cells have a clear killing effect on target cells and a single V H H CAR-T cells had a clear killing effect on target cells, and the single domain antibody G5V was more effective than Mock T cells. HH1 CAR-T, single domain antibody G5V H H2 CAR-T, double epitope target OriCAR-017, and positive control ET150-8 all showed significant killing effects on target cells. In particular, when the effector-to-target ratio was 1:9, the killing rate of OriCAR-017 against MM.1S target cells was significantly higher than that of single domain antibody CAR-T and positive control ET150-8, reaching 70%.

[0240] Example 7 Cytokine secretion after in vitro killing of GPRC5D CAR-T cells See Example 6, the target cell killing experiment was carried out in a 96-well plate, the effector cell to target cell ratio was 1:1, and then the effector cell number in each 96-well was increased to 2×10 4 The wells were then fixed in individual wells, and target and effector cells were added sequentially. Two parallel wells were set up in each group at each effector to target ratio, and two effector cell-only wells were also set up in each group to detect the amount of background factors secreted by the effector cells.

[0241] Approximately 24 hours after target cell killing, the 96-well plate was centrifuged and the supernatant from each well was collected, and the effector cells were centrifuged, the supernatant discarded, and 3 × 10 5 The cells were diluted to 100 μL per well and used. Both non-targeted cells (i.e., CAR-T cells not co-cultured with tumor cells) and MM.1S cells were mixed in a round-bottom 96-well plate with an effector-target ratio of 1:1, and after co-incubation for 24 hours, the supernatant was aspirated and the contents of IL-2 and IFN-γ in the supernatant were detected by R&D Cytokine Kit.

[0242] As shown in Figures 6A-6B, under stimulation of MM.1S target cells, the double-binding domain GPRC5D double V HH The IL-2 and IFN-γ secretion levels of CAR-T cells were all higher than those of the Mock T cell group, and the IFN-γ secretion of each CAR-T group was significantly higher than that of the Mock T cell group. In addition, compared with the two single domain antibody CAR-T cell groups and the positive control ET150-8 group, the double epitope targeting OriCAR-017 had a higher IFN-γ secretion level, indicating that OriCAR-017 has a stronger antigen-specific activation effect. In addition, the IL-2 secretion of each CAR-T group was significantly higher than that of Mock T cells, but the IL-2 secretion level of OriCAR-017 was lower than that of single domain antibody CAR-T cells, which also indicated that OriCAR-017 had a certain activation and proliferation property.

[0243] Example 8 Targeted proliferation ability of GPRC5D CAR-T cells In this example, to detect the target proliferation ability of CAR-T cells, untreated MM.1S cells were selected as activated target cells.

[0244] Generally, this is done when CAR-T cells are cultured for about 9 to 12 days. The CAR positivity rate of each group must be detected in advance, and the CAR positivity rate of each group must be adjusted to match with blank effector T cells (the medium is X-VIVO, which does not contain any additives). 15 (the medium).

[0245] The first round of target stimulation was performed on CAR-T cells and MM.1S cells at an effector-to-target ratio of 1:1. After co-incubation for 5 days, all cells were collected, sampled and counted. The second round of target stimulation was performed at a ratio of 1:3 (total CAR-T cells: new MM.1S cells), and after co-incubation for another 4-5 days, the third round of target stimulation was performed (the ratio between the two types of cells was the same as in the second round of target stimulation). During the three rounds of target stimulation, an appropriate amount of X-VIVO was administered every 1-2 days depending on the cell growth status. 15 The medium was replenished.

[0246] As shown in Figure 7A, the double-binding domain GPRC5D double V H H CAR-T cells have different degrees of target proliferation ability after stimulation with target cells, and the proliferation fold of two rounds of repeated stimulation is between 50-200 times, which is significantly different from the control Mock T cells. The double binding domain GPRC5D double V H These results suggest that H CAR-T cells can be efficiently and rapidly expanded against specific tumors in vivo. As shown in Figure 7B, the CAR-T cells in each group showed strong cell proliferation ability after stimulation with MM.1S target cells, and the proliferation fold could reach 1000-3500-fold after three rounds of target cell stimulation. Compared with the control mock T cells, the proliferation activity was obviously increased, where G5V H H1 CAR T cells had the highest expansion efficiency under target cell stimulation. In addition, the expansion efficiency of target proliferation of OriCAR-017 cells was higher than that of the positive control ET150-8 CAR T cells. These results indicated that OriCAR-017 CAR T cells could rapidly proliferate under target cell stimulation and could be efficiently and rapidly expanded against specific tumors in vivo.

[0247] Example 9 Analysis of the Cellular Phenotype of GPRC5D CAR-T Cells CD3 activated by lentivirus infection + After the T cells obtained positive results for GPRC5D CAR expression, the cells were examined for memory, activation, and exhaustion phenotypes on day 12 after coculture. H H1 CAR-T, G5V H H2 CAR-T, OriCAR-017 cells, and positive control ER150-8 were collected and analyzed by flow cytometry to determine the CD3+, CD45RO+, CCR7+, and CD62L+ cell ratios in each group. + We detected memory markers in T cells, activation markers in CD3+ T cells in each group, including the cell ratios of CD25 and CD69, and exhaustion markers in CD3+ T cells in each group, including the cell ratios of PD-1, LAG-3, and TIM-3.

[0248] As shown in Table 3 and Figures 8 and 9, when CAR-T cells were cultured for 12 days, G5V H H2(T SCM :7.79%, T CM :51.1%) and ET150-8 CAR-T cells (T SCM :3.91%, T CM 43.1%) compared with OriCAR-017 (T SCM :22.7%, T CM :25.7%) and G5V H H1 CAR-T cells (T SCM :22.8%, T CM :34.4%), which has a relatively high stem cell memory (T SCM ) phenotype, relatively low central memory T cell (T CM ) phenotype and a low degree of T cell differentiation. As shown in FIG. 10, when CAR T cells were cultured for 12 days, OriCAR-017 and G5V H The expression levels of the activation markers CD25 and CD69 in H1 CAR T cells were significantly higher in G5V H H2 and ET150-8 CAR T cells, i.e., the degree of T cell activation was relatively low, and OriCAR-017 T cells had the advantage of being less activated cell phenotype. As shown in Figure 11, when CAR-T cells were cultured for 12 days, the expression levels of OriCAR-017 T cell exhaustion markers LAG-3 and TIM-3 were significantly higher than those of G5V. H Compared to H2 and ET150-8 CAR-T cells, i.e., the degree of T cell depletion was relatively low, OriCAR-017 T cells had the advantage of a less exhausted cell phenotype.

[0249] TIFF2025504803000006.tif51170

[0250] Example 10 Study of immune synapse mechanism of GPRC5D CAR-T cells The immune synapse (IS) is a structure formed after binding with a specific activating receptor, which is the CAR-T cell's identifying antigen and is a key step in proliferation and activation, and is an important component of the body's cellular and humoral immune responses. The formation of the immune synapse is related to the effector function of CAR-T cells. At the CAR-T level, the polarization of the microtubule-organizing center (MTOC) of the immune synapse structure was measured to further evaluate the functional benefits of CAR-T cells. CAR-T cells and target cells were co-cultured at a 1:1 ratio for 5 min, then transferred to Nunc chamber slides, incubated for 20 min, fixed with 4% PFA at room temperature, washed three times with PBS, incubated at room temperature with immunostaining permeabilization solution (Triton X-100), incubated at room temperature with 10% horse serum / immunostaining permeabilization solution (Triton X-100), incubated at room temperature with primary antibody diluted in 2% horse serum / immunostaining permeabilization solution (Triton X-100), incubated at room temperature with secondary antibody diluted in 2% horse serum / immunostaining permeabilization solution (Triton X-100), and incubated at room temperature. The slides were separated, sealed with ProLong Diamond, dried and solidified, and then 3D images and videos were acquired by overlaying using a laser confocal 100x oil lens, ultraviolet, 488 nm, 543 nm, 1024 × 1024, Z-axis scan 0.4 μm.

[0251] As shown in Figure 12, G5V H H1, G5V H Confocal imaging of H2 and OriCAR-017 CAR T cells was performed, where the cell nuclei were stained blue, γ-microtubule protein was stained green, and T cells were stained red. The white arrows indicate the polarized position of the MTOC at the microtubule organization center of the IS, and the closer the distance between the MTOC and the IS, the more stable the IS structure. As shown in Figure 13, G5V H H1 and G5V HCompared with H2 CAR-T cells, the MTOC polarization level of OriCAR-017 cells toward the IS was obviously increased, manifested as a decrease in the distance between the MTOC and the IS, which resulted in a better and more stable IS structure, thus having better functional advantages and suggesting that OriCAR-017 cell-mediated cytotoxicity is more potent than that of single-targeted GPRC5D V. H H1 and GPRC5DV H It was shown to be superior to H2 CAR-T cells.

[0252] Example 11. In vivo experiment of GPRC5D CAR-T cells in mice The inventors further verified the tumor elimination and tumor suppression effects of OriCAR-017 CAR-T cells in NSG mice. Specifically, the experiment was conducted in five mice per group, using Mock T cells, G5V H H1 CAR-T, G5V H The mice were divided into five groups: H2 CAR-T, ET150-8 CAR-T, and OriCAR-017 CAR-T cell groups. First, NSG mice were subcutaneously injected with 1 × 10 7 MM1S cells were inoculated at a dose of 100 cells / animal, and approximately 2 weeks later (when the tumor size was 50–150 mm 3 ) and inject CAR-T (or T) cells cultured for 10 to 12 days into the tail vein. The dose was 1.6 × 10 6 The dose was 100 CAR-T cells / mouse (the total number of cells inoculated into each mouse was adjusted to match the T cells). After inoculation of CAR-T (or T) cells, cytokine secretion in the mouse peripheral blood was detected on the 7th and 14th days after inoculation of CAR-T (or T) cells, and the mouse tumor size and body weight were measured three times a week and observed for 3 weeks.

[0253] The experimental flow of tumor suppression in NSG mice in vivo is shown in Figure 14. As shown in Figure 15A, the cytokine IFN-γ secreted was detected 7 days after injection, and the IFN-γ produced by the OriCAR-017 CAR-T cell group was significantly higher than that of the G5V H H1 CAR-T, G5V HThe body weight of mice was significantly higher than that of H2 CAR-T and the positive control ET150-8 CAR-T. Regarding safety, as shown in FIG. 15B, the body weight of mice showed a stable increasing trend over time, and all CAR-T cell experimental groups were consistent with the weight curve trend of mice in the Mock T group. In addition, the fur of mice after injection of OriCAR-017 was bright, and vital signs such as mouse behavior were normal, demonstrating that OriCAR-017 has good safety in mice. Regarding the tumor suppression effect in vivo, as shown in FIG. 15C, the single binding domain G5V H H1 CAR-T and G5V H Compared with H2 CAR-T cells and the positive control ET150-8 CAR-T cells, the OriCAR-017 cell group showed a very significant antitumor effect, with the tumor inhibition rate reaching nearly 100%. As shown in Figure 15D, the OriCAR-017 cell group achieved a 100% mouse survival rate during the study period, and the mouse survival rate was significantly higher than that of the single binding domain V. H This was much higher than that of the H CAR T cell group and the positive control ET150-8 CAR-T cells, indicating that OriCAR-017 may exhibit relatively superior antitumor activity clinically.

[0254] In order to further simulate the distribution of blood tumors in the human body, the inventors further verified the tumor elimination and tumor suppression effects of OriCAR-017 cells in B-NSG mice based on in vivo imaging technology. MM1S-Luciferase target cells were injected into the tail vein of B-NSG mice, and the mice were randomly divided into groups of 5 mice according to their bioluminescence intensity. Different doses of pre-frozen OriCAR-017 CAR-T or Mock T cells were injected into the tail vein. After re-injection of CAR-T, the changes in bioluminescence in the mice were observed every Tuesday and Thursday using an animal in vivo imaging system, and the weight and health status of the mice were observed. During the observation period, blood was collected from the eyes or tail vein of the mice three times to detect cytokines, and CD3+ was detected by BD flow cytometry. + CD8 + The T cell ratio was detected.

[0255] As shown in Figure 16, compared with Mock T, OriCAR-017 cryopreserved cells at different doses all showed obvious antitumor activity and significantly reduced the tumor burden in mice. However, the low-dose group showed an efficient tumor elimination effect on the 7th day after reinjection, while the OriCAR-017 medium-dose and high-dose groups showed obvious tumor-suppressing activity within a short period (4 days) after reinjection, and especially the high-dose group was able to rapidly eliminate tumors after reinjection and continuously inhibit tumor growth, and the complete tumor elimination rate of the high-dose group reached 60% on the 21st day.

[0256] Example 12. Clinical study of OriCAR-017 CAR-T in patients with refractory / relapsed multiple myeloma A single-arm, open-label, Phase I multicenter clinical study was conducted to determine the safety and efficacy of OriCAR-017 CAR-T cells in treating human patients diagnosed with refractory or relapsed multiple myeloma ("r / r MM"). Clinical trial registration information is at www.clinicaltrials.gov, identifier NCT05016778.

[0257] In this study, patients with refractory / relapsed multiple myeloma were treated with OriCAR-017 CAR-T cells derived from the patient's autologous T cells. 6 / kg, 3×10 6 / kg, 6×10 6 / kg CAR +The T cell dose was administered to each patient by intravenous injection. Patients were monitored for adverse events and patient samples were obtained for evaluation on days 1-30 of the study. All patients were followed up for at least 36 months after CAR T administration. The primary outcome of the study measured the occurrence of treatment-related adverse events assessed by the Common Terminology Criteria for Adverse Events (CTCAE) v4.0 within 1-30 days after OriCAR-017 CAR-T cell injection, and secondary outcomes evaluated CAR-T-induced anti-myeloma responses, including abnormal immunoglobulin levels in serum and the number of multiple myeloma cells in patients' bone marrow before and after administration of OriCAR-017 CAR-T cells. Efficacy objectives of the study included the rate of pathological complete response, 3-year disease-free survival, and 3-year progression-free survival. We presented this study in detail at the 2022 ASCO Annual Meeting (https: / / ascopubs.org / doi / abs / 10.1200 / JCO.2022.40.16_suppl.8004). As of April 30, 2022, 12 patients had been enrolled in the study and had undergone apheresis, with 10 patients completing OriCAR-017 CAR-T infusion, median age 64 years (range, 41-71), and median number of prior lines of therapy 5.5 (range, 3-17). Five (50.0%) patients had received prior anti-BCMA CAR-T therapy. No dose-limiting toxicities occurred. The most common treatment-emergent adverse events (TEAEs) were neutropenia, thrombocytopenia, leukopenia, lymphopenia, and anemia. No neurotoxicity was reported. All patients developed cytokine release syndrome (CRS), 9 with grade 1 and 1 with grade 2. All CRS rapidly resolved after conventional intervention (tocilizumab and steroids). Median time to onset was 2 days (range, 1-9), median duration was 6 days (range, 3-9). Objective response rate (ORR) was 100%, MRD was 100% negative, and complete response / strictly complete response (CR / sCR) rate was 60%.Treatment efficacy was evaluable for 5 patients who had previously received anti-BCMA CAR-T therapy, with 2 achieving sCR, 2 achieving VGPR, and 1 achieving PR, as shown in Tables 4 and 5.

[0258] TIFF2025504803000007.tif179170

[0259] TIFF2025504803000008.tif165170

[0260] ORR = (number of CR + sCR + VGPR + PR cases) / number of subjects in the population evaluable for treatment effect × 100%. DCR = (number of CR + sCR + VGPR + PR + MR cases) / number of subjects in the population evaluable for treatment effect × 100%. Percentages were calculated based on the number of subjects enrolled in each dose group within the treatment-evaluable population. [1] Two-sided matched CIs were calculated based on the Clopper Pearson method.

[0261] Example 13. Two V in GPRC5D CAR-T cells H Binding experiments of different epitopes of the H domain OriCAR-017 CAR-T GPRC5D V H We identified an epitope in the H domain. Two different anti-GPRC5D V antibodies with different epitopes that specifically bind to GPRC5D H An exemplary bivalent GPRC5D CAR with an H domain was constructed. H H1 and G5V H The bivalent / double epitope CAR containing H2 was named OriCAR-017 and is an exemplary GPRC5D double VH2 double binding domain listed in Table 2. H It was H CAR.

[0262] 13.1 FORTEBIO antibody measurement G5V H H1 and G5V HThe H2 antibody sequence was cloned into the eukaryotic carrier p3.4-hIgG1Fc, which contains the human IgG1 Fc fragment (hIgG1Fc) sequence, and H The recombinant expression of H-hIgG1Fc was promoted to obtain and purify the recombinant protein. H H1-hIgG1Fc and G5V H The affinity of H2-hIgG1Fc for GPRC5D and for the N-terminal peptide (biotin) was measured. Briefly, the N-terminal peptide (>sp|Q9NZD1|1-27:MYKDCIESTGDYFLLCDAEGPWGIILE) protein of human GPRC5D was labeled with biotin (EZ-Link Sulfo-NHS-LC-Biotin, Pierce, 21327). The binding kinetics between the antigen and the antibody was analyzed by biolayer interferometry (BLI) using a molecular interaction analyzer fortebiooctetRED384 (PALL) (both antigen and antibody were diluted in PBS buffer containing 0.1% BSA and 0.02% Tween 20). The antigen bound to biotin was fixed to the SA sensor at a concentration of 20 nM, bound for 3 min, and then diluted antibody (i.e. G5V H H1 and G5V H The antibody was bound to the H2 antibody solution for 5 min and then dissociated for 5 min. The results were analyzed using Octet Data Analysis 9.0 software (fortebio) to calculate the binding strength between the antigen and the antibody, and the KD, Ka (1 / Ms) and Kd (1 / s) values ​​were obtained. H H1-hIgG1Fc and G5V H The binding affinity results of H2-hIgG1Fc are shown in Figure 17, and G5V H In the regression equation for H1 antibody detection, the fitting goodness of fit of the sample data points was low (R2 value: 0.7914), and the model was not reliable. H H1 does not bind to the N-terminal peptide of GPRC5D, but H The H2 antibody bound to the N-terminal peptide and the KD was found to be 7.69E-08M. H H1, G5V HWe further demonstrated that there is a difference in the epitope that H2 binds to on the GPRC5D target.

[0263] 13.2 Competitive Binding Measurements Cell-based competitive binding assays revealed that G5V H H1 and G5V H To further verify the distinct epitope binding of H2, a stable CHO cell line overexpressing human GPRC5D (CHO-GPRC5D) was used for the assay. H H1 and G5V H H2 was cloned into the p3.4-hIgG1Fc eukaryotic vector containing the human IgG1 Fc fragment (hIgG1Fc) sequence and an expression vector containing a 6xHis label, and GPRC5D V H H-hIgG1Fc and GPRC5D V H Promote recombinant expression of H-His and purify it to G5V H H1-hIgG1Fc, G5V H H2-hIgG1Fc, G5V H H1-His, G5V H The H2-His recombinant protein was obtained.

[0264] Use PBS buffer to obtain a concentration of 1 x 10 6 Prepare CHO-GPRC5D target cells at 300 cells / mL and add 30 μL / well to a conical bottom 96-well plate (corning 3894). Add detection antibody G5V to a concentration of 30 μg / mL using PBS buffer. H H1-hIgG1Fc, G5V HH2-hIgG1Fc was prepared, the antibody was diluted at a 3-fold ratio to form an 8-concentration gradient (final concentration 0), and the prepared antibodies at different concentrations were added to the seeded target cells at 30μL / well and mixed evenly, incubated in a 4℃ refrigerator for 1 hour, buffer was added at 150μL / well, centrifuged at 300g for 5 minutes, the supernatant was discarded, washing was repeated once, goat anti-human IgG (Fc specificity, ab98593) fluorescent secondary antibody was prepared at a mixing ratio of 1:200 using buffer, added to the cells at 30μL / well and mixed evenly, incubated in a 4℃ refrigerator for 30 minutes, buffer was added at 170μL / well, centrifuged at 300g for 5 minutes, the supernatant was discarded, washing was repeated once, buffer was added at 35μL / well and mixed evenly, and then detected by iQue Screener flow cytometer (purchased from IntelliCyt). According to the analysis results of the flow cytometry affinity binding experiment, G5V H H1-hIgG1Fc, G5V H The binding activity EC80 value of H2-hIgG1Fc was found and is shown in FIG.

[0265] According to the antibody concentration corresponding to EC80, 0.65ng / mL of G5V H H2-hIgG1Fc was measured using a total of 3 × 10 cells. 4 The cells were incubated with CHO-GPRC5D cells for 1 h and the detection antibody G5V was diluted to a concentration of 80 μg / mL in PBS buffer. H Prepare H1-His, dilute the antibody in a 3-fold ratio, form a 12-concentration gradient (final concentration 0), and then serially dilute G5V HThe H1-His recombinant antibody was added to the well plate and incubated at 4°C for another 1h. After incubation, 140μL / well of buffer was added, centrifuged at 300g for 5 minutes, the supernatant was discarded, washing was repeated once, and goat anti-human IgG (Fc specificity, ab98593) fluorescent secondary antibody was prepared at a mixing ratio of 1:200 using the buffer, added to the cells at 30μL / well and mixed evenly, incubated in a refrigerator at 4°C for 30 minutes, 170μL / well of buffer was added, centrifuged at 300g for 5 minutes, the supernatant was discarded, washing was repeated once, and 35μL / well of buffer was added and mixed evenly, and then detected by iQue Screener flow cytometer (purchased from IntelliCyt). As a measurement control, G5V was detected by following the same procedure as above. H G5V was incubated directly with CHO-GPRC5D cells in the absence of H2-hIgG1Fc and serially diluted H H1-His, G5V H It was used to detect the binding of H1-His to CHO-GPRC5D cells.

[0266] As shown in Figure 19, G5V H When H2-hIgG1Fc binds to cells at saturation, G5V H H1-His still has antibody gradient dependence and G5V H G5V in the absence of H2-hIgG1Fc H Close to H1-His binding activity, i.e. G5V H The presence of H2-hIgG1Fc is G5V H It did not affect the binding to H1-His. H When H2-hIgG1Fc binds to cells at saturation, G5V H When H1-His is added and the target cell is bound to G5V H H2-hIgG1Fc is G5V H G5V in the absence of H1-His H H2-hIgG1Fc binding activity is close to that of G5V H The presence of H1-His is G5V H It was found that the binding to H1-His was not affected.H H1 and G5V H There was a difference in the epitope that H2 bound to the GPRC5D target, and the two did not affect each other.

[0267] Example 14. Cross-binding studies of CAR antibody sequences with target antigens of different species Stably transfected CHO cells expressing human GPRC5D, cynomolgus monkey GPRC5D (SEQ ID NO: 36), mouse GPRC5D (SEQ ID NO: 37), and the human GPRC5D control subtype GPRC5A (SEQ ID NO: 38) were used to evaluate the binding of different anti-GPRC5D antibodies to human, cynomolgus monkey, and mouse GPRC5D by flow cytometry identification. Briefly, 1×10 antibodies were diluted in PBS buffer at a concentration of 1×10 6 Prepare CHO-HuGPRC5D, CHO-CyGPRC5D, CHO-MuGPRC5D, and CHO-HuGPRC5A target cells at 30 μL / well in a V-bottom 96-well plate, and dilute the detection antibody Double-V with PBS buffer to a concentration of 30 μg / mL. H H hFc was prepared, the antibody was diluted at a 3-fold ratio to form an 8-concentration gradient (final concentration 0), the prepared antibodies at different concentrations were added to the seeded target cells at 30 μL / well and mixed evenly, incubated in a refrigerator at 4 ° C for 1 hour, buffer was added at 150 μL / well, centrifuged at 300 g for 5 minutes, the supernatant was discarded, washing was repeated once, Dyna650-goat anti-human IgG (Fc specificity, ab98593) fluorescent secondary antibody was prepared at a mixing ratio of 1:200 using buffer, added to the cells at 30 μL / well and mixed evenly, incubated in a refrigerator at 4 ° C for 30 minutes, buffer was added at 170 μL / well, centrifuged at 300 g for 5 minutes, the supernatant was discarded, washing was repeated once, buffer was added at 35 μL / well and mixed evenly, and then detected by iQue Screener flow cytometer (purchased from IntelliCyt).

[0268] The flow cytometry binding activity detection results are shown in Figure 20, and a summary of the binding characteristics is shown in Table 6. Dual Binding Domain V H In H-hFc antibodies, the double-binding domain VH The H-hFc antibody showed strong binding activity to human GPRC5D and relatively weak binding activity to mouse GPRC5D and cynomolgus monkey GPRC5D. H No binding of H-hFc antibody was detected, and the dual-binding domain V of GPRC5A, which expresses human GPRC5D and a different subtype of the same family, was bound to CHO cells. H No binding of H-hFc antibody was detected, and the double-binding domain V applied to this product H It was found that the high specificity of the H-hFc antibody can reduce the risk of various off-target side effects.

[0269] TIFF2025504803000009.tif31170

[0270] The foregoing detailed description is provided by way of interpretation and example, and is not intended to limit the scope of the appended claims. Various variations of the embodiments recited herein will be apparent to those skilled in the art, and are intended to fall within the scope of the appended claims and their equivalents.

Claims

1. A chimeric antigen receptor, a first antigen-binding domain that targets a G protein-coupled receptor class C group 5 member D (GPRC5D) protein; and a second antigen-binding domain that targets the GPRC5D protein. Chimeric antigen receptor.

2. the first antigen-binding domain and / or the second antigen-binding domain comprises an antibody or an antigen-binding fragment thereof; The chimeric antigen receptor of claim 1.

3. The antigen-binding fragment may be Fab, Fab', F(ab) ２ , Fv fragment, F(ab') ２ , scFv, di-scFv, VHH and / or dAb; The chimeric antigen receptor of claim 2.

4. the antigen-binding fragment comprises a VHH; The chimeric antigen receptor according to any one of claims 2 to 3.

5. The first antigen-binding domain comprises at least one CDR of an antibody heavy chain variable region VH, and the VH comprises an amino acid sequence represented by any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO:

30. The chimeric antigen receptor according to any one of claims 1 to 4.

6. The first antigen-binding domain comprises an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33; The chimeric antigen receptor according to any one of claims 1 to 5.

7. The first antigen-binding domain comprises an HCDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO: 27; The chimeric antigen receptor according to any one of claims 1 to 6.

8. The first antigen-binding domain is represented by SEQ ID NO: 34 (IX １ X ２ X ３ X ４ GX ５ T, but X １ is N or T, X ２ is S, W, X ３ is G, S, or X ４ D or G, X ５ N, S or T), The chimeric antigen receptor according to any one of claims 1 to 7.

9. The first antigen-binding domain comprises an HCDR2 comprising an amino acid sequence as set forth in any one of SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21, and SEQ ID NO:26; The chimeric antigen receptor according to any one of claims 1 to 8.

10. The first antigen-binding domain comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35; The chimeric antigen receptor according to any one of claims 1 to 9.

11. The first antigen-binding domain comprises an HCDR1 comprising an amino acid sequence as set forth in any one of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:20, and SEQ ID NO:25; The chimeric antigen receptor according to any one of claims 1 to 10.

12. The first antigen-binding domain comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 34 (IX １ X ２ X ３ X ４ GX ５ T, but X １ is N or T, X ２ is S, W, X ３ is G, S, or X ４ D or G, X ５ and an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33; The chimeric antigen receptor according to any one of claims 1 to 11.

13. The first antigen-binding domain comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 8, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 10; (2) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 17, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 18; (3) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 21, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 22; and (4) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27; Any set of amino acid sequences selected from the group consisting of: The chimeric antigen receptor according to any one of claims 1 to 12.

14. The first antigen-binding domain comprises an H-FR1 comprising the amino acid sequence shown in SEQ ID NO: 11, and the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1. The chimeric antigen receptor according to any one of claims 10 to 13.

15. The first antigen-binding domain comprises an H-FR2 having an amino acid sequence represented by any one of SEQ ID NO: 12, SEQ ID NO: 28, and SEQ ID NO: 23, and the H-FR2 is located between the HCDR1 and the HCDR2. The chimeric antigen receptor according to any one of claims 10 to 14.

16. The first antigen-binding domain comprises an H-FR3 comprising an amino acid sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 29, and the H-FR3 is located between the HCDR2 and the HCDR3. The chimeric antigen receptor according to any one of claims 8 to 15.

17. The first antigen-binding domain comprises an H-FR4 comprising the amino acid sequence set forth in SEQ ID NO: 14, and the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3. The chimeric antigen receptor according to any one of claims 6 to 16.

18. The first antigen-binding domain comprises a VH comprising an amino acid sequence as set forth in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 30; The chimeric antigen receptor according to any one of claims 1 to 17.

19. The first antigen-binding domain is a VHH comprising an amino acid sequence shown in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO:

30. The chimeric antigen receptor according to any one of claims 1 to 18.

20. The second antigen-binding domain comprises at least one CDR of an antibody heavy chain variable region VH, and the VH comprises an amino acid sequence represented by any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO:

30. The chimeric antigen receptor according to any one of claims 1 to 19.

21. The second antigen-binding domain comprises an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33; The chimeric antigen receptor according to any one of claims 1 to 20.

22. The second antigen-binding domain comprises an HCDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO: 27; The chimeric antigen receptor according to any one of claims 1 to 21.

23. The second antigen-binding domain is represented by SEQ ID NO: 34 (IX １ X ２ X ３ X ４ GX ５ T, but X １ is N or T, X ２ is S, W, X ３ is G, S, or X ４ D or G, X ５ N, S or T), The chimeric antigen receptor according to any one of claims 1 to 22.

24. The second antigen-binding domain comprises an HCDR2 comprising an amino acid sequence as set forth in any one of SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:21, and SEQ ID NO:26; The chimeric antigen receptor according to any one of claims 1 to 23.

25. The second antigen-binding domain comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35; The chimeric antigen receptor according to any one of claims 1 to 24.

26. The second antigen-binding domain comprises an HCDR1 comprising an amino acid sequence as set forth in any one of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:20, and SEQ ID NO:25; The chimeric antigen receptor according to any one of claims 1 to 25.

27. The second antigen-binding domain comprises an HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 35, an HDR2 comprising the amino acid sequence shown in SEQ ID NO: 34 (IX １ X ２ X ３ X ４ GX ５ T, but X １ is N or T, X ２ is S, W, X ３ is G, S, or X ４ D or G, X ５ and an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33; The chimeric antigen receptor according to any one of claims 1 to 26.

28. The second antigen-binding domain comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 8, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 10; (2) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 17, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 18; (3) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 21, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 22; and (4) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27; Any set of amino acid sequences selected from the group consisting of: The chimeric antigen receptor according to any one of claims 1 to 27.

29. The second antigen-binding domain comprises an H-FR1 comprising the amino acid sequence shown in SEQ ID NO: 11, and the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1. The chimeric antigen receptor according to any one of claims 25 to 28.

30. The second antigen-binding domain comprises an H-FR2 having an amino acid sequence represented by any one of SEQ ID NO: 12, SEQ ID NO: 28, and SEQ ID NO: 23, and the H-FR2 is located between the HCDR1 and the HCDR2. The chimeric antigen receptor according to any one of claims 25 to 29.

31. The second antigen-binding domain comprises an H-FR3 comprising the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 29, and the H-FR3 is located between the HCDR2 and the HCDR3. The chimeric antigen receptor according to any one of claims 23 to 30.

32. The second antigen-binding domain comprises an H-FR4 comprising the amino acid sequence set forth in SEQ ID NO: 14, and the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3. The chimeric antigen receptor according to any one of claims 21 to 31.

33. The second antigen-binding domain comprises a VH comprising an amino acid sequence as set forth in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 30; The chimeric antigen receptor according to any one of claims 1 to 32.

34. The second antigen-binding domain is a VHH comprising an amino acid sequence shown in any one of SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO:

30. The chimeric antigen receptor according to any one of claims 1 to 33.

35. The first antigen-binding domain and the second antigen-binding domain each comprise HCDR1, HCDR2, and HCDR3, and the sequences of HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain and the second antigen-binding domain are (1) A first antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; a second antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; (2) a first antigen-binding domain in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; a second antigen-binding domain in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; (3) A first antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; a second antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; (4) A first antigen-binding domain in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; a second antigen-binding domain in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; (5) A first antigen-binding domain in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; a second antigen-binding domain in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; (6) A first antigen-binding domain in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; a second antigen-binding domain in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; (7) A first antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; a second antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; (8) A first antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; a second antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; (9) A first antigen-binding domain in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; a second antigen-binding domain in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; (10) A first antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; a second antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; (11) A first antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; a second antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; (12) A first antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; a second antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; (13) A first antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; a second antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10; (14) A first antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; a second antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; (15) A first antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; a second antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 22; and (16) A first antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; a second antigen-binding domain, in which the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27; Any set of amino acid sequences selected from the group consisting of: The chimeric antigen receptor according to any one of claims 1 to 34.

36. The first antigen-binding domain and the second antigen-binding domain target the same epitope of GPRC5D. The chimeric antigen receptor according to any one of claims 1 to 35.

37. The first antigen-binding domain and the second antigen-binding domain comprise the same or different amino acid sequences; The chimeric antigen receptor according to any one of claims 1 to 36.

38. The first antigen-binding domain and the second antigen-binding domain are directly or indirectly linked. The chimeric antigen receptor according to any one of claims 1 to 37.

39. The first antigen-binding domain and the second antigen-binding domain are linked via a linker. The chimeric antigen receptor according to any one of claims 1 to 38.

40. The linker comprises an amino acid sequence of (GGGGS)n, where n is any positive integer from 1 to 10; The chimeric antigen receptor of claim 39.

41. The linker comprises an amino acid sequence of (EAAAK)n, where n is any positive integer from 1 to 10; The chimeric antigen receptor of claim 39.

42. a costimulatory signal region, the costimulatory signal region comprising 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 chimeric antigen receptor according to any one of claims 1 to 40.

43. The costimulatory signal region is an intracellular costimulatory signal region derived from 4-1BB.

42. The chimeric antigen receptor of claim 41.

44. The costimulatory signal region comprises the amino acid sequence set forth in SEQ ID NO:

4. The chimeric antigen receptor according to any one of claims 42 to 43.

45. an intracellular signaling domain, the intracellular signaling domain comprising 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 herpesvirus (HSKV), DAP10, DAP-12, and at least one ITAM-containing domain; The chimeric antigen receptor according to any one of claims 1 to 44.

46. The intracellular signaling domain is a signaling domain derived from CD3ζ. The chimeric antigen receptor of claim 45.

47. The intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:

5. The chimeric antigen receptor according to any one of claims 45 to 46.

48. a 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; The chimeric antigen receptor according to any one of claims 1 to 47.

49. The transmembrane domain is a transmembrane domain derived from CD8.

49. The chimeric antigen receptor of claim 48.

50. The transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:

3. The chimeric antigen receptor according to any one of claims 48 to 49.

51. The present invention further comprises a hinge region, the hinge region being 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 according to any one of claims 1 to 50.

52. The hinge region is a hinge region derived from CD8.

52. The chimeric antigen receptor of claim 51.

53. The hinge region comprises the amino acid sequence set forth in SEQ ID NO:2; The chimeric antigen receptor according to any one of claims 51 to 52.

54. Further comprising a low density lipoprotein receptor-related protein or a fragment thereof. A chimeric antigen receptor according to any one of claims 1 to 53.

55. The low density lipoprotein receptor-related protein or a fragment thereof is located at the C-terminus of the intracellular signal region.

55. The chimeric antigen receptor of claim 54.

56. The low density lipoprotein receptor-related protein or a fragment thereof includes one or more selected from the group consisting of low density lipoprotein receptor-related proteins 1 to 12 and functional fragments thereof. The chimeric antigen receptor according to any one of claims 54 to 55.

57. The low density lipoprotein receptor-related protein or a fragment thereof is low density lipoprotein receptor-related protein 5 and / or 6 or a fragment thereof; The chimeric antigen receptor according to any one of claims 54 to 56.

58. The low density lipoprotein receptor-related protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:

7. The chimeric antigen receptor according to any one of claims 54 to 57.

59. Further comprising a signal peptide, A chimeric antigen receptor according to any one of claims 1 to 58.

60. The signal peptide is derived from the signal peptide of the CD8 protein.

60. The chimeric antigen receptor of claim 59.

61. The signal peptide comprises the amino acid sequence shown in SEQ ID NO:

1. The chimeric antigen receptor according to any one of claims 59 to 60.

62. One or more isolated nucleic acid molecules, Encoding the chimeric antigen receptor according to any one of claims 1 to 61. Nucleic acid molecule.

63. Further comprising a promoter, 63. The nucleic acid molecule of claim 62.

64. The promoter is a constitutive promoter.

64. The nucleic acid molecule of claim 63.

65. The promoter is an EF1α promoter. A nucleic acid molecule according to any one of claims 63 to 64.

66. A vector comprising: The nucleic acid molecule according to any one of claims 62 to 65, vector.

67. It is a viral vector.

67. The vector of claim 66.

68. A lentiviral vector, 68. The vector according to any one of claims 66 to 67.

69. The chimeric antigen receptor according to any one of claims 1 to 61, the nucleic acid molecule according to any one of claims 62 to 65, and / or the vector according to any one of claims 66 to 68. cell.

70. Immune effector cells, 70. The cell of claim 69.

71. including 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; A cell according to any one of claims 69 to 70.

72. T cells, A cell according to any one of claims 69 to 71.

73. A method for producing the chimeric antigen receptor of any one of claims 1 to 61, comprising: The method comprises culturing the cell according to any one of claims 69 to 72 under conditions allowing expression of the chimeric antigen receptor according to any one of claims 1 to 61. method.

74. 1. A method for producing modified immune effector cells, comprising: The method comprises introducing a vector according to any one of claims 66 to 68 into an immune effector cell. method.

75. 1. A pharmaceutical composition comprising: A chimeric antigen receptor according to any one of claims 1 to 61, a nucleic acid molecule according to any one of claims 62 to 65, a vector according to any one of claims 66 to 68, and / or a cell according to any one of claims 69 to 72, and optionally a pharma- ceutically acceptable carrier. Pharmaceutical compositions.

76. 13. Use of a chimeric antigen receptor according to any one of claims 1 to 61, a nucleic acid molecule according to any one of claims 62 to 65, a vector according to any one of claims 66 to 68, a cell according to any one of claims 69 to 72 and / or a pharmaceutical composition according to claim 75 in the manufacture of a medicament for the prevention, treatment and / or amelioration of a disease and / or a pathology.

77. The disease and / or condition includes a disease and / or condition associated with abnormal expression of GPRC5D; 77. The use according to claim 76.

78. The disease and / or condition associated with the abnormal expression of GPRC5D includes tumors.

78. The use according to claim 77.

79. The tumor comprises a solid tumor.

79. The use according to claim 78.

80. The tumor comprises a non-solid tumor.

79. The use according to claim 78.

81. The tumor comprises a hematological tumor and / or a lymphoma; Use according to any one of claims 79 to 80.

82. The tumor comprises a myeloma.

82. Use according to any one of claims 78 to 81.

83. The myeloma includes refractory / relapsed multiple myeloma; 83. The use according to claim 82.

84. 1. A method for preventing, treating and / or ameliorating a disease and / or condition, comprising: Administering to a subject in need thereof a cell according to any one of claims 69 to 72 and / or a pharmaceutical composition according to claim 75. method.

85. The disease and / or condition includes a disease and / or condition associated with abnormal expression of GPRC5D; 85. The method of claim 84.

86. The disease or condition associated with abnormal expression of GPRC5D includes tumors.

86. The method of claim 85.

87. The tumor comprises a solid tumor.

87. The method of claim 86.

88. The tumor comprises a non-solid tumor.

87. The method of claim 86.

89. The tumor comprises a hematological tumor and / or a lymphoma; 89. The method according to any one of claims 87 to 88.

90. The tumor comprises a myeloma.

90. The method according to any one of claims 87 to 89.

91. The myeloma includes refractory / relapsed multiple myeloma; 91. The method of claim 90.

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