GPRC5D ANTIBODIES AND USES THEREOF

Abstract

The present application relates to antibodies that bind to GPRC5D and uses thereof, as well as cells, pharmaceutical compositions, and drug combinations that contain the antibodies that bind to GPRC5D according to the present application, and methods for preparing the antibodies.

Description

Related Applications

[0001] This patent application claims priority to Chinese patent application No. 202210111504.5 filed on January 29, 2022, and claims priority to Chinese patent application No. 202210985594.0 filed on August 17, 2022. Concurrently submitted sequence listing file

[0002] The entire contents of the following XML file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Format (CRF) (Name: FF00719PCT-sequence listing.xml, Date: 20230122, Size: 95.6KB). [Technical field]

[0003] The present application relates to the field of tumor immunotherapy or diagnosis, and more particularly to antibodies that specifically bind to GPRC5D and uses thereof. [Background technology]

[0004] Currently, clinical targeted therapy for multiple myeloma mainly includes immunomodulators (thalidomide, lenalidomide, pomalidomide), proteasome inhibitors (bortezomib, carfilzomib, isazomib), and anti-CD38 monoclonal antibodies. Although up-front therapy has shown good results, the efficacy of final treatment is very limited. CAR-T cell therapy targeting BCMA has shown good efficacy in clinical trials, but there is an urgent need to develop new treatments for cases with low BCMA expression or cases that have relapsed after BCMA CAR-T treatment. Studies have shown that 65% of multiple myeloma patients express GPRC5D, so the clinical value of developing a therapeutic pathway targeting GPRC5D is remarkable. GPRC5D is a relatively new target and functions as a multi-transmembrane receptor, so there are certain technical barriers to the development of its antibodies. Therefore, the GPRC5D antibody developed in the present application, in particular the fully human GPRC5D antibody, has high application value. DISCLOSURE OF THEINVENTION

[0005] [Summary of the Invention] The present application aims to provide an antibody that specifically recognizes GPRC5D. The present application also relates to a method for preparing an anti-GPRC5D specific antibody, including phage display technology and hybridoma technology. The present application also relates to a study of the properties and specificity of anti-GPRC5D antibodies (including but not limited to Fab, scFv, scFv-Fc forms). The present application also provides a chimeric antigen receptor (CAR) targeting GPRC5D and a method for preparing the same. The present application also provides an isolated nucleic acid encoding the anti-GPRC5D antibody and the chimeric antigen receptor targeting GPRC5D of the present application. The present application also provides a host cell comprising the nucleic acid of the present application. The method further includes culturing the host cell of the present application to prepare the antibody or the CAR. The antibody and / or CAR of the present application are used for tumor treatment or tumor diagnosis.

[0006] In a first aspect, the present application provides an antibody that recognizes GPRC5D, the antibody being selected from any one of the following groups: (1) An antibody comprising a heavy chain variable region comprising HCDR1 set forth in any one of SEQ ID NOs: 1, 11, 17, 70, or 71, and / or HCDR2 set forth in any one of SEQ ID NOs: 2, 12, 18, 72, or 73, and / or HCDR3 set forth in any one of SEQ ID NOs: 3, 7, 9, 13, or 19; (2) an antibody comprising a light chain variable region comprising an LCDR1 set forth in any of SEQ ID NOs: 4, 14, 20, or 74, and / or an LCDR2 set forth in any of SEQ ID NOs: 5, 15, 21, or 75, and / or an LCDR3 set forth in any of SEQ ID NOs: 6, 8, 10, 16, or 22; (3) An antibody comprising the heavy chain variable region of the antibody according to (1) and the light chain variable region of the antibody according to (2); (4) An antibody which is a mutant of the antibody according to any one of (1) to (3), and has the same or similar activity as the antibody according to any one of (1) to (3). is selected from.

[0007] In certain embodiments, the antibody comprises at least one CDR of a heavy chain variable region comprising an amino acid sequence set forth in any of SEQ ID NOs: 23, 27, 31, 35, 39, 76 or 80, or a variant thereof, or an amino acid sequence that has at least 80% identity to any one of the aforementioned sequences; and / or at least one CDR of a light chain variable region comprising an amino acid sequence set forth in any of SEQ ID NOs: 25, 29, 33, 37, 41 or 78, or a variant thereof, or an amino acid sequence that has at least 80% identity to any one of the aforementioned sequences.

[0008] In certain embodiments, the antibody comprises HCDR1, HCDR2, HCDR3 of a heavy chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, 35, 39, 76 or 80 or a variant thereof, or an amino acid sequence having at least 80% identity with any one of the said sequences; and / or LCDR1, LCDR2, LCDR3 of a light chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, 41 or 78 or a variant thereof, or an amino acid sequence having at least 80% identity with any one of the said sequences.

[0009] In certain embodiments, the CDR regions of the heavy chain variable region and / or the CDR regions of the light chain variable region of the antibody are selected from any one of the following groups: (1) an antibody comprising an HCDR1 as set forth in SEQ ID NO:1, an HCDR2 as set forth in SEQ ID NO:2, and an HCDR3 as set forth in SEQ ID NO:3; an LCDR1 as set forth in SEQ ID NO:4, an LCDR2 as set forth in SEQ ID NO:5, and an LCDR3 as set forth in SEQ ID NO:6; or (2) an antibody comprising an HCDR1 as set forth in SEQ ID NO:1, an HCDR2 as set forth in SEQ ID NO:2, and an HCDR3 as set forth in SEQ ID NO:7; an LCDR1 as set forth in SEQ ID NO:4, an LCDR2 as set forth in SEQ ID NO:5, and an LCDR3 as set forth in SEQ ID NO:8; or (3) an antibody comprising an HCDR1 as set forth in SEQ ID NO:1, an HCDR2 as set forth in SEQ ID NO:2, and an HCDR3 as set forth in SEQ ID NO:9; an LCDR1 as set forth in SEQ ID NO:4, an LCDR2 as set forth in SEQ ID NO:5, and an LCDR3 as set forth in SEQ ID NO:10; or (4) an antibody comprising an HCDR1 as set forth in SEQ ID NO: 11, an HCDR2 as set forth in SEQ ID NO: 12, and an HCDR3 as set forth in SEQ ID NO: 13; an LCDR1 as set forth in SEQ ID NO: 14, an LCDR2 as set forth in SEQ ID NO: 15, and an LCDR3 as set forth in SEQ ID NO: 16; or (5) an antibody comprising HCDR1 as set forth in SEQ ID NO: 17, HCDR2 as set forth in SEQ ID NO: 18, and HCDR3 as set forth in SEQ ID NO: 19; LCDR1 as set forth in SEQ ID NO: 20, LCDR2 as set forth in SEQ ID NO: 21, and LCDR3 as set forth in SEQ ID NO: 22; or (6) An antibody comprising an HCDR1 as set forth in SEQ ID NO: 70, an HCDR2 as set forth in SEQ ID NO: 72, and an HCDR3 as set forth in SEQ ID NO: 3; an LCDR1 as set forth in SEQ ID NO: 4, an LCDR2 as set forth in SEQ ID NO: 5, and an LCDR3 as set forth in SEQ ID NO: 6; or (7) An antibody comprising an HCDR1 as set forth in SEQ ID NO: 71, an HCDR2 as set forth in SEQ ID NO: 73, and an HCDR3 as set forth in SEQ ID NO: 3; an LCDR1 as set forth in SEQ ID NO: 4, an LCDR2 as set forth in SEQ ID NO: 5, and an LCDR3 as set forth in SEQ ID NO: 6; or (8) An antibody comprising an HCDR1 set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 2, and an HCDR3 set forth in SEQ ID NO: 3; an LCDR1 set forth in SEQ ID NO: 74, an LCDR2 set forth in SEQ ID NO: 75, and an LCDR3 set forth in SEQ ID NO: 6; (9) An antibody which is a mutant of the antibody according to any one of (1) to (8) and has the same or similar activity as the antibody according to any one of (1) to (8). or a variant thereof.

[0010] In some embodiments, the antibody is one of the following groups: (1) an antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:23 or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:23, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:25 or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:25; (2) an antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:27 or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:27, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:29 or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:29; (3) an antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:31, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:31, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:33, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:33; (4) an antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:35, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:35, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:37, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:37; (5) an antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:39, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:39, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:41, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:41; (6) An antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:76, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:76, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:25, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:25; (7) An antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:80 or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:80, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:25 or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:25; (8) An antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:23, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:23, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:78, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:78; (9) An antibody which is a mutant of the antibody according to any one of (1) to (8) and has the same or similar activity as the antibody according to any one of (1) to (8). or a variant thereof.

[0011] In certain embodiments, the antibody is selected from a whole antibody, an scFv, a single domain antibody, a Fab fragment, a Fab' fragment, an Fv fragment, an F(ab')2 fragment, an Fd fragment, a dAb fragment, a multifunctional antibody or an scFv-Fc antibody, a hybridoma antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a monoclonal antibody.

[0012] In certain embodiments, the antibody is (1) an antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:35, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:35, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:37, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:37; (2) an antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:39, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:39, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:41, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:41; (3) An antibody which is a mutant of the antibody according to any one of (1) to (2) and has the same or similar activity as the antibody according to any one of (1) to (2). The hybridoma antibody is selected from the group consisting of:

[0013] In certain embodiments, the antibody is (1) an antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:23 or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:23, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:25 or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:25; (2) an antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:27 or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:27, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:29 or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:29; (3) an antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:31, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:31, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:33, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:33; (4) an antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:76 or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:76, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:25 or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:25; (5) an antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:80 or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:80, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:25 or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:25; (6) An antibody wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:23, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:23, and the light chain variable region has an amino acid sequence set forth in SEQ ID NO:78, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:78; (7) An antibody which is a mutant of the antibody according to any one of (1) to (6) and has the same or similar activity as the antibody according to any one of (1) to (6). The fully human antibody is selected from the group consisting of:

[0014] In certain embodiments, the antibody is (1) an antibody wherein the heavy chain has an amino acid sequence set forth in SEQ ID NO:45, or an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:45, and the light chain has an amino acid sequence set forth in SEQ ID NO:47, or an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:47; (2) an antibody wherein the heavy chain has an amino acid sequence set forth in SEQ ID NO:49, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:49, and the light chain has an amino acid sequence set forth in SEQ ID NO:51, or comprises an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:51; (3) an antibody wherein the heavy chain has an amino acid sequence set forth in SEQ ID NO:53, or an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:53, and the light chain has an amino acid sequence set forth in SEQ ID NO:55, or an amino acid sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:55; (4) An antibody which is a mutant of the antibody according to any one of (1) to (3), and has the same or similar activity as the antibody according to any one of (1) to (3). is selected from.

[0015] In certain embodiments, the antibody according to any one of the first aspects is a hybridoma antibody, a chimeric antibody, a humanized antibody, or a fully human antibody, or said antibody is a monoclonal antibody, or said antibody is a whole antibody, an scFv, a single domain antibody, a Fab fragment, a Fab' fragment, an Fv fragment, an F(ab')2 fragment, an Fd fragment, a dAb fragment, a multifunctional antibody, or an scFv-Fc antibody.

[0016] In certain embodiments, the antibody of any of the first aspects comprises: (1) An antibody having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 35 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 37; (2) An antibody having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 39 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 41; (3) An antibody which is a mutant of the antibody according to any one of (1) to (2) and has the same or similar activity as the antibody according to any one of (1) to (2). The hybridoma antibody is selected from the group consisting of:

[0017] In certain embodiments, the antibody of any of the first aspects comprises: (1) An antibody having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:23 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:25; (2) An antibody having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:27 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:29; (3) An antibody having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 33; (4) An antibody which is a mutant of the antibody according to any one of (1) to (3), and has the same or similar activity as the antibody according to any one of (1) to (3). The fully human antibody is selected from the group consisting of:

[0018] In certain embodiments, the antibody of any of the first aspects comprises: (1) An antibody having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:45 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:47; (2) An antibody having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 49 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 51; (3) An antibody having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 55; (4) An antibody which is a mutant of the antibody according to any one of (1) to (3), and has the same or similar activity as the antibody according to any one of (1) to (3). The fully human antibody is selected from the group consisting of:

[0019] In a second aspect, the present application provides an immunoconjugate comprising an antibody according to any of the first aspects and a functional molecule linked thereto.

[0020] In a third aspect, the present application provides a chimeric receptor, wherein the extracellular domain of said chimeric receptor comprises an antibody according to any of the first aspects, and wherein said chimeric receptor comprises a chimeric antigen receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC), or a combination thereof.

[0021] In a particular embodiment, the chimeric receptor is a chimeric antigen receptor (CAR).

[0022] In certain embodiments, the CAR comprises an antibody according to any of the first aspects, a transmembrane domain, and an intracellular signaling domain.

[0023] In certain embodiments, the chimeric receptor comprises an antibody according to any of the first aspects, a transmembrane domain, and an intracellular signaling domain linked in sequence.

[0024] In a specific embodiment, the intracellular signaling region is selected from the intracellular signaling region sequences of CD3ζ, FcεRIγ, CD27, CD28, CD137, CD134, MyD88, CD40, or a combination thereof, and / or the transmembrane region comprises the transmembrane region of CD8 or CD28.

[0025] In a specific embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:60.

[0026] In a specific embodiment, the CD137 intracellular signaling region comprises the amino acid sequence set forth in SEQ ID NO:61.

[0027] In a specific embodiment, the CD3 zeta intracellular signaling region comprises the amino acid sequence set forth in SEQ ID NO:62.

[0028] In certain embodiments, the chimeric receptor comprises an antibody according to any of the first aspect, a transmembrane region of CD8 / CD28, and CD3ζ; or an antibody according to any of the first aspect, a transmembrane region of CD8 / CD28, an intracellular signalling region of CD137, and CD3ζ; or an antibody according to any of the first aspect, a transmembrane region of CD8 / CD28, an intracellular signalling region of CD28, and CD3ζ; or an antibody according to any of the first aspect, a transmembrane region of CD8 / CD28, an intracellular signalling region of CD28, CD137 and CD3ζ.

[0029] In certain embodiments, the chimeric receptor further comprises a hinge region.

[0030] In a specific embodiment, the hinge region comprises a CD8 hinge region.

[0031] In a specific embodiment, the CD8 hinge region comprises the amino acid sequence set forth in SEQ ID NO:59.

[0032] In certain embodiments, the chimeric receptor further comprises a signal peptide.

[0033] In a specific embodiment, the signal peptide comprises a CD8 signal peptide.

[0034] In a specific embodiment, the CD8 signal peptide comprises the amino acid sequence set forth in SEQ ID NO:58.

[0035] In certain embodiments, the chimeric receptor comprises the amino acid sequence set forth in any one of SEQ ID NOs: 43, 82, 84, or 86.

[0036] In certain embodiments, the amino acid sequence of the chimeric receptor is set forth in any one of SEQ ID NOs: 63, 88, 89, or 90.

[0037] In a fourth aspect, the application provides a nucleic acid encoding an antibody according to any of the first aspect, an immunoconjugate according to the second aspect, or a chimeric receptor according to the third aspect.

[0038] In certain embodiments, the nucleic acid comprises a sequence set forth in any of SEQ ID NOs: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 77, 79, or 81, or a combination thereof.

[0039] In certain embodiments, the nucleic acid comprises the sequence set forth in any of SEQ ID NOs: 46, 48, 50, 52, 54, or 56, or a combination thereof.

[0040] In certain embodiments, the nucleic acid comprises the sequence set forth in any of SEQ ID NOs: 44, 83, 85, or 87.

[0041] In a fifth aspect, the present application provides a vector comprising a nucleic acid according to the fourth aspect.

[0042] In a sixth aspect, the present application provides a virus comprising an expression vector according to the fifth aspect.

[0043] In a seventh aspect, the present application provides a composition comprising an immunoconjugate according to the second aspect, and / or a chimeric receptor according to the third aspect, wherein the composition is cytotoxic to cells expressing GPRC5D.

[0044] In certain embodiments, the cells expressing GPRC5D are tumor cells and / or pathogen cells.

[0045] In an eighth aspect, the present application provides a cell comprising an antibody according to the first aspect, an immunoconjugate according to the second aspect, a chimeric receptor according to the third aspect, a nucleic acid according to the fourth aspect, and / or a vector according to the fifth aspect.

[0046] In certain embodiments, the cell is a host cell comprising a vector according to the fifth aspect or having a nucleic acid according to the fourth aspect integrated into its genome.

[0047] In a particular embodiment, the cell / host cell expresses a chimeric receptor according to the third aspect.

[0048] In certain embodiments, the cell / host cell is a T cell, a natural killer cell, a natural killer T cell, an NK92 cell, a stem cell derived immune effector cell, or a combination thereof.

[0049] In certain embodiments, the T cells comprise cytotoxic T lymphocytes, DNT cells, and / or regulatory T cells.

[0050] In certain embodiments, the T cells comprise natural T cells and / or T cells induced by pluripotent stem cells.

[0051] In a specific embodiment, said T cells are autologous / allogeneic T cells.

[0052] In a specific embodiment, the T cells are primary T cells.

[0053] In a specific embodiment, the T cells comprise autologous human T cells.

[0054] In certain embodiments, the T cells comprise stem cell-like memory T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Treg), effector memory T cells (Tem), γδ T cells, or a combination thereof.

[0055] In certain embodiments, the host cells bind to cells that express GPRC5D, but do not significantly bind to cells that do not express GPRC5D.

[0056] In certain embodiments, the host cell further comprises a coding sequence for an exogenous cytokine, and / or expresses a chimeric receptor that does not target GPRC5D, and / or expresses a chemokine receptor, and / or expresses a safety switch.

[0057] In certain embodiments, the host cell also comprises a coding sequence for an exogenous cytokine, including IL-7, IL-12, IL-15, IL-18, IL-21, or a type I interferon.

[0058] In certain embodiments, the host cells also express chemokines, including CCL19 or CCL21.

[0059] In certain embodiments, the host cell also expresses a chemokine receptor, including CCR2, CCR4, CCR5, CXCR2, CXCR4, or CXCR5.

[0060] In certain embodiments, the host cell further expresses a safety switch comprising iCaspase-9, Truncated EGFR, or RQR8.

[0061] In a ninth aspect, the present application provides a pharmaceutical composition comprising an antibody according to any of the first aspect, an immunoconjugate according to the second aspect, a chimeric receptor according to the third aspect, a nucleic acid according to the fourth aspect, a vector according to the fifth aspect, a virus according to the sixth aspect, a composition according to the seventh aspect and / or a cell according to the eighth aspect, and a pharma- ceutically acceptable adjuvant.

[0062] In a tenth aspect, the present application provides a pharmaceutical combination, in which an antibody according to any of the first aspect, an immunoconjugate according to the second aspect, a chimeric receptor according to the third aspect, a composition according to the seventh aspect, a cell according to the eighth aspect, a pharmaceutical composition according to the ninth aspect is administered in combination with an agent that enhances their function, preferably in combination with a chemotherapeutic agent, and / or in combination with an agent that ameliorates one or more side effects associated therewith, and / or in combination with a host cell expressing a chimeric antigen receptor that targets other than GPRC5D.

[0063] In an eleventh aspect, the present application provides a method for preparing an antibody according to any of the first aspect, an immunoconjugate according to the second aspect, a chimeric receptor according to the third aspect, a composition according to the seventh aspect, a pharmaceutical composition according to the ninth aspect, said method comprising culturing a host cell according to the eighth aspect under conditions suitable for expression of said antibody, immunoconjugate, chimeric receptor, composition and / or pharmaceutical composition expressed by said host cell.

[0064] In a twelfth aspect, the present application provides a kit comprising an antibody according to any of the first aspect, an immunoconjugate according to the second aspect, a chimeric receptor according to the third aspect, a nucleic acid according to the fourth aspect, a vector according to the fifth aspect, a virus according to the sixth aspect, a composition according to the seventh aspect, a cell according to the eighth aspect, and / or a pharmaceutical composition according to the ninth aspect.

[0065] In certain embodiments, the kit comprises: a container, and a pharmaceutical composition according to the ninth aspect disposed within the container; or a container; and disposed within the container an antibody according to any of the first aspect or a nucleic acid encoding the antibody; or an immunoconjugate according to the second aspect or a nucleic acid encoding the immunoconjugate; or a chimeric receptor according to the third aspect or a nucleic acid encoding the chimeric receptor; or a host cell according to the eighth aspect.

[0066] In a thirteenth aspect, the present application provides a method of treating / diagnosing a disease comprising administering to a subject in need thereof an effective amount of an antibody according to any of the first aspect, or an immunoconjugate according to the second aspect, or a cell according to the eighth aspect, or a pharmaceutical composition according to the ninth aspect, or a kit according to the twelfth aspect.

[0067] In certain embodiments, the disease is selected from an inflammatory disease, an infectious disease, an autoimmune disease, and a tumor; In certain embodiments, the subject is a human, In certain embodiments, the host cells are autologous or allogeneic T cells to the subject.

[0068] In a 14th aspect, the present application provides the use of an antibody according to any of the first aspect, or an immunoconjugate according to the second aspect, or a host cell according to the eighth aspect, or a pharmaceutical composition according to the ninth aspect, or a kit according to the 12th aspect, in the preparation of a medicament for treating / diagnosing a disease, wherein said disease comprises expression of GPRC5D, preferably said disease is selected from inflammatory diseases, infectious diseases, autoimmune diseases and tumors.

[0069] It should be understood that within the scope of this application, each of the above-mentioned technical features of this application and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions, which will not be described one by one here due to space limitations. [Brief description of the drawings]

[0070] [Figure 1] FIG. 1 shows the EC50 of hybridoma antibodies AB1 and AB2 binding to CHOK1-huGPRC5D cells. [Diagram 2] FIG. 1 shows that both hybridoma antibodies AB1 and AB2 significantly bind to human GPRC5D and mouse GPRC5D. [Diagram 3]FIG. 1 shows that recombinant hybridoma antibodies AB1 (scFv-mFc) and AB2 (scFv-mFc) specifically bind to cell lines expressing human GPRC5D. [Figure 4] FIG. 1 shows that antibodies AB3, AB4, and AB5 all specifically bind to human GPRC5D. [Diagram 5] FIG. 1 shows that antibodies AB3, AB4, and AB5 all specifically bind to 293T cells overexpressing human GPRC5D. [Figure 6] FIG. 1 shows the EC50 of antibodies AB3, AB4, and AB5 binding to human GPRC5D. [Figure 7] FIG. 1 shows that antibodies AB3, AB4, and AB5 all significantly bind to cells expressing human GPRC5D (CHOK1-huGPRC5D, 293T-huGPRC5D, MM.1S, and NCI-H929), but not to cells that do not express GPRC5D (CHOK1, 293T, and Daudi). [Figure 8] FIG. 1 shows the EC50 of antibodies AB3, AB4, and AB5 binding to CHOK1-huGPRC5D cells. [Figure 9] FIG. 1 shows the EC50 of antibodies AB3, AB4, and AB5 binding to MM.1S cells. [Figure 10] FIG. 1 shows binding of antibody AB3 to CHOK1-muGPRC5D cells. [Figure 11] Figure 1 shows the in vitro killing effect of AB3-BBZ CAR T cells on target cells. [Figure 12] Figure 1 shows the effect of AB3-BBZ CAR T cells and 18-BBZ CAR T cells in inhibiting tumor growth in tumor-bearing mice. [Figure 13] Figure 1 shows the body weight changes of tumor-bearing mice treated with AB3-BBZ CAR T cells and 18-BBZ CAR T cells. [Figure 14] FIG. 1 shows the binding results of antibodies AB6, AB7 and AB8 to human GPRC5D. [Figure 15]FIG. 1 shows the detection of antibodies AB3, AB6, AB7 and AB8 binding to GPRC5D cell lines of different species. [Figure 16] FIG. 1 shows the EC50 detection results of antibodies AB3, AB6, AB7 and AB8 binding to 293T-huGPRC5D cells and MM.1S cells. [Figure 17] FIG. 1 shows the results of affinity detection between antibodies AB3, AB7 and Bio-GPRC5D. [Figure 18] Figure 1 shows the in vitro killing effect of AB3-BBZ, AB6-BBZ, AB7-BBZ, and AB8-BBZ CAR T cells on target cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] After intensive research and screening, the applicant of the present application has obtained a hybridoma antibody and a fully human antibody that specifically recognizes GPRC5D. The antibody of the present application can be used to prepare targeted anti-tumor agents and agents for diagnosing tumors.

[0072] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of gene therapy, biochemistry, genetics and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of this application, and suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. Furthermore, unless otherwise specified, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0073] Unless otherwise indicated, the practice of the present application will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, all of which are within the skill of the art and are fully described in the literature. For example, Current Protocols in Molecular Biology (FrederickM.AUSUBEL,2000,Wileyand sonInc,Library of Congress,USA);Molecular Cloning: A Laboratory Manual,Third Edition,(Sambrooketal,2001,Cold Spring Harbor,NewYork: Cold Spring Harbor Laboratory Press);Oligonucleotide Synthesis (MJGaited.,1984);Mullis et al. al.USPat.No.4,683,195;Nucleic Acid Hybridization (BDHarries & SJHigginseds.1984);Transcription And Translation (BDHames & SJHigginseds.1984);Culture Of Animal Cells (RIFreshney,Alan R.Liss,Inc.,1987);Immobilized Cells And Enzymes (IRL Press,1986);B.Perbal,A Practical Guide To Molecular Cloning (1984); the series,Methods In ENZYMOLOGY (J. Abelson & M. Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols. 154 and 155 (Wuetal. eds.) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JHMiller and MPCaloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer & Walker, eds., Academic Press, London, 1987); Hand book Of Experimental Immunology, Vols. I-IV (DM Weir & CC Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0074] In this disclosure, various aspects of the subject matter for which protection is sought are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the subject matter for which protection is sought. Thus, the description of a range should be considered to specifically disclose all possible subranges and individual values ​​within such range. For example, when a range of values ​​is provided, it is understood that all values ​​between the upper and lower limits of the range, as well as other stated or intermediate values ​​within the stated range, are included within the subject matter for which protection is sought, and that the upper and lower limits of the stated range also belong to the scope of the subject matter for which protection is sought. The upper and lower limits of such smaller ranges are independently included within the stated smaller ranges, and are included within the scope of the subject matter for which protection is sought, unless the upper and lower limits of the stated ranges are expressly excluded. When a stated range includes one or two limit values, the subject matter for which protection is sought also includes the range excluding the one or two ranges of said limit values. This applies regardless of the breadth of the range.

[0075] The term "about" refers to the normal error range of each value, which is readily known to those of ordinary skill in the art. Reference to "about" a value or parameter herein includes (and describes) an embodiment that is directed to the value or parameter itself. For example, a statement about "about X" also includes a statement about "X". For example, "about" or "including" may mean within 1 or more than 1, based on the practical standard deviation in the field. Alternatively, "about" or "including" may mean a range of up to 10% (i.e., ±10%). For example, about 5 μM may include any number between 4.5 μM and 5.5 μM. When a particular value or composition is provided in an application or patent application, unless otherwise specified, "about" or "including" shall be considered to be within the acceptable error range of that particular value or composition.

[0076] Unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range described herein should be understood to include any integer within the described range, and fractions thereof, where appropriate (e.g., tenths and hundredths of integers).

[0077] To facilitate understanding of this application, some terms are first defined.

[0078] The term "GPRC5D" refers to group 5 of the G protein-coupled receptor family C, and is one of the human GPCR proteins newly discovered by homology search of the EST database using a series of amino acid sequences of human GPCRs. This protein is registered under Genbank accession numbers: AF209923, NM_018654, and NP_0611124. GPRC5D is an orphan receptor with no known ligand or function in humans and human cancers. For example, the full-length amino acid sequence of human GPRC5D is shown in SEQ ID NO: 65, and the full-length amino acid sequence of mouse GPRC5D is shown in SEQ ID NO: 66.

[0079] The terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably and refer to polymers of amino acids of any length. The polymers may be linear, cyclic or branched, may contain modified amino acids, particularly conservatively modified amino acids, and may be interrupted by non-amino acids. The term also includes modified amino acid polymers, such as those modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, isoprenylation, racemization, selenoacylation, transfer-RNA mediated amino additions, such as arginylation and ubiquitination, or other manipulations, such as conjugation with a labeling component. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics. A polypeptide or amino acid sequence "derived from" a particular protein refers to the source of the polypeptide. The term also includes polypeptides expressed from a particular nucleic acid sequence.

[0080] The term "antibody" is used herein in the broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the requisite antigen-binding activity.

[0081] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains, including Fab' and Fab'-SH, (ii) an Fd fragment consisting of the VH and CH1 domains, (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a single variable region (Ward et al., 1989, Nature 341:544-546); (v) an F(ab')2 fragment, which contains a bivalent fragment containing two linked Fab fragments; (vi) an antigen-binding site of a single chain Fv molecule; (vii) a bispecific single chain Fv dimer (PCT / US92 / 09965); (viii) "dibodies" or "tribodies", multivalent or multispecific fragments constructed by genetic fusion, and (ix) scFv genetically fused to the same or different antibodies.

[0082] The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain has. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (allotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different types of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0083] The term "variable region or variable domain" refers to the domain of an antibody heavy or light chain involved in antibody antigen binding. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved FRs and three CDRs. (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman & Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen can be isolated by using the VH or VL domain of an antibody that binds to that antigen to screen a library of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0084] The term "hypervariable region" or "complementarity determining region" or "CDR" refers to each region in an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops") and / or contains residues that contact the antigen ("antigen contact points"). In certain embodiments, the CDRs may be determined by a numbering system selected from Kabat, Chothia, IMGT, Gelfand, Aho, and Martin. For example, they may be determined through the Kabat numbering system. For example, an antibody may contain six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3).

[0085] The term "Fc region" or "Fc" is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions.

[0086] "Framework (FR)" refers to variable domain residues different from hypervariable region (CDR) residues. The FR of a variable domain is usually composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, in a VH (or VL), the CDR and FR sequences are usually displayed in the following order: FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

[0087] Unless otherwise indicated, CDR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0088] The term "natural antibody" refers to naturally occurring immunoglobulin molecules with various structures. For example, natural IgG antibodies are heterotetrameric proteins of about 150,000 daltons composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a light chain constant (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ).

[0089] The terms "whole antibody," "full length antibody," and "intact antibody" are used interchangeably and include complete, full-length antibodies having a structure substantially similar to that of a native antibody, having a heavy chain containing an Fc region as defined herein, or having an antigen-binding region.

[0090] The term "single domain antibody (sdAb)" refers to a type of antibody that lacks the light chain and has only the variable region of the heavy chain, and is also called a nanobody due to its small molecular weight.

[0091] The term "single domain antibody" refers to an antibody that comprises all or a portion of a heavy chain variable domain, or all or a portion of a light chain variable domain. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).

[0092] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the population includes individual antibodies that are identical and / or bind to the same epitope, excluding variant antibodies that, for example, include naturally occurring mutations or that may arise during the preparation of a monoclonal antibody preparation, such variants usually being present in small amounts. In contrast to polyclonal antibody preparations, which usually include different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the designation "monoclonal" indicates the nature of the antibody being obtained from a substantially homogeneous antibody population and is not to be considered as requiring that the antibody be prepared by any particular method. For example, they can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals that contain all or part of the human immunoglobulin loci.

[0093] By way of illustration, the monoclonal antibodies of the present application can be produced by the hybridoma method, which can be formed by isolating stimulated immune cells, such as immune cells from the spleen of a vaccinated animal. These cells, such as myeloma cells or transformed cells, can be fused with immortalized cells that can replicate indefinitely in cell culture, thereby generating immortal immunoglobulin-secreting cell lines. The immortal cell line used is selected (whether it lacks an enzyme required for the utilization of a particular nutrient). Many such cell lines (such as myelomas) are known to those skilled in the art, for example, thymidine kinase (TK) or hypoxanthine-guanine phosphoribosyltransferase (HGPRT). These defects make it possible to select fused cells based on their ability to grow, for example, on hypoxanthine aminopterin thymidine medium (HAT).

[0094] The term "chimeric antibody" refers to an antibody in which a portion of the antibody's heavy and / or light chain is derived from a particular source or species, and the remaining portion of the heavy and / or light chain is derived from a different source or species. In certain embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further embodiments, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof. In certain embodiments, a chimeric antibody is a "humanized antibody."

[0095] The term "humanized" is used for non-human antibodies, such as rodent or primate, that are hybrid immunoglobulins, immunoglobulin chains, or fragments thereof that contain minimal sequence derived from a non-human immunoglobulin. A "humanized antibody" refers to a chimeric antibody that contains amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody contains substantially all of at least one (and typically two) variable domains, with all or substantially all of the CDRs corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding to those of a human antibody. A humanized antibody can optionally contain at least a portion of an antibody constant region derived from a human antibody.

[0096] In some embodiments, a "humanized antibody" may contain mutations such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo.

[0097] The term "fully human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes a human antibody library or other human antibody coding sequence. The definition of a fully human antibody specifically excludes humanized antibodies, which comprise non-human antigen-binding residues. In certain embodiments, the antibodies provided herein are "fully human antibodies" generated by phage display technology.

[0098] Antibodies of the present application can be isolated by screening combinatorial libraries of antibodies with the desired activity. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. For such methods, see, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al. ed., Human Press, Totowa, NJ, 2001), and further see, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0099] In some phage display methods, VH and VL gene libraries can be cloned separately by polymerase chain reaction (PCR), randomly recombined into phage libraries, and then screened for antigen-binding phages, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide antibodies with high affinity to immunogens without constructing hybridomas. Alternatively, naive libraries (e.g., from humans) can be cloned to provide a single source of antibodies against multiple non-self and self antigens without the need for immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, natural libraries can also be prepared synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions to achieve rearrangement in vitro, as described in Hoogenboom, J. Mol. Biol. 227:381-388 (1992).

[0100] As used herein, antibodies or antibody fragments isolated from a fully human antibody library are considered to be fully human antibodies or fully human antibody fragments.

[0101] In certain embodiments, amino acid sequence variants of the antibodies are provided herein. The term "parent antibody" refers to an antibody provided herein, or an antibody obtained by processes such as mutation, affinity maturation, etc. based on an antibody provided herein. The parent antibody may be a naturally occurring antibody, or a variant or engineered version of a naturally occurring antibody. The parent antibody may refer to the antibody itself, a composition comprising the parent antibody, or its encoded amino acid sequence.

[0102] The term "affinity matured" antibody refers to an antibody that contains one or more changes in one or more hypervariable regions (CDRs) compared to a parent antibody, which changes increase the affinity of the antibody for antigen.

[0103] The term "variant" refers to a polypeptide having one or more activities encoded by substantially the same amino acid sequence or substantially the same nucleotide sequence as the sequence of an antibody provided herein. The variant has the same or similar activity as the antibody provided in the examples of the present application. For example, the variant may be a variant antibody or antibody variant based on the amino acid sequence of the antibody provided herein.

[0104] The term "variant antibody" or "antibody variant" includes antibody sequences that differ from the parent antibody sequence by at least one amino acid modification compared to the parent. A variant antibody sequence herein preferably has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity with the parent antibody sequence. An antibody variant may refer to the antibody itself or a composition comprising an antibody variant. An amino acid sequence variant of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding said antibody or by peptide synthesis. The term "amino acid modification" includes amino acid substitution, addition and / or deletion. An "amino acid substitution" or "amino acid replacement" refers to replacing an amino acid at a particular position in a parent polypeptide sequence with another amino acid. An "amino acid insertion" refers to adding an amino acid at a particular position in a parent polypeptide sequence. "Amino acid deletion" refers to the removal of an amino acid at a particular position in a parent polypeptide sequence. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, so long as the final construct possesses the desired properties, such as binding to an antigen.

[0105] The term "modification" refers to a change in the state or structure of a protein or polypeptide of the present application. Modifications may be chemical, structural, or functional.

[0106] The term "conservative modification" or "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding characteristics of an antibody containing the recited amino acid sequence. Such conservative modifications include amino acid substitutions, insertions, and deletions. Modifications can be introduced into the antibodies of the present application by standard techniques known in the art, such as site-directed mutagenesis, PCR-mediated mutagenesis, and the like. Families of amino acid residues having similar side chains have been defined in the art, as shown in Table 1.

[0107] [Table 1]

[0108] Thus, one or more amino acid residues in the CDR or framework regions of the antibodies of the present application can be replaced with other amino acid residues from the same side chain family and the modified antibody (mutant antibody) can be tested for retained function.

[0109] Non-conservative substitutions involve exchanging a member of one of these groups for a member of another group.

[0110] Substitutional variants include the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variants selected for further study will have altered (e.g., improved) certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which can be routinely prepared using, for example, phage display-based affinity maturation techniques as described herein. Briefly, one or more CDR residues are mutated and the mutated antibodies are displayed on phage and screened for specific biological activity (such as binding affinity).

[0111] For example, changes (e.g., substitutions) can be made in the CDR regions to increase antibody affinity. Such changes can be made in CDR "hot spots," i.e., codon-encoded residues that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or antigen-unlocked residues, and the resulting mutant VH or VL are tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis) are used to introduce diversity into the selection of mature variable gene species to generate secondary libraries that are then screened to identify antibody variants with the desired affinity. Alternative methods for introducing diversity include CDR-directed methods in which several CDR residues (e.g., 4-6 residues at a time) are randomized.

[0112] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such changes do not significantly reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative modifications as described herein) that do not significantly reduce binding affinity may be made in the CDRs. Such changes may, for example, be outside the residues of the CDRs that contact the antigen. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than one, no more than two, or no more than three amino acid substitutions.

[0113] The terms "anti-GPRC5D antibody", "antibody that binds to GPRC5D", "GPRC5D antibody", and "antibody that recognizes GPRC5D" refer to an antibody that can bind to GPRC5D with sufficient affinity to be useful as a diagnostic and / or therapeutic agent for targeting GPRC5D. In one embodiment, the anti-GPRC5D antibody binds to an unrelated non-GPRC5D protein to an extent of less than about 10% of that of an antibody that binds to GPRC5D, as measured by enzyme-linked immunosorbent assay (ELISA). In certain embodiments, the anti-GPRC5D antibody binds to an epitope of GPRC5D that is conserved among GPRC5D from different species.

[0114] The term "chimeric T cell receptor" includes recombinant polypeptides derived from the various polypeptides that make up the TCR, which can bind to surface antigens on target cells and interact with other polypeptides of the complete TCR complex that are normally co-localized on the T cell surface. Chimeric T cell receptors consist of a TCR subunit and an antigen-binding domain that is composed of a human or humanized antibody domain. The TCR subunit includes at least a portion of the TCR extracellular domain, a transmembrane domain, and a stimulatory domain of the intracellular signaling domain of the TCR intracellular domain. The TCR subunit and the antibody domain are operatively connected, and the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3ε or CD3γ, and the chimeric T cell receptor is incorporated into a TCR expressed on a T cell.

[0115] The term "T cell antigen coupler (TAC)" includes three functional domains: 1, an antigen-binding domain that may include a single-chain antibody, designed ankyrin repeat protein (DARPin) or other targeting group; 2, an extracellular domain that brings the TAC receptor into close proximity to the TCR receptor by the single-chain antibody that binds to CD3; and 3, a transmembrane domain and an intracellular domain of the CD4 co-receptor, where the intracellular domain is linked to the protein kinase LCK, which catalyzes the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR complex as the first step in T cell activation.

[0116] The term "chimeric antigen receptor" (CAR) comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain comprises a functional signaling domain of a stimulatory molecule (sometimes referred to as a stimulatory molecule, a primary signaling molecule) and / or a costimulatory molecule. For example, the stimulatory molecule can be the zeta chain associated with the T cell receptor complex. For example, the cytoplasmic signaling domain further comprises a functional signaling domain of one or more costimulatory molecules, such as 4-1BB (i.e., CD137), CD27, and / or CD28.

[0117] In this application, in one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule, and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at the amino acid (ND-terminus) of the CAR fusion protein. In one aspect, the CAR also comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, which is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

[0118] The term "primary signaling molecule" or "stimulatory molecule" stimulatorily regulates the initial activation of the TCR complex. Typically, a primary signal is initiated, for example, by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, thereby mediating a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. Primary signaling molecules that act in a stimulatory manner may contain immunoreceptor tyrosine-based activation motifs or ITAM signaling motifs. Examples of functional signaling domains (primary signaling domains) of ITAM-containing primary signaling molecules that are particularly useful in this application include, but are not limited to, the sequences of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), and CD66d. In a particular example of the CAR of the present application, the intracellular signaling domain in any one or more of the CARs of the present application includes an intracellular signaling sequence (such as a primary signaling domain such as CD3ζ).

[0119] The term "costimulatory signaling domain" generally refers to the intracellular domain of a costimulatory molecule that can bind to a cell stimulatory signaling molecule, such as TCR / CD3, and combine signals that cause T cell proliferation and / or upregulation or downregulation of key molecules. Costimulatory molecules are typically associated binding partners on T cells that specifically bind to costimulatory ligands, thereby mediating T cell costimulatory responses, such as, but not limited to, proliferation. Costimulatory molecules are non-antigen receptor cell surface molecules or their ligands that are required for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137).

[0120] The term "CD3ζ (also known as CD3 zeta)" is defined as the protein provided by GenBan Accession No. BAG36664.1, or the equivalent residues from a non-human species, such as mouse, rodent, monkey, ape, etc. A "CD3ζ domain" is defined as the amino acid residues of the cytoplasmic domain of the ζ chain sufficient to functionally transmit the initial signal required for T cell activation. In one embodiment, the cytoplasmic domain of ζ comprises residues 52-164 of GenBan Accession No. BAG36664.1, or a functional ortholog thereof that is the equivalent residues from a non-human species, such as mouse, rodent, monkey, ape, etc.

[0121] Detailed Description of the Invention antibody Herein, we describe the immunization of mice to obtain hybridoma antibodies using conventional hybridoma antibody preparation techniques in the art, the construction of recombinant hybridoma antibodies (scFv-Fc), and also describe antigen-binding proteins, including antibody Fabs, with Fab (fragment-binding antigen)-based antigen-binding regions. Herein, human GPRC5D antigen (Kactus) was used to select Fabs from a fully human natural Fab phage library. These molecules show exquisite specificity. For example, this antibody only recognizes GPRC5D, and 293T cells, CHO-K1 cells, MM.1S cells, and NCI-H929 cells that overexpress GPRC5D, but not cells that do not express GPRC5D. Unless otherwise specified in this application, GPRC5D in this specification refers to human GPRC5D or murine GPRC5D.

[0122] In some embodiments, the present application includes antibodies with Fab sequences fused to one or more heavy chain constant regions, forming the antibody with a human immunoglobulin Fc region to generate a bivalent protein, thereby increasing the overall affinity and stability of the antibody. Additionally, the Fc portion allows other molecules (including but not limited to fluorochromes, cytotoxins, radioisotopes, etc.) to be directly attached to the antibody for use in antigen quantification studies, etc., for immobilization of the antibody for affinity measurements, directed delivery of therapeutic agents, testing of Fc-mediated cytotoxicity using immune effector cells, and many other applications.

[0123] The results provided herein highlight the specificity, sensitivity, and utility of the antibodies of the present application in targeting GPRC5D.

[0124] The antibody or antibody fragment of the present application is based on the use of phage display to identify and select antigen-binding fragments (Fabs), the amino acid sequence of which confers specificity to the antibody or antibody fragment against GPRC5D and forms the basis of all antigen-binding proteins of the present disclosure. The Fabs can therefore be used to design a series of different "antibodies or antibody fragments", including, for example, full-length antibodies, fragments thereof such as F(ab')2, fusion proteins, multivalent antibodies, scFv-Fc antibodies, i.e., antibodies with multiple specificities against the same or different antigens, e.g., bispecific T cell binding antibodies (BiTEs), tribodies, etc. (Cuesta et al., Multivalent antibodies: when design surpasses evolution, Trends in Biotechnology 28:355-362, 2010).

[0125] In certain embodiments, the application provides full-length antibodies, in which the heavy and light chains may be full length (e.g., the antibody may comprise at least one, preferably two complete heavy chains, and at least one, preferably two complete light chains) or may comprise an antigen-binding portion (Fab, F(ab')2, Fv, or scFv). In other embodiments, the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. The choice of antibody type is determined by the immune effector function that the engineered antibody is intended to elicit. In constructing recombinant immunoglobulins, the appropriate amino acid sequences of the constant regions of various immunoglobulin isotypes and methods for generating a wide variety of antibodies are known to those skilled in the art.

[0126] The present application provides an antibody that recognizes GPRC5D, comprising at least one CDR of a heavy chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 23, 27, 31, 35, 39, 76 or 80 or a variant thereof, or an amino acid sequence having at least 80% identity with any of the above sequences; and / or at least one CDR of a light chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 25, 29, 33, 37, 41 or 78 or a variant thereof, or an amino acid sequence having at least 80% identity with any of the above sequences. For example, the heavy chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 23, 27, 31, 35 or 39 or a variant thereof, or an amino acid sequence having at least 80% identity with any of the above sequences, and / or the light chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 25, 29, 33, 37 or 41 or a variant thereof, or an amino acid sequence having at least 80% identity with any of the above sequences. For example, the heavy chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 23, 76 or 80, or a variant thereof, or an amino acid sequence having at least 80% identity with any of the above sequences, and / or the light chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 25 or 78, or a variant thereof, or an amino acid sequence having at least 80% identity with any of the above sequences. Here, the CDRs can be determined by a numbering system selected from Kabat, Chothia, IMGT, Gelfand, Aho, and Martin. The antibody recognizing GPRC5D provided in the present application may comprise a specific CDR sequence determined by any of the above numbering systems or a combination thereof, but the CDRs of the heavy and / or light chains constituting the antibody do not necessarily need to be determined by the same numbering. For example, one or some CDRs in the antibody can be determined by the Kabat system, and other CDRs can be determined by any one or combination of the above numbering systems.In certain embodiments, the CDRs of the antibody can be determined by a numbering system, for example, by the Kabat numbering system, for example, by the Chothia numbering system, for example, by the IMGT numbering system, for example, by the Gelfand numbering system, for example, by the Aho numbering system, for example, by the Martin numbering system.

[0127] In certain embodiments, the antibody may comprise one, two, or three CDRs of the heavy chain variable region.

[0128] In certain embodiments, the antibody may comprise one, two, or three CDRs of the light chain variable region.

[0129] In certain embodiments, the antibody may comprise one, two, or three CDRs of a heavy chain variable region and one, two, or three CDRs of a light chain variable region. For example, it may comprise one CDR of a heavy chain variable region and one, two, or three CDRs of a light chain variable region. For example, it may comprise two CDRs of a heavy chain variable region and one, two, or three CDRs of a light chain variable region. For example, it may comprise three CDRs of a heavy chain variable region and one, two, or three CDRs of a light chain variable region. For example, it may comprise three CDRs of a heavy chain variable region and one, two, or three CDRs of a light chain variable region.

[0130] In certain embodiments, the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, 35, 39, 76 or 80, or a variant thereof.

[0131] In certain embodiments, the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, 41, or 78, or a variant thereof.

[0132] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, 35, 39, 76 or 80, or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, 41 or 78, or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 23, or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, 41 or 78, or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 27, or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, 41 or 78, or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31 or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, 41, or 78 or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 35 or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, 41, or 78 or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 39 or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, 41, or 78 or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76 or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, 41, or 78 or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80 or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, 41, or 78 or a variant thereof.

[0133] In certain embodiments, the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, 35, or 39, or a variant thereof.

[0134] In certain embodiments, the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, or 41, or a variant thereof.

[0135] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, 35, or 39, or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, or 41, or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 23, or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, or 41, or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 27, or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, or 41, or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31, or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, or 41, or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 35 or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, or 41, or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 39, or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, or 41, or a variant thereof.

[0136] In certain embodiments, the heavy chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 23, 76, or 80, or a variant thereof.

[0137] In a specific embodiment, the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25 or 78, or a variant thereof.

[0138] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 23, 76, or 80, or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25 or 78, or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 23, or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25 or 78, or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76, or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25 or 78, or a variant thereof. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80, or a variant thereof, and the light chain variable region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25 or 78, or a variant thereof.

[0139] For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 23, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 25. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 29. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 33. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 37. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 39, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 41. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 25. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 25. For example, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:23, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:78.

[0140] In certain embodiments, the antibody may comprise the heavy chain variable region and / or the light chain variable region. For example, the heavy chain variable region may comprise a heavy chain CDR1 (HCDR1), which may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1, 11, 17, 70, or 71. For example, the heavy chain variable region may comprise a heavy chain CDR2 (HCDR2), which may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 2, 12, 18, 72, or 73. For example, the heavy chain variable region may comprise a heavy chain CDR3 (HCDR3), which may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 13, or 19. For example, the heavy chain variable region may comprise HCDR1 and HCDR3, wherein HCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1, 11, 17, 70 or 71, and wherein HCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 13 or 19. For example, the heavy chain variable region may comprise HCDR1 and HCDR2, wherein HCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1, 11, 17, 70 or 71, and wherein HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 2, 12, 18, 72 or 73. For example, the heavy chain variable region may comprise HCDR2 and HCDR3, wherein HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 2, 12, 18, 72 or 73, and wherein HCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 13 or 19. For example, the heavy chain variable region may comprise HCDR1, HCDR2, and HCDR3, wherein the HCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1, 11, 17, 70, or 71, the HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 2, 12, 18, 72, or 73, and the HCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 13, or 19. For example, the light chain variable region may comprise a light chain CDR1 (LCDR1), wherein the LCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 4, 14, 20, or 74.For example, the light chain variable region may comprise a light chain CDR2 (LCDR2), wherein the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5, 15, 21, or 75. For example, the light chain variable region may comprise a light chain CDR3 (LCDR3), wherein the LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 16, or 22. For example, the light chain variable region may comprise an LCDR1 and an LCDR3, wherein the LCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 4, 14, 20, or 74, and the LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 16, or 22. For example, the light chain variable region may comprise LCDR1 and LCDR2, where LCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 4, 14, 20, or 74, and where LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5, 15, 21, or 75. For example, the light chain variable region may comprise LCDR2 and LCDR3, where LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5, 15, 21, or 75, and where LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 16, or 22. For example, the light chain variable region may comprise LCDR1, LCDR2, and LCDR3, wherein the LCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 4, 14, 20, or 74; the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5, 15, 21, or 75; and the LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 16, or 22.

[0141] In certain embodiments, the antibody may comprise the heavy chain variable region and / or the light chain variable region. For example, the heavy chain variable region may comprise a heavy chain CDR1 (HCDR1), wherein the HCDR1 may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 1, 11, or 17. For example, the heavy chain variable region may comprise a heavy chain CDR2 (HCDR2), wherein the HCDR2 may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 2, 12, or 18. For example, the heavy chain variable region may comprise a heavy chain CDR3 (HCDR3), wherein the HCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 13, or 19. For example, the heavy chain variable region may comprise HCDR1 and HCDR3, wherein the HCDR1 may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 1, 11, or 17, and wherein the HCDR3 may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 13, or 19. For example, the heavy chain variable region may comprise HCDR1 and HCDR2, wherein HCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1, 11, or 17, and HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 2, 12, or 18. For example, the heavy chain variable region may comprise HCDR2 and HCDR3, wherein HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 2, 12, or 18, and HCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 13, or 19. For example, the heavy chain variable region may comprise HCDR1, HCDR2, and HCDR3, wherein HCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1, 11, or 17, wherein HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 2, 12, or 18, and HCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 13, or 19. For example, the light chain variable region may comprise a light chain CDR1 (LCDR1), which may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 4, 14, or 20. For example, the light chain variable region may comprise a light chain CDR2 (LCDR2), which may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5, 15, or 21.For example, the light chain variable region may comprise a light chain CDR3 (LCDR3), and the LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 16, or 22. For example, the light chain variable region may comprise an LCDR1 and an LCDR3, and the LCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 4, 14, or 20, and the LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 16, or 22. For example, the light chain variable region may comprise an LCDR1 and an LCDR2, and the LCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 4, 14, or 20, and the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5, 15, or 21. For example, the light chain variable region may comprise LCDR2 and LCDR3, wherein the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5, 15, or 21, and the LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 16, or 22. For example, the light chain variable region may comprise LCDR1, LCDR2, and LCDR3, wherein the LCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 4, 14, or 20, the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5, 15, or 21, and the LCDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 16, or 22.

[0142] In certain embodiments, the antibody may comprise the heavy chain variable region and / or the light chain variable region. For example, the heavy chain variable region may comprise a heavy chain CDR1 (HCDR1), wherein the HCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1, 70, or 71. For example, the heavy chain variable region may comprise a heavy chain CDR2 (HCDR2), wherein the HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 2, 72, or 73. For example, the heavy chain variable region may comprise a heavy chain CDR3 (HCDR3), wherein the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 3. For example, the heavy chain variable region may comprise HCDR1 and HCDR3, wherein the HCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1, 70, or 71, and the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 3. For example, the heavy chain variable region may comprise HCDR1 and HCDR2, wherein HCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1, 70, or 71, and wherein HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 2, 72, or 73. For example, the heavy chain variable region may comprise HCDR2 and HCDR3, wherein HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 2, 72, or 73, and wherein HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 3. For example, the heavy chain variable region may comprise HCDR1, HCDR2, and HCDR3, wherein HCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1, 70, or 71, wherein HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 2, 72, or 73, and wherein HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 3. For example, the light chain variable region may comprise a light chain CDR1 (LCDR1), which may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 4 or 74. For example, the light chain variable region may comprise a light chain CDR2 (LCDR2), which may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5 or 75. For example, the light chain variable region may comprise a light chain CDR3 (LCDR3), which may comprise the amino acid sequence set forth in SEQ ID NO: 6.For example, the light chain variable region may comprise LCDR1 and LCDR3, wherein the LCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 4 or 74, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 6. For example, the light chain variable region may comprise LCDR1 and LCDR2, wherein the LCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 4 or 74, and the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5 or 75. For example, the light chain variable region may comprise LCDR2 and LCDR3, wherein the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5 or 75, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 6. For example, the light chain variable region may comprise LCDR1, LCDR2, and LCDR3, wherein the LCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 4 or 74, the LCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5 or 75, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 6.

[0143] For example, the antibody may comprise the heavy chain variable region and / or the light chain variable region, the heavy chain variable region may comprise the HCDR1, the HCDR2, and the HCDR3, and the light chain variable region may comprise the LCDR1, the LCDR2, and the LCDR3. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 2, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 3, and the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 4, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 5, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 6. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 7, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 4, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 8. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 9, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 4, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 10. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:11, the HCDR12 may comprise the amino acid sequence set forth in SEQ ID NO:2, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:13, and the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:14, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO:15, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:16.For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 17, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 19, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 20, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 21, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 22. For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 70, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 72, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 4, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 5, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 6. For example, the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 71, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 73, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 3, and the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 4, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 5, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 6.

[0144] In certain embodiments, the antibody comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1, 11, or 17, and / or a heavy chain CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2, 12, or 18, and / or a heavy chain CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 13, or 19. In certain embodiments, the present application provides an antibody recognizing GPRC5D comprising a light chain CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4, 14, or 20, and / or a light chain CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5, 15, or 21, and / or a light chain CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 16, or 22. In a particular embodiment, the present application provides an antibody recognizing GPRC5D, comprising a heavy chain CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 1, 11 or 17, and / or a heavy chain CDR2 comprising the amino acid sequence shown in any one of SEQ ID NOs: 2, 12 or 18, and / or a heavy chain CDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 3, 7, 9, 13 or 19, and / or a light chain CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 4, 14 or 20, and / or a light chain CDR2 comprising the amino acid sequence shown in any one of SEQ ID NOs: 5, 15 or 21, and / or a light chain CDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 6, 8, 10, 16 or 22. For example, the antibody that recognizes GPRC5D comprises a heavy chain CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 1, 11 or 17, a heavy chain CDR2 comprising the amino acid sequence shown in any one of SEQ ID NOs: 2, 12 or 18, and a heavy chain CDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 3, 7, 9, 13 or 19, and / or a light chain CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 4, 14 or 20, a light chain CDR2 comprising the amino acid sequence shown in any one of SEQ ID NOs: 5, 15 or 21, and a light chain CDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 6, 8, 10, 16 or 22.For example, the antibody that recognizes GPRC5D comprises a heavy chain CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 1, 11 or 17, a heavy chain CDR2 comprising the amino acid sequence shown in any one of SEQ ID NOs: 2, 12 or 18, and a heavy chain CDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 3, 7, 9, 13 or 19, and a light chain CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 4, 14 or 20, a light chain CDR2 comprising the amino acid sequence shown in any one of SEQ ID NOs: 5, 15 or 21, and a light chain CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 6, 8, 10, 16 or 22. For example, the antibody recognizing GPRC5D comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 3; LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6; or HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 7; LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 8; or HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 9; LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 10.

[0145] In certain embodiments, the antibody may comprise the heavy chain variable region and / or the light chain variable region. For example, the heavy chain variable region may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, 35, 39, 76, or 80. For example, the light chain variable region may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, 41, or 78.

[0146] In certain embodiments, the antibody may comprise the heavy chain variable region and / or the light chain variable region. For example, the heavy chain variable region may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, 35, or 39. For example, the light chain variable region may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, or 41.

[0147] In certain embodiments, the antibody may comprise the heavy chain variable region and / or the light chain variable region. For example, the heavy chain variable region may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 23, 76, or 80. For example, the light chain variable region may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 25 or 78.

[0148] For example, the antibody may comprise the heavy chain variable region and the light chain variable region. For example, the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 23, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 29. For example, the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 31, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 33. For example, the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 35, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 37. For example, the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 39, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 41. For example, the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 76, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 80, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 25. For example, the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 23, and the light chain variable region may comprise the amino acid sequence set forth in SEQ ID NO: 78.

[0149] In another aspect, the application provides an antibody recognizing GPRC5D, comprising a heavy chain variable region comprising the amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, 35 or 39, or a variant of the above sequences.

[0150] In another aspect, the application provides an antibody recognizing GPRC5D, comprising a light chain variable region comprising the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37 or 41, or a variant of the above sequences.

[0151] In another aspect, the present application provides an antibody recognizing GPRC5D comprising the above-mentioned heavy chain variable region and light chain variable region.

[0152] Given that each of these heavy and light chain variable region sequences can bind to GPRC5D, the heavy and light chain variable region sequences can be "mixed and matched" to create the anti-GPRC5D binding molecules of the present application.

[0153] In another aspect, the present application provides a variant of an antibody or fragment thereof that binds to GPRC5D. Thus, the present application provides an antibody or fragment thereof having a heavy and / or light chain variable region that is at least 80% identical to the heavy or light chain variable region sequence. Preferably, the amino acid sequence identity of the heavy and / or light chain variable region is at least 85%, more preferably at least 90%, most preferably at least 95%, particularly 96%, more particularly 97%, even more particularly 98%, and most particularly 99%, including, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. The variant can be obtained by methods such as yeast library screening, phage library screening, point mutation, etc., using the antibody described in the present application as the parent antibody.

[0154] In another aspect, the present application provides an antibody that recognizes the same epitope as the aforementioned anti-GPRC5D antibody.

[0155] In another aspect, the present application provides an antibody that binds to GPRC5D competitively with the aforementioned anti-GPRC5D antibodies.

[0156] In another aspect, the present application provides an antibody that specifically binds to GPRC5D, wherein the antibody is a complete antibody, an scFv, a single domain antibody, a Fab fragment, a Fab' fragment, an Fv fragment, an F(ab')2 fragment, an Fd fragment, a dAb fragment, a multifunctional antibody, or an scFv-Fc antibody.

[0157] Antibody assays Anti-GPRC5D antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art, including, for example, ELISA, Biacore, Western blotting, flow cytometry analysis, etc. Suitable assays are described in detail in the Examples.

[0158] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1:1 interaction between binding partner members (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be expressed as a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including determining the affinity of an antibody using Biacore as described herein. The "affinity" of an antibody for GPRC5D herein is represented by the KD of the antibody. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The higher the KD value of an antibody for binding to an antigen, the weaker its binding affinity for that particular antigen.

[0159] The term "EC50" refers to the half-maximal concentration (the concentration for 50% of the maximal effect, EC50), the concentration that produces 50% of the maximal effect.

[0160] antigen The term "antigen" refers to a substance that is recognized and specifically bound by an antigen-binding unit. Antigens can include peptides, proteins, glycoproteins, polysaccharides, and lipids, portions thereof, and combinations thereof. Non-limiting exemplary antigens include tumor antigens or pathogen antigens. "Antigen" can also refer to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immunologically-competent cells, or both. One of skill in the art will appreciate that any macromolecule, including virtually any protein or peptide, can function as an antigen.

[0161] The term "epitope" refers to an antigen or a part of an antigen that is recognized by an antibody, B cell, T cell, or engineered cell. For example, an epitope can be a tumor epitope or a pathogen epitope that is recognized by an antibody; an antibody recognizes multiple epitopes within an antigen. Epitopes can also be mutated.

[0162] The term "antigenic determinant" is also known as "antigenic epitope" or "epitope" or "antigenic determinant" and includes any determinant or region to which an antibody can bind. An antigenic epitope is a region of an antigen to which an antibody that targets that antigen binds, and includes specific amino acids that directly contact the antibody. For example, an antigenic epitope may be composed of a continuous sequence of the GPRC5D protein sequence, or may be composed of a discontinuous three-dimensional structure of the GPRC5D protein sequence. Exemplarily, the antigen used herein is human or mouse GPRC5D.

[0163] immune complex The present application also provides an immunoconjugate comprising the antibody described herein and a functional molecule linked thereto. The antibody provided in the present application is described above, and the immunoconjugate provided in the present application includes all technical solutions thereof. The antibody and the functional molecule can form a conjugate by covalent bond, coupling, attachment, crosslinking, etc.

[0164] "Linked" or "fused" are used interchangeably herein. These terms refer to the joining of two or more chemical elements or components by any means, including chemical conjugation or recombinant means. "In-frame fusion" refers to the joining of two or more open reading frames (ORFs) to form a continuous, longer ORF in a manner that maintains the correct reading frame of the original ORFs. The resulting recombinant fusion protein is thus a single protein that contains two or more segments that correspond to the polypeptides encoded by the original ORFs (these segments are not normally so joined in nature). In this way, the reading frame is continuous throughout the fusion segments, but the segments may be physically or spatially separated, for example, by an in-frame linker sequence (e.g., "Flexon").

[0165] Another aspect of the present application provides a nucleic acid molecule encoding at least one antibody, functional variant thereof, or immunoconjugate of the present application. Once the relevant sequence is obtained, recombinant methods can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, transforming it into a cell, and then isolating the relevant sequence from the host cell grown by conventional methods.

[0166] The present application also relates to vectors that contain the appropriate DNA sequences as described above and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to allow the expression of proteins. The host cells can be prokaryotic cells, such as bacterial cells, lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells.

[0167] Chimeric Receptors The present application also provides chimeric receptors. Chimeric receptors generally refer to the expression product of a fusion molecule formed by linking DNA fragments or cDNA corresponding to proteins from different sources using recombinant gene technology, and can include an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes the antibody provided by the present application described in detail above, and the chimeric receptor provided by the present application includes all of its technical solutions. The chimeric receptors include, but are not limited to, chimeric antigen receptors (CARs), chimeric T cell receptors, and T cell antigen couplers (TACs).

[0168] In some embodiments, the chimeric receptor of the present application is a chimeric antigen receptor (CAR). In certain embodiments, the extracellular antigen-binding region (or extracellular domain) of the CAR is derived from a mouse monoclonal antibody, a humanized monoclonal antibody, or a human monoclonal antibody.

[0169] The chimeric antigen receptor typically comprises an extracellular antigen binding region or an antibody. In some embodiments, the extracellular antigen binding region may be fully human. In other cases, the extracellular antigen binding region may be humanized. In other cases, the extracellular antigen binding region may be of murine origin, or the chimera in the extracellular antigen binding region may be composed of amino acid sequences from at least two different animals. In some embodiments, the extracellular antigen binding region may be non-human.

[0170] In certain embodiments, the chimeric antigen receptor can also be designed to include multiple antigen-binding regions, including single chain variable fragments (scFv) derived from an antibody, fragment antigen-binding regions (Fab) selected from a library, single domain fragments, or natural ligands that bind to their cognate receptors. In some embodiments, the extracellular antigen-binding region can include scFv, Fab, or natural ligand, as well as any derivatives thereof. The extracellular antigen-binding region can refer to a molecule other than an intact antibody that includes a portion of an intact antibody and can bind to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bifunctional antibodies, linear antibodies; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. The extracellular antigen-binding region, such as scFv, Fab, or natural ligand, can be part of a CAR that determines the antigen specificity. The extracellular antigen-binding region can bind to any complementary target. The extracellular antigen-binding region can be derived from an antibody of known variable region sequence. The extracellular antigen-binding region may be derived from antibody sequences obtained from available mouse hybridomas. Alternatively, the extracellular antigen-binding region may be obtained by total exo-cleavage sequencing of tumor cells or primary cells such as tumor infiltrating lymphocytes (TILs).

[0171] In some embodiments, the binding specificity of the extracellular antigen-binding region of the CAR can be determined by a complementarity determining region or CDR, such as a light chain CDR or a heavy chain CDR. In many cases, the binding specificity can be determined by a light chain CDR and a heavy chain CDR.

[0172] In some embodiments, the extracellular antigen binding region of the CAR comprises a hinge or spacer region, and the hinge region and spacer region may be used interchangeably. The hinge may be considered as a part of the CAR that provides flexibility to the extracellular antigen binding region. In some embodiments, the hinge may be used to detect the CAR on the cell surface of the cell, especially when antibodies that detect the extracellular antigen binding region are ineffective or unavailable. In some embodiments, the hinge may not belong to an immunoglobulin, but to another molecule, such as the natural hinge of the CD8α molecule. The CD8α hinge may contain cysteine ​​and proline residues that are known to play a role in the interaction of the CD8 coreceptor with the MHC molecule. The hinge may be adjusted depending on the extracellular antigen binding region used. The hinge may be of any length. For example, the hinge may comprise the amino acid sequence shown in SEQ ID NO:59.

[0173] The transmembrane domain (or structural region) of the CAR can anchor the CAR to the plasma membrane of the cell. The native transmembrane portion of CD28 can be used in the CAR. In other cases, the native transmembrane portion of CD8α can also be used in the CAR. "CD8" can be a protein having at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to NCBI Accession No.: NP_001759 or a fragment thereof having stimulatory activity. A "CD8 nucleic acid molecule" can be a polynucleotide encoding a CD8 polypeptide, and in certain cases, the transmembrane region can be the native transmembrane portion of CD28, and "CD28" can be a protein having at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to NCBI Accession No.: NP_006130 or a fragment thereof having stimulatory activity. A "CD28 nucleic acid molecule" can be a polynucleotide encoding a CD28 polypeptide. In some embodiments, the transmembrane portion can include a CD8α region. For example, the transmembrane domain may comprise the amino acid sequence set forth in SEQ ID NO:60.

[0174] The intracellular signaling domain of a CAR may be involved in the activation of at least one of the effector functions of an immune response cell that contains said CAR. A CAR can induce effector functions of T cells. For example, said effector functions are cytolytic or auxiliary activities, including secretion of cytokines such as IL-2, TNF-α, γ-IFN, etc. Thus, the term intracellular signaling domain refers to a portion of a protein that transmits an effector function signal and instructs a cell to perform a specific function. Usually, the entire intracellular signaling region can be used, but in many cases it is not necessary to use the entire chain of the signaling domain. In some embodiments, a truncated portion of the intracellular signaling region is used. Thus, in some embodiments, the term intracellular signaling region is intended to include any truncated portion of the intracellular signaling region sufficient to transmit an effector function signal.

[0175] Preferred examples of signaling domains (or structural regions) used in CARs may include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act synergistically to initiate signal transduction following target-receptor binding, as well as any derivative or mutant sequences thereof and any synthetic sequences having the same functions of these sequences.

[0176] In some embodiments, the intracellular signaling region of the CAR may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs that contain cytoplasmic signaling sequences include those derived from CD3zeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. However, in preferred embodiments, the intracellular signaling domain is derived from the CD3epsilon chain.

[0177] An example of a T cell signaling domain that contains one or more ITAM motifs is the CD3ζ domain, also known as the T cell receptor CD3ζ chain or CD247. This domain is part of the T cell receptor-CD3 complex and plays a key role in coupling antigen recognition with several intracellular signaling pathways and activation of the primary effect of T cells. As used herein, CD3ζ refers primarily to human CD3ζ and its isoforms, as known from Swissprot entry P20963, and includes proteins with essentially identical sequences. As part of the chimeric antigen receptor, the complete T cell receptor CD3ζ chain is not required, and any derivative (including its functional equivalent) that contains the signaling domain of the T cell receptor CD3ζ chain is suitable. For example, the signaling domain of the CD3ζ chain may include the amino acid sequence shown in SEQ ID NO:62.

[0178] In certain embodiments, the intracellular signaling domain (or structural region) of the CAR can be selected from any of the costimulatory domains in Table 2. In some embodiments, the domain can be modified to have about 50% to about 100% identity with the reference domain. Any of the domains in Table 2 can be modified such that the modified version can include about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or at most about 100% identity.

[0179] In certain embodiments, the intracellular signaling region of the CAR may further comprise one or more costimulatory domains. The intracellular signaling region may comprise a single costimulatory domain, such as the zeta chain (first generation CAR) or CD28 or 4-1BB (second generation CAR). In other examples, the intracellular signaling domain may comprise two costimulatory domains, such as CD28 / OX40 or CD28 / 4-1BB (third generation). For example, the costimulatory domain of 4-1BB may comprise the amino acid sequence shown in SEQ ID NO:61.

[0180] In certain embodiments, these costimulatory domains, together with intracellular signaling domains such as CD8, can cause downstream activation of kinase pathways, thereby supporting gene transcription and functional cellular responses. The costimulatory domain of a CAR can activate proximal signaling proteins associated with the activation of the CD28 (phosphatidylinositol-4,5-bisphosphate 3-kinase) or 4-1BB / OX40 (TNF receptor associated factor adaptor protein) pathways, as well as MAPK and Akt signaling.

[0181] In some cases, the signal generated through the CAR may be combined with an auxiliary signal or a costimulatory signal. In the case of a costimulatory signaling domain, the chimeric antigen receptor-like complex can be designed to include several possible costimulatory signaling domains. It is well known in the art that in naive T cells, T cell receptor binding alone is insufficient to induce full activation of T cells into cytotoxic T cells. A second costimulatory signal is required for full productive T cell activation. Several receptors have been reported that provide costimulation for T cell activation, including but not limited to CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1BBL, MyD88, and 4-1BB. Any of the signaling pathways used by these costimulatory molecules act synergistically with the main signal of T cell receptor activation. The signals provided by these costimulatory signaling domains can act synergistically with the main effect activation signal derived from one or more ITAM motifs (e.g., CD3zeta signaling domain) to meet the requirements of T cell activation.

[0182] In some embodiments, the addition of a costimulatory domain to the chimeric antigen receptor-like complex can increase the efficacy and durability of the engineered cells, hi other embodiments, the T cell signaling domain and the costimulatory domain are fused to each other to form the signaling domain.

[0183] [Table 2]

[0184] In some embodiments, cells (e.g., T cells) are transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the cells can stably express the CAR.

[0185] In certain embodiments, the GPRC5D binding portion of the CAR is an scFv that retains the same affinity avidity as the Fab antibody from which it is derived, e.g., binds to the same antigen with the same efficacy. The antibody fragment is functional, providing a biochemical response, such as activating an immune response, inhibiting the initiation of signal transduction from a target antigen, inhibiting kinase activity, etc. For example, the scFv can comprise any of the sequences set forth in SEQ ID NO: 43, 82, 84, or 86.

[0186] In certain embodiments, the anti-GPRC5D antigen-binding domain of the CAR is a fully human antibody fragment.

[0187] In certain embodiments, the CAR of the present application combines the antigen-binding domain of a particular antibody with an intracellular signaling molecule, including but not limited to the CD3 zeta chain, 4-1BB, and CD28 signaling modules, and combinations thereof.

[0188] In certain embodiments, the GPRC5D-CAR comprises at least one intracellular signaling domain selected from a CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3ζ signaling domain, and any combination thereof. In one aspect, the GPRC5D-CAR comprises at least one intracellular signaling domain derived from one or more costimulatory molecules other than CD137 (4-1BB) or CD28.

[0189] As an example, the sequence of GPRC5D-CAR is as follows: an extracellular domain set forth in SEQ ID NO: 43, a hinge domain set forth in SEQ ID NO: 59, a transmembrane domain set forth in SEQ ID NO: 60, a costimulatory signal domain set forth in SEQ ID NO: 61, and a primary signal domain set forth in SEQ ID NO: 62 (AB3-BBZ). An extracellular domain set forth in SEQ ID NO: 82, a hinge domain set forth in SEQ ID NO: 59, a transmembrane domain set forth in SEQ ID NO: 60, a costimulatory signal domain set forth in SEQ ID NO: 61, and a primary signal domain set forth in SEQ ID NO: 62 (AB6-BBZ). An extracellular domain set forth in SEQ ID NO:84, a hinge domain set forth in SEQ ID NO:59, a transmembrane domain set forth in SEQ ID NO:60, a costimulatory signal domain set forth in SEQ ID NO:61, and a primary signal domain set forth in SEQ ID NO:62 (AB7-BBZ). An extracellular domain set forth in SEQ ID NO: 86, a hinge domain set forth in SEQ ID NO: 59, a transmembrane domain set forth in SEQ ID NO: 60, a costimulatory signal domain set forth in SEQ ID NO: 61, and a primary signal domain set forth in SEQ ID NO: 62 (AB8-BBZ).

[0190] The amino acid sequence of the chimeric antigen receptor is shown, for example, in any one of SEQ ID NOs: 63, 88, 89, or 90.

[0191] Those skilled in the art can select conventional transmembrane and intracellular domains to replace the above-mentioned transmembrane and intracellular domains of the chimeric antigen receptor, all of which fall within the scope of protection of the present application.

[0192] Nucleic acids, vectors, viruses, and host cells The terms "nucleic acid molecule coding," "encoding a DNA sequence," and "encoding DNA" refer to the order or sequence of deoxyribonucleotides along a deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Thus, a nucleic acid sequence codes for an amino acid sequence.

[0193] As used herein, the term "sequence," when used to refer to a nucleotide sequence, includes DNA or RNA, and can be single-stranded or double-stranded.

[0194] The term "target sequence" refers to a sequence that has complementarity with guide sequence, and the complementary pairing between target sequence and guide sequence promotes the formation of CRISPR complex.Target sequence can include any polynucleotide, such as DNA or RNA polynucleotide.In some embodiments, target sequence is located in the nucleus or cytoplasm of a cell.

[0195] The term sequence "identity" refers to a percentage of identity determined by comparing two optimally matched sequences over a comparison window (e.g., at least 20 positions), where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps), e.g., a gap of 20% or less (e.g., 5-15%, or 10-12%) for the two optimally matched sequences compared to the reference sequence (not including additions or deletions). Typically, the percentage is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in the two sequences, thereby generating the number of correct pairing positions, and dividing the number of correct pairing positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to generate the percentage of sequence identity.

[0196] The term "transfection" refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection can be accomplished by a variety of means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0197] The term "expression vector" as used herein refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operatively linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as plasmids (and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0198] The term "vector" as used herein refers to a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term can also include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc.

[0199] The term "lentivirus" as used herein refers to a genus of the Retroviridae family. Retroviruses are unique among retroviruses in that they can infect non-dividing cells. They can deliver large amounts of genetic information to the DNA of host cells, making them one of the most effective methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

[0200] The term "endogenous" refers to a nucleic acid molecule or polypeptide, etc., that originates from the organism itself.

[0201] The term "exogenous" as used herein refers to a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not endogenously expressed in the organism itself or the expression level is insufficient to achieve the function when overexpressed.

[0202] As used herein, the term "exogenous protein" may refer to a protein exogenously introduced into a cell that recognizes a target antigen, such as an exogenous receptor (i.e., a "chimeric receptor" as referred to herein).

[0203] The term "host" as used herein refers to the recipient of the graft, which in some embodiments can be an individual, such as a human, into which the exogenous cells are transplanted.

[0204] The term "isolated" means that the polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof is separated from cellular or other components with which it is normally associated in the natural state. As will be appreciated by those skilled in the art, a non-natural polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof does not need to be "isolated" to be distinguished from its naturally occurring counterpart. Furthermore, a "concentrated", "isolated" or "diluted" polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof may be distinguished from its naturally occurring counterpart because the concentration or number of molecules per volume is greater than that of its naturally occurring counterpart ("concentrated") or less than that of its naturally occurring counterpart ("diluted"). The degree of enrichment may be measured in absolute terms, such as weight per volume of solution, or relative to another potential interfering substance present in the source mixture. In some embodiments, the preferred degree of enrichment of the technical solution of the present application is higher. Thus, for example, 2-fold enrichment is preferred, 10-fold enrichment is more preferred, 100-fold enrichment is more preferred, and 1000-fold enrichment is more preferred. An "isolated" material can also be provided by methods of artificial construction, such as chemical synthesis or recombinant expression.

[0205] The present application provides isolated nucleic acids, vectors, and host cells comprising said nucleic acids or vectors encoding antibodies or fragments thereof that recognize GPRC5D. The nucleic acids may be from intact cells, cell lysates, or in a partially purified or substantially purified form.

[0206] The nucleic acids of the present application can be obtained using standard molecular biology techniques, for example, cDNAs encoding the light and heavy chains or VH and VL segments of the antibody can be obtained by standard PCR amplification or cDNA cloning techniques. In the case of antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), one or more nucleic acids encoding the antibody can be recovered from the library. Methods for introducing exogenous nucleic acids into host cells are generally known in the art and may vary depending on the host cell used.

[0207] Preferably, the nucleic acid molecule of the present application is selected from SEQ ID NOs: 24, 28, 32, 36, 40, 77 or 81 encoding a heavy chain variable region, and / or selected from SEQ ID NOs: 26, 30, 34, 38, 42 or 79 encoding a light chain variable region. More preferably, the nucleic acid molecule comprises a heavy chain variable region sequence comprising SEQ ID NO:24 and a light chain variable region sequence comprising SEQ ID NO:26, or comprises a heavy chain variable region sequence comprising SEQ ID NO:28 and a light chain variable region sequence comprising SEQ ID NO:30, or comprises a heavy chain variable region sequence comprising SEQ ID NO:32 and a light chain variable region sequence comprising SEQ ID NO:34, or comprises a heavy chain variable region sequence comprising SEQ ID NO:36 and a light chain variable region sequence comprising SEQ ID NO:38, or comprises a heavy chain variable region sequence comprising SEQ ID NO:40 and a light chain variable region sequence comprising SEQ ID NO:42, or comprises a heavy chain variable region sequence comprising SEQ ID NO:77 and a light chain variable region sequence comprising SEQ ID NO:26, or comprises a heavy chain variable region sequence comprising SEQ ID NO:81 and a light chain variable region sequence comprising SEQ ID NO:26, or comprises a heavy chain variable region sequence comprising SEQ ID NO:24 and a light chain variable region sequence comprising SEQ ID NO:79.

[0208] In one embodiment, one or more vectors (eg, expression vectors) are provided that contain the above-described nucleic acids.

[0209] The term "cell" refers to a cell of human or non-human animal origin.

[0210] The term "host cell" refers to a cell into which exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny (regardless of the number of passages). The nucleic acid content of the progeny will not be identical to that of the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0211] The term "GPRC5D-positive host cells" refers to host cells that express GPRC5D on their cell surface, and these cells can be detected, for example, by flow cytometry using an antibody that specifically recognizes an epitope on GPRC5D.

[0212] In some embodiments, the host cell is an immune effector cell.

[0213] The term "immune effector cell" refers to a cell that participates in an immune response and produces an immune effect, such as a T cell, a B cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a dendritic cell, a CIK cell, a macrophage, a mast cell, and the like. In some embodiments, the immune effector cell is a T cell, a NK cell, or a NKT cell. In some embodiments, the T cell can be an autologous T cell, a heterologous T cell, or an allogeneic T cell. In some embodiments, the NK cell can be an allogeneic NK cell. "Immune effector function or immune effector response" refers to a function or response of an immune effector cell that enhances or promotes an immune attack against a target cell, and the like. For example, an immune effector function or response refers to a property of a T cell or an NK cell that promotes the killing or inhibits the growth or proliferation of a target cell.

[0214] The term "artificially modified cells with immune effector cell function" refers to cells that have no immune effect or cells that acquire immune effector cell function after the cell line is artificially modified or stimulated by a stimulant. For example, 293T cells are artificially modified to have immune effector cell function. For example, stem cells are induced in vitro to differentiate into immune effector cells.

[0215] In some cases, "T cells" are pluripotent stem cells derived from bone marrow that can differentiate and mature in the thymus into mature T cells with immunological activity. In some cases, "T cells" can be a cell population with a specific phenotypic characteristic or a mixed cell population with different phenotypic characteristics, for example, "T cells" can be cells that include at least one T cell subpopulation from stem cell-like memory T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs) and / or effector memory T cells (Tem). In some cases, "T cells" can be a specific subtype of T cell, such as γδ T cells.

[0216] T cells can be obtained from multiple sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural fluid, spleen tissue, and tumor tissue. In some cases, T cells can be obtained from blood collected from an individual using any number of techniques known to those skilled in the art, such as Ficoll™ isolation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. Apheresis products typically contain lymphocytes, such as T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis collection can be washed to remove plasma molecules and placed in an appropriate buffer or medium for subsequent processing steps. In one embodiment, T cells can be obtained from healthy donors or from cells derived from patients diagnosed with tumors.

[0217] The term "peripheral blood mononuclear cells" (PBMCs) refers to mononuclear cells in peripheral blood, including lymphocytes, monocytes, etc.

[0218] The terms "activate" and "activating" can refer to the process by which cells change from a resting state to an active state. This process can include a response to an antigen, migration, and / or phenotypic or genetic changes to a functionally active state. For example, the term "activation" can refer to the process of stepwise activation of NK cells, T cells.

[0219] The term "T cell activation" or "activating a T cell" can refer to the state of a T cell that is sufficiently stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector function.

[0220] The term "chemokine" refers to a polypeptide with a molecular weight of 8-10 kDa, which is the largest family of cytokines, whose main function is to recruit monocytes, neutrophils, lymphocytes, etc. in the blood to invade specific lymphoid organs and tissues and sites where infection occurs. "Chemokine receptor" is a type of seven-transmembrane G protein-coupled receptor (GPCR) that mediates the function of chemokines and is usually expressed on the cell membrane of immune cells, neutrophils, endothelial cells, etc. Chemokines and chemokine receptors play important roles in mediating cell migration, proliferation, and defense against pathogen invasion, and are closely related to inflammation in the immune environment and the development and progression of cancer.

[0221] The term "safety switch" is used to improve the safety of CAR-T therapy by engineering a rapid and reversible "off" or "on" safety switch in CAR-T cells to minimize treatment-related toxicity. Although CAR-T cell therapy has excellent clinical characteristics, it may cause fatal side effects such as severe CRS when tumor burden is unpredictable and T cell activity is uncontrolled. Controlling toxicity requires proper monitoring using a CRS grading system with severe CRS as a guide and precise modulation using small molecule-based safety switches.

[0222] In another embodiment, a host cell is provided that comprises the above-described nucleic acid. The host cell comprises (e.g., is transduced with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and an amino acid sequence comprising an antibody VH, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell, a 293T cell, an NIH3T3 cell, or a lymphocyte (e.g., YO, NSO, Sp20 cell).

[0223] In another embodiment, the host cell expresses a chimeric receptor described herein.

[0224] In another embodiment, the host cells include T cells, natural killer cells, cytotoxic T lymphocytes, natural killer T cells, DNT cells, regulatory T cells, NK92 cells, and / or stem cell derived immune effector cells.

[0225] In another embodiment, the T cells comprise natural T cells and / or T cells derived from pluripotent stem cells. Preferably, the T cells are autologous / allogeneic T cells. Preferably, the T cells are primary T cells. Preferably, the T cells comprise human autologous T cells.

[0226] In another embodiment, the T cells comprise stem cell-like memory T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Treg), effector memory T cells (Tem), γδ T cells, or a combination thereof.

[0227] In another embodiment, the host cell binds to cells that express GPRC5D, but does not significantly bind to cells that do not express GPRC5D.

[0228] In another embodiment, the host cell also has a coding sequence for an exogenous cytokine, and / or expresses a chimeric receptor that does not target GPRC5D, and / or expresses a chemokine, and / or expresses a chemokine receptor, and / or expresses a safety switch.

[0229] In another embodiment, the host cell further comprises a coding sequence for an exogenous cytokine comprising IL-7, IL-12, IL-15, IL-18, IL-21, or a type I interferon.

[0230] In another embodiment, the host cell may express, in addition to the GPRC5D-binding receptors described above, a chimeric receptor that binds another antigen.

[0231] In another embodiment, the host cell further expresses a chemokine comprising CCL19 or CCL21. In another embodiment, the host cell further expresses a chemokine receptor comprising CCR2, CCR4, CCR5, CXCR2, CXCR4 or CXCR5. In another embodiment, the host cell further expresses a safety switch comprising iCaspase-9, truncated EGFR, or RQR8.

[0232] In one embodiment, a method for preparing an anti-GPRC5D antibody is provided, the method comprising culturing a host cell comprising a nucleic acid encoding said antibody under conditions suitable for expression of said antibody, and optionally recovering the antibody from the host cell (or from the host cell culture medium).

[0233] To express the protein, the nucleic acid encoding the antibody of the present application can be incorporated into an expression vector. A variety of expression vectors are available for protein expression. Expression vectors may include autonomously replicating extrachromosomal vectors or vectors that integrate into the host genome. Expression vectors for use in the present application include, but are not limited to, those that allow for expression of proteins in mammalian cells, bacteria, insect cells, yeast, and in vitro systems. As is known in the art, a variety of expression vectors are commercially available or otherwise available. They can be used to express the antibodies in the present application.

[0234] In a preferred embodiment, the host cells are administered in combination with an agent that enhances its function, preferably in combination with a chemotherapeutic agent, and / or the host cells are administered in combination with an agent that ameliorates one or more associated side effects, and / or the host cells are administered in combination with host cells expressing a chimeric antigen receptor that targets other than GPRC5D.

[0235] Pharmaceutical Compositions The antibodies, immunoconjugates comprising the antibodies, chimeric receptors, and host cells of the present application can be used to prepare pharmaceutical compositions or diagnostic reagents. In addition to an effective amount of the antibody, immunoconjugate, chimeric receptor, nucleic acid, or host cell, the composition may also include a pharma- ceutically acceptable carrier.

[0236] The term "pharmaceutical acceptable" means that molecular entities and compositions do not produce adverse, allergic, or other adverse reactions when administered appropriately to animals or humans.

[0237] In some embodiments, the composition includes another therapeutic agent. In some embodiments, the other therapeutic agent is a chemotherapeutic agent, such as those described in U.S. Patent Application Publication No. 20140271820, and / or a pharma- ceutically acceptable salt or analog thereof. In some embodiments, the therapeutic agent includes mitotic inhibitors (vinca alkaloids), including vincristine, vinblastine, vindesine, and Novivin™ (vinorelbine, 5'-sulfide dehydrogenase); topoisomerase I inhibitors, including Camptosar™ (irinotecan HCL), Hycamtin™ (topotecan HCL), and other compounds derived from camptothecin and its analogs; podophyllotoxin derivatives, such as etoposide, teniposide, and midozozide; alkylating agents, cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphamide, carmustine, busulfan, chloramphenicol, cyclophosphamide ... These include, but are not limited to, mubucil, brexinidine, uracil mustard, cloprofen, and dacarbazine; antimetabolites such as cytarabine, 5-fluorouracil, methotrexate, mercaptopurine, azathioprine, and procarbazine; antibiotics including, but not limited to, doxorubicin, bleomycin, dactinomycin, daunorubicin, mycomycin, mitomycin, sarcomycin C, daunomycin; and other chemotherapeutic agents including, but not limited to, antitumor antibodies, dacarbazine, azacitidine, amzacam, melphalan, ifosfamide, and mitoxantrone. In some embodiments, the additional therapeutic agent is selected from one or more of epirubicin, oxaliplatin, and 5-fluorouracil. In some embodiments, the additional therapeutic agents include, but are not limited to, anti-angiogenic agents, including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides), and other vascular inhibitors, such as angiostatin, endostatin, interferons, interleukin-1 (including alpha and beta), interleukin-12, retinoic acid, tissue inhibitors of metalloproteinases 1 and 2.

[0238] Some specific examples of substances that can be used as pharma- ceutically acceptable carriers or components thereof include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut, cottonseed, sesame, olive, corn, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffers.

[0239] The pharmaceutical compositions described herein may contain one or more pharma-ceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not produce any harmful toxic effects (see, for example, Berge, SM et al., 1977, J.Pharm.Sci.66:1-19). Examples of such salts include acid addition salts and base addition salts.

[0240] Acid addition salts include salts derived from non-toxic inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc., and salts derived from non-toxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base addition salts include salts derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, etc., and from non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucosamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.

[0241] The pharmaceutical compositions described herein may also contain antioxidants. Examples of antioxidants include, but are not limited to, water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, and sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0242] The compositions of the present application can be made into various dosage forms as needed, and can be administered by a physician who determines the beneficial dose for the patient based on factors such as the type, age, weight, and general disease state of the patient, and the method of administration, such as parenteral administration (such as injection) and other treatment methods.

[0243] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0244] In some embodiments, the compositions may be isotonic, i.e., they may have the same osmotic pressure as blood and tears. The desired isotonicity of the compositions of the present application may be achieved using sodium chloride or other pharma- ceutically acceptable agents, such as glucose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. If desired, a pharma-ceutically acceptable thickening agent may be used to maintain the viscosity of the composition at a selected level. Suitable thickening agents include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The preferred concentration of the thickening agent will vary depending on the reagent selected. Obviously, the selection of the appropriate carrier and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., a liquid dosage form.

[0245] kit The application also provides kits comprising the antibodies, immunoconjugates, chimeric receptors, nucleic acids, or host cells described herein. In some embodiments, the kits may include a therapeutic or prophylactic composition comprising an effective amount of the antibodies, chimeric receptors, nucleic acids, or host cells described herein in one or more unit dosage forms. In some embodiments, the kits include a sterile container that may contain the therapeutic or prophylactic composition, such a container may be a box, an ampoule, a bottle, a vial, a tube, a bag, a blister pack, or other suitable container known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceutical agents. In some embodiments, the kits include the antibodies, immunoconjugates, chimeric receptors, nucleic acids, or host cells described herein, and instructions for administering the antibodies, immunoconjugates, chimeric receptors, nucleic acids, or host cells described herein to an individual. The instructions generally include a method of treating or preventing cancer or tumors using the antibodies, immunoconjugates, chimeric receptors, nucleic acids, or host cells described herein. In some embodiments, the kits include the host cells described herein, and further include about 1×10 4 Approximately 1 × 10 cells 6 In some embodiments, the kit comprises at least about 1 x 10 cells. 5 of cells, at least about 1 x 10 6 of cells, at least about 1 x 10 7 of cells, at least about 4 x 10 7 of cells, at least about 5 x 10 7 of cells, at least about 6 x 10 7 of cells, at least about 6 x 10 7 of cells, at least about 8 x 10 7 of cells, at least about 9 × 10 7 of cells, at least about 1 x 10 8 of cells, at least about 2 x 10 8 of cells, at least about 3 x 10 8 of cells, at least about 4 x 10 8 of cells, at least about 5 x 10 8 of cells, at least about 6 x 10 8of cells, at least about 6 x 10 8 of cells, at least about 8 x 10 8 of cells, at least about 9 × 10 8 of cells, at least about 1 x 10 9 of cells, at least about 2 x 10 9 of cells, at least about 3 x 10 9 of cells, at least about 4 x 10 9 of cells, at least about 5 x 10 9 of cells, at least about 6 x 10 9 of cells, at least about 8 x 10 9 of cells, at least about 9 × 10 9 of cells, at least about 1 x 10 10 of cells, at least about 2 x 10 10 of cells, at least about 3 x 10 10 of cells, at least about 4 x 10 10 of cells, at least about 5 x 10 10 of cells, at least about 6 x 10 10 of cells, at least about 7 x 10 10 of cells, at least about 8 x 10 10 of cells, at least about 9 × 10 10 of cells, at least about 1 x 10 11 of cells, at least about 2 x 10 11 of cells, at least about 3 x 10 11 of cells, at least about 4 x 10 11 of cells, at least about 5 x 10 11 of cells, at least about 8 x 10 11 of cells, at least about 9 × 10 11 cells, or at least about 1 × 10 12 For example, the kit may contain about 5×10 10 In another example, the kit can include 3×10 6 The cells can contain approximately 5 × 10 10 The cells can be multiplied and administered to the test subject.

[0246] In some embodiments, the kits may include allogeneic cells. In some embodiments, the kits may include cells that may include genomic modifications. In some embodiments, the kits may include "off-the-shelf" cells. In some embodiments, the kits may include cells that can be expanded for clinical use. In some cases, the kits may include content for research purposes.

[0247] In some embodiments, the instructions include at least one of the following: description of therapeutic agent; dosing schedule and administration to treat or prevent a tumor or a symptom thereof; precautions, warnings, contraindications, overdose information, side effects, animal pharmacology, clinical studies, and / or literature references. The instructions may be printed directly on the container (if available), may be a label on the container, or may be provided in or on the container as a separate sheet, pamphlet, card, or folder. In some embodiments, the instructions provide a method of administering an antibody described herein to treat or prevent a tumor. In some cases, the instructions provide a method of administering an antibody of the present application before, after, or simultaneously with administration of a chemotherapeutic agent.

[0248] Methods for diagnosis / detection / treatment The term "modulation" refers to a positive or negative alteration. Examples of modulation include a 1%, 2%, 10%, 25%, 50%, 75%, or 100% alteration. In certain embodiments, it refers to a negative alteration.

[0249] The term "treatment" refers to an intervention that seeks to modify the course of a disease, either preventively or in a clinical pathological process. Therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of direct or indirect pathological consequences of a disease, prevention of metastasis, delay of disease progression, amelioration or alleviation of a condition, alleviation or improvement of a prognosis, etc.

[0250] The term "prophylaxis" refers to an intervention that seeks to prevent a disease (such as rejection of a cell transplant) before it develops.

[0251] "Tumor antigen" refers to an antigen that is newly emerged or overexpressed during the development and development of a hyperproliferative disease. In certain embodiments, the hyperproliferative disorder of the present invention refers to a tumor.

[0252] The tumor antigen according to the present invention may be a solid tumor antigen or a hematoma antigen.

[0253] The tumor antigens of the present invention include thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin-13 receptor subunit alpha (IL-13Rα); interleukin-11 receptor alpha (IL-11Rα); prostate stem cell antigen (PSCA); prostate-specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor 20 receptor family and its variants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin type A receptor receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; TGS5; high molecular weight melanin tumor-associated antigen (HMWMAA); O-acetyl-GD2 ganglioside (OAcGD2); folate receptor; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); Claudin6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B-cell maturation antigen (BCMA); CA9; kappa light chain (kappa light chain); CSPG4; EGP2, EGP40; FAP; FAR; FBP;embryonic AchR;HLA-A1, HLA-A2;MAGEA1, MAGE3;KDR;MCSP;NKG2D ligand;PSC1;ROR1;Sp17;SURVIVIN;TAG72;TEM1;fibronectin;tenascin;tumor necrotic zone carcinoembryonic variant;G protein-coupled receptor class C group 5 member D (GPRC5D);X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta specific 1 (PLAC1); hexose moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AM L); sperm protein 17 (SPA17); X-antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; P53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma transition breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); pairing box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen recognized by T cells 3 (SART3); pairing box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchor protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79B; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2);The tumor antigens include, but are not limited to, CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like 2 containing EGF-like module (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like peptide 1 (IGLL1). Preferably, the tumor antigen is CS1, Claudin18.2, GPC3, BCMA or CD19.

[0254] The pathogen antigen is selected from a viral, bacterial, fungal, protozoan, or parasitic antigen. The viral antigen is selected from a cytomegalovirus antigen, an Epstein-Barr virus antigen, a human immunodeficiency virus antigen, or an influenza virus antigen.

[0255] The term "individual" refers to any animal, such as a mammal or marsupial. Individuals of the present invention include, but are not limited to, humans, non-human primates (such as rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any type of poultry.

[0256] As used herein, the term "effective amount" refers to an amount that provides a therapeutic or prophylactic benefit.

[0257] Any of the anti-GPRC5D antibodies, immunoconjugates, chimeric receptor modified host cells, pharmaceutical compositions, or kits provided herein may be used in therapeutic methods.

[0258] In one aspect, any anti-GPRC5D antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition or kit is provided for use as a medicament. In another aspect, any anti-GPRC5D antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition or kit is provided for treating a disease. In a particular embodiment, any anti-GPRC5D antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition or kit is provided for use in a method of treatment. In a particular embodiment, the present application provides any anti-GPRC5D antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition or kit for use in a method of treating an individual suffering from a disease, the method comprising administering to the individual an effective amount of any anti-GPRC5D antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition or kit. In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. The "individual" is preferably a human.

[0259] In another aspect, the application provides the use of any of the anti-GPRC5D antibodies, immunoconjugates, chimeric receptor modified host cells, pharmaceutical compositions or kits in the preparation or formulation of a medicament. In one embodiment, the medicament is used to treat a disease. In another embodiment, the medicament is used in a method of treating a disease, the method comprising administering an effective amount of the medicament to an individual suffering from the disease. In one embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual. The "individual" is preferably a human.

[0260] In another aspect, the present application provides a method for treating a disease. In one embodiment, the method comprises administering to an individual suffering from a disease expressing GPRC5D an effective amount of any anti-GPRC5D antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition, or kit. In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. The "individual" is preferably a human.

[0261] In another aspect, the application provides a pharmaceutical formulation comprising any of the anti-GPRC5D antibodies, immunoconjugates, chimeric receptor modified host cells, pharmaceutical compositions or kits provided herein, e.g., for use in any of the above methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-GPRC5D antibodies, immunoconjugates, chimeric receptor modified host cells, pharmaceutical compositions or kits provided herein, and a pharmaceutical carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-GPRC5D antibodies, immunoconjugates, chimeric receptor modified host cells, pharmaceutical compositions or kits provided herein, and at least one additional therapeutic agent.

[0262] In another aspect, the pharmaceutical preparation is used to treat a disease. In one embodiment, the pharmaceutical preparation is administered to an affected individual. An "individual" according to any of the above embodiments is preferably a human.

[0263] In another aspect, the application provides a method for preparing a medicament or pharmaceutical formulation, the method comprising combining any of the anti-GPRC5D antibodies, immunoconjugates, chimeric receptor modified host cells, pharmaceutical compositions or kits provided herein with a pharmaceutical carrier, e.g., for use in any of the above-mentioned methods of treatment. In one embodiment, the method for preparing the medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.

[0264] Any of the anti-GPRC5D antibodies, immunoconjugates, chimeric receptor modified host cells, pharmaceutical compositions, or kits of the present application can be used in therapy alone or in combination with other agents. Alternatively, any of the anti-GPRC5D antibodies, immunoconjugates, chimeric receptor modified host cells, pharmaceutical compositions, or kits of the present application can be administered in combination with at least one additional therapeutic agent.

[0265] Combination treatment as described above includes combined administration (two or more therapeutic agents in the same or separate formulations) and separate administration, where administration of an anti-GPRC5D antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition, or kit of the present application may occur before, simultaneously with, and / or after administration of the additional therapeutic agent or agent. In one embodiment, administration of any anti-GPRC5D antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition, or kit of the present application and administration of the additional therapeutic agent occur within about one month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other.

[0266] Any anti-GPRC5D antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition or kit (and any additional therapeutic agent) of the present application may be administered by any suitable means, including parenteral, intrapulmonary or intranasal administration, and, if therapeutically necessary, intralesional administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether administration is temporary or chronic. Various dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses at multiple time points, bolus administration, and pulse infusion.

[0267] The formulation containing the population of immunoreactive cells administered to an individual contains a plurality of immunoreactive cells effective to treat and / or prevent a particular indication or disease. Thus, a therapeutically effective population of immunoreactive cells can be administered to an individual. Typically, the population is about 1×10 4 ~Approx. 1×10 10 A preparation containing approximately 1×10 immune reactive cells is administered. In most cases, the preparation contains approximately 1×10 immune reactive cells. 5 ~Approx. 1×10 9 Immunoreactive cells, approximately 5 x 10 5 ~Approx. 5×10 8 immunoreactive cells, or approximately 1 x 10 6 ~Approx. 1×10 7The individual will contain 100 immune reactive cells. However, the number of CAR immune reactive cells administered to an individual will vary widely depending on the location, origin, identity, extent and severity of the tumor, the age and physical condition of the individual being treated, etc. The physician will ultimately determine the appropriate dosage.

[0268] In some embodiments, the chimeric receptor is used to stimulate a host cell-mediated immune response. For example, a T cell-mediated immune response is an immune response involving the activation of T cells. Activated antigen-specific cytotoxic T cells can induce apoptosis in target cells that present foreign antigen epitopes on their surface, such as cancer cells that present tumor antigens. In other embodiments, the chimeric antigen receptor is used to provide anti-tumor immunity to a mammal. The recipient develops anti-tumor immunity as a result of the T cell-mediated immune response.

[0269] In some cases, a method for treating a subject with a tumor may include administering to a subject in need of treatment one or more host cells described in the present application. The host cells can bind to tumor targeting molecules and induce cancer cell death. As mentioned above, the present application also provides a method for treating a pathogen infection in an individual, comprising administering to the individual a therapeutically effective amount of the host of the present application.

[0270] The frequency of administration of the immune reactive cells of the present application will depend on factors including the disease being treated, the components of the particular immune reactive cells, and the mode of administration. For example, the agent can be administered 4 times, 3 times, 2 times, or once a day, every other day, every 3rd, 4th, 5th, or 6th day, once a week, once every 8th, 9th, or 10th day, once a week, or twice a month. As described herein, the immune response cells of the present application have improved viability and therefore can be administered at a lower therapeutically effective amount as well as at a lower administration frequency than similar immune response cells that do not express exogenous type I interferon to achieve at least a similar, and preferably more significant, therapeutic effect.

[0271] Advantages of this application: The present application provides a fully human antibody that specifically recognizes GPRC5D, and CAR T cells prepared from this antibody show excellent killing effect on target cells both in vivo and in vitro.

[0272] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application, and do not limit the scope of the present application. In the following embodiments, the experimental method that does not show specific conditions usually follows conventional conditions, such as the conditions described in J.Sambrook et al., Molecular Cloning Experimental Guide, Third Edition, Science Press, 2002, or follows the recommended conditions of the manufacturer.

[0273] Example 1. Preparation of cell lines expressing GPRC5D Using conventional molecular biology techniques, human GPRC5D (amino acid sequence shown in SEQ ID NO: 65) was transfected via lentivirus into CHOK1 cells (ATCC), 293T cells (ATCC), and NIH3T3 cells (ATCC), which do not express endogenous GPRC5D, respectively, and positive single clones were picked up by limiting dilution to construct cell lines stably transfected with CHOK1-huGPRC5D, 293T-huGPRC5D, and NIH3T3-huGPRC5D.

[0274] Using conventional molecular biology techniques, mouse GPRC5D (amino acid sequence shown in SEQ ID NO: 66) was transfected into CHOK1 cells via lentivirus, and positive clones were picked up by limiting dilution to construct a cell line stably transfected with CHOK1-muGPRC5D.

[0275] Example 2. Preparation of anti-GPRC5D hybridoma cell lines Anti-GPRC5D hybridoma antibodies were prepared using conventional hybridoma antibody preparation techniques known in the art.

[0276] The full-length gene encoding human GPRC5D (SEQ ID NO: 65) was constructed into the eukaryotic expression plasmid pCAGGS to form the eukaryotic expression plasmid pCAGGS-GPRC5D. Plasmid pCAGGS-GPRC5D and NIH3T3-huGPRC5D cells were then used to immunize Balb / c mice (Vital River, female, 6-8 weeks old) four times. For the first three immunizations, 100 μg of plasmid pCAGGS-GPRC5D (diluted in saline, total volume 2 mL) was bolus-injected into the tail vein to complete the immunization within 5-8 seconds, and for the fourth immunization, 2 × 10 7 NIH3T3-huGPRC5D cells were used for intraperitoneal boost, and 3 days later, mouse spleens were harvested and fused according to published standard protocols (Kohler and Milstein, 1975). After specific screening by HAT and flow cytometry, two hybridoma antibodies, mIgG:AB1 (IgG2b-k) and AB2 (IgG3-k), that bind to human GPRC5D were obtained.

[0277] Example 3. EC50 measurement of binding of anti-GPRC5D hybridoma antibodies to CHOK1-huGPRC5D cells 2 x 10 cells in a 96-well round-bottom culture plate 5 Cells / well of CHOK1-huGPRC5D cells were taken, washed twice with PBS containing 1% FBS, and hybridoma antibodies were added at a starting concentration of 100 μg / mL, followed by 11 three-fold gradient dilutions, incubated at 4°C for 45 min, centrifuged at 500g for 5 min, discarded the supernatant, and washed twice with PBS containing 1% FBS. Next, 100 μL / well of goat anti-mouse FITC (KANFCHEN, used at 1:200) was added, incubated at 4°C for 45 min, centrifuged at 500g for 5 min, discarded the supernatant, and washed twice with PBS containing 1% FBS, after which 200 μL of PBS containing 1% FBS was added to resuspend the cells, and detected using a flow cytometer. Results were statistically analyzed using FlowJo vX0.7 and graphed using GraphPad Prism8.0.

[0278] The results are shown in Figure 1. The two hybridoma antibodies AB1 and AB2 each have strong binding activity to CHOK1-huGPRC5D cells, with EC50 of 1.692 nM and 1.930 nM, respectively, indicating that both AB1 and AB2 can bind significantly to human GPRC5D.

[0279] Example 4. Cross-reactivity assay of anti-GPRC5D hybridoma antibodies with human GPRC5D and mouse GPRC5D CHOK1 cells (as negative control cells), CHOK1-huGPRC5D cells, and CHOK1-muGPRC5D cells were collected, and the cross-reactivity of AB1 and AB2 with human GPRC5D and mouse GPRC5D was measured according to the method of Example 3.

[0280] The results are shown in Figure 2. Both hybridoma antibodies AB1 and AB2 bind significantly to human and mouse GPRC5D.

[0281] Example 5. Acquisition of gene sequence of anti-GPRC5D hybridoma antibody Using conventional molecular biology techniques, RNA was extracted from hybridoma cells AB1 and AB2, and reverse transcribed to synthesize cDNA. PCR was performed using UPM, mIgG2b-outer (SEQ ID NO: 67), mIgG3-outer (SEQ ID NO: 68), and mK-outer (SEQ ID NO: 69) primers, and cloning and sequencing were performed to obtain the AB1-VH gene sequence (SEQ ID NO: 36), AB1-VL gene sequence (SEQ ID NO: 38), AB2-VH gene sequence (SEQ ID NO: 40), and AB2-VL gene sequence (SEQ ID NO: 42).

[0282] The amino acid sequence of HCDR1 of AB1 is shown in SEQ ID NO: 11, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 12, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 13, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 14, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 15, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 16. The amino acid sequence of the heavy chain variable region of AB1 is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 37.

[0283] The amino acid sequence of HCDR1 of AB2 is shown in SEQ ID NO: 17, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 18, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 19, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 20, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 21, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 22. The amino acid sequence of the heavy chain variable region of AB2 is shown in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 41.

[0284] Example 6. Binding activity assay of recombinant hybridoma scFv-mFc antibodies to cell lines expressing GPRC5D Using molecular cloning technology, the heavy and light chain gene sequences of the AB1 and AB2 antibodies were cloned into a eukaryotic expression vector containing mFc (sequence number 64), respectively, to construct V152S-AB1-mFc and V152S-AB2-mFc plasmids, which were transfected into 293F cells (Thermo Company), cultured for 7 days, and purified by affinity chromatography to obtain the anti-GPRC5D recombinant antibodies AB1 (scFv-mFc) and AB2 (scFv-mFc).

[0285] CHOK1-huGPRC5D, 293T-huGPRC5D, NIH3T3-huGPRC5D, and CHOK1 cells were harvested, respectively, and the binding activity of recombinant antibodies AB1 (scFv-mFc) and AB2 (scFv-mFc) against cell lines expressing human GPRC5D was measured according to the method of Example 3.

[0286] The results are shown in Figure 3. The two recombinant antibodies AB1 (scFv-mFc) and AB2 (scFv-mFc) specifically bind to a cell line expressing human GPRC5D.

[0287] Example 7. Screening and identification of GPRC5D antibodies 1. Screening for antibodies that bind to human GPRC5D using a fully human phage display library The phage display library used in this application is a phage library constructed by our company, with a library capacity of 1E+11. Using human GPRC5D antigen (Kactus), Fab fragments that specifically bind to human GPRC5D were obtained by screening methods known to those skilled in the art. Finally, three antibodies that specifically bind to human GPRC5D were obtained, and named AB3, AB4, and AB5.

[0288] The amino acid sequence of HCDR1 of AB3 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6. The amino acid sequence of the heavy chain variable region of AB3 is shown in SEQ ID NO: 23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 25. The amino acid sequence of the heavy chain of AB3 is shown in SEQ ID NO: 45, and the amino acid sequence of the light chain is shown in SEQ ID NO: 47.

[0289] The amino acid sequence of HCDR1 of AB4 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 7, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 8. The amino acid sequence of the heavy chain variable region of AB4 is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29. The amino acid sequence of the AB4 heavy chain is shown in SEQ ID NO: 49, and the amino acid sequence of the light chain is shown in SEQ ID NO: 51.

[0290] The amino acid sequence of HCDR1 of AB5 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 9, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 10. The amino acid sequence of the heavy chain variable region of AB5 is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 33. The amino acid sequence of the AB5 heavy chain is shown in SEQ ID NO: 53, and the amino acid sequence of the light chain is shown in SEQ ID NO: 55.

[0291] 2. Assaying Antibody Specificity by Standard ELISA 50μl of the induced expression supernatant of each clone producing antibodies AB3, AB4, and AB5 was taken and incubated with 2μg / ml human GPRC5D antigen at room temperature for 1 hour, and the bound antibody was detected by incubating with secondary antibody anti-Flag-HRP (Sigma, used at 1:4000 dilution) at room temperature for 1 hour. Then, TMB was added for color development, and the OD450 value was read with a microplate reader.

[0292] The results are shown in FIG. 4 (blank group was no Fab antibody added, ie, NA) and show that antibodies AB3, AB4, and AB5 all specifically bind to the human GPRC5D antigen.

[0293] 3. Assaying the specificity of antibody binding to target cells using FAC 2 x 10 50 μl of the expression supernatant of each clone producing antibodies AB3, AB4, and AB5 was added to the wells of the 50-well plate. 5 The antibodies bound to the cells were then fluorescently labeled and detected using secondary antibody anti-F(ab')2-488 (Jackson ImmunoResearch, used at a dilution of 1:200). Mean fluorescence intensity (MFI) was calculated for the experimental data using FlowJo analysis software.

[0294] The results are shown in Figure 5 (blank group was no Fab antibody added, i.e., NA) and show that antibodies AB3, AB4, and AB5 all specifically bind to 293T cells overexpressing human GPRC5D, but do not bind to 293T cells.

[0295] Example 8. Anti-GPRC5D antibody and antigen binding EC50 assay using ELISA The clones producing antibodies AB3, AB4, and AB5 in Example 7 were subjected to purification of prokaryotic expression by nickel column, respectively, to obtain antibodies AB3, AB4, and AB5. The antibodies were then diluted 3-fold (8 gradient) from a concentration of 10 μg / ml, incubated with human GPRC5D antigen (coating concentration: 2 μg / ml) at room temperature for 1 hour, and the secondary antibody anti-Flag-HRP (Sigma, used at a dilution of 1:4000) was incubated at room temperature for 1 hour to detect the bound antibodies. Then, TMB was added for color development, and the OD450 value was read with a microplate reader. Using GraphPad Prism5 software, a four-parameter fitting was performed with the primary antibody concentration on the horizontal axis and the OD450 value on the vertical axis to calculate the EC50 value.

[0296] The results are shown in Figure 6. Antibodies AB3, AB4, and AB5 all significantly bound to the human GPRC5D antigen with EC50 values ​​of 0.07382 μg / ml, 0.1424 μg / ml, and 0.1396 μg / ml, respectively.

[0297] Example 9. Specific binding activity assay of anti-GPRC5D antibody to target cells Human lymphoma cells Daudi (Chinese Academy of Sciences Cell Bank) are cells that do not express endogenous GPRC5D, while multiple myeloma cells MM.1S (Chinese Academy of Sciences Cell Bank) and NCI-H929 (ATCC) are cells that express endogenous GPRC5D.

[0298] The AB3, AB4, and AB5 antibodies (10 μg / ml) described in Example 7 were incubated with CHOK1, CHOK1-huGPRC5D, 293T, 293T-huGPRC5D, MM.1S, NCI-H929, and Daudi cells, respectively (blank group was not added with Fab antibody, i.e., NA), and then the secondary antibody anti-F(ab')2-488 (Jackson ImmunoResearch, used at a dilution of 1:200) was used to fluorescently label the antibody bound to the cells, and the fluorescence intensity was detected by a flow cytometer. For the experimental data, the mean fluorescence intensity (MFI) was calculated using FlowJo analysis software.

[0299] The test results are shown in Figure 7. The AB3, AB4, and AB5 antibodies all significantly bind to cells expressing human GPRC5D (CHOK1-huGPRC5D, 293T-huGPRC5D, MM.1S, and NCI-H929), but not to cells not expressing human GPRC5D (CHOK1, 293T, and Daudi).

[0300] Example 10. EC50 binding activity assay of anti-GPRC5D antibodies to cells expressing human GPRC5D Count CHOK1-huGPRC5D and MM.1S cells and place them at approximately 2 × 10 per well. 5 Each cell was spread on a U-shaped bottom plate, then incubated sequentially with a primary antibody (AB3, AB4 or AB5 antibody described in Example 7: starting from 30 μg / mL, 3-fold gradient dilution, 8 gradients) and a secondary antibody (Anti-Fab-FITC: 1:200, Jackson ImmunoResearch), and the fluorescence intensity was detected using a flow cytometer. For the experimental data, the mean fluorescence intensity (MFI) was calculated using FlowJo analysis software, and then the EC50 value was calculated using GraphPad Prism5 software by performing four-parameter fitting with the primary antibody concentration as the horizontal axis and the calibrated mean fluorescence intensity (MFI) as the vertical axis.

[0301] The results are shown in Figures 8 and 9. The EC50 of AB3, AB4, and AB5 antibodies binding to CHOK1-huGPRC5D cells was 3.869 μg / ml, 5.064 μg / ml, and 4.196 μg / ml, respectively, and the EC50 of AB3, AB4, and AB5 binding to MM.1S cells was 0.8512 μg / ml, 0.6578 μg / ml, and 0.8047 μg / ml, respectively.

[0302] Example 11. Binding activity assay of anti-GPRC5D antibody to CHOK1-muGPRC5D cells The AB3, AB4 and AB5 antibodies (5 μg / ml) described in Example 7 were incubated with CHOK1-muGPRC5D cells respectively (blank group was no antibody added, i.e. NA), then fluorescently labeled with secondary antibody anti-F(ab')2-488 (Jackson ImmunoResearch, used at a dilution of 1:200), and the fluorescence intensity was detected by a flow cytometer. The mean fluorescence intensity (MFI) of the experimental data was calculated using FlowJo analysis software.

[0303] The detection results are shown in Figure 10. The AB3 antibody can bind to mouse GPRC5D.

[0304] Example 12. Preparation of GPRC5D CAR-T cells 1. Construction of CAR Vector PRRLSIN-cPPT.EF-1α was used as a vector to construct a second generation chimeric antigen receptor lentiviral plasmid expressing AB3 scFv (SEQ ID NO: 43) or control antibody 18 scFv (SEQ ID NO: 57, sequence from patent CN107428829A), which contains CD8α signal peptide (SEQ ID NO: 58), CD8 hinge region (SEQ ID NO: 59) and transmembrane region (SEQ ID NO: 60), CD137 intracellular signaling domain (SEQ ID NO: 61) and CD3ζ (SEQ ID NO: 62). The amino acid sequence of AB3-CAR is shown in SEQ ID NO: 63.

[0305] 2. Preparation of GPRC5D CAR-T Cells The lentivirus was packaged using the calcium phosphate method, and the viral supernatant was purified with PEG8000 / NaCl, after which T cells activated with CD3 / CD28 magnetic beads for 48 hours were infected to obtain AB3-BBZ CAR-T cells expressing AB3-BBZ and 18-BBZ CAR-T cells expressing 18-BBZ, respectively, and T cells not transfected with the virus were considered as UTD. Five days after infection, the CAR positivity rate was detected using the FACS method. The primary antibody for detection was Biotin-anti-F(ab')2-488 (Jackson ImmunoResearch, used at 1:100), and the secondary antibody was SA-PE (eBioscience, used at 1:200).

[0306] Example 13. In vitro killing assay of target cells by GPRC5D CAR-T cells First, the target cells, MM.1S cells, 293T-huGPRC5D cells, and 293T cells, were each diluted to 0.2 × 10 6 The density was adjusted to 100 μg / mL, and 50 μl was added to each well of a 96-well culture plate, followed by 50 μl of effector cells (CAR-T cells, UTD as control) according to effector / target ratios of 3:1, 1:1, and 1:3, respectively, and after co-incubation at 37 degrees for 16 hours, the supernatant was collected and detected using an LDH kit. Finally, the OD490 was read using a microplate reader.

[0307] The results are shown in Figure 11. AB3-BBZ CAR-T showed no killing effect on 293T cells, but showed a clear killing effect on 293T-huGPRC5D cells and MM.1S cells expressing GPRC5D (the killing rate was as high as about 60% when the effector / target ratio was 3:1), and the killing effect was positively correlated with the effector / target ratio. The results indicate that AB3-BBZ CAR-T has a specific in vitro killing effect on cells expressing GPRC5D.

[0308] Example 14. In vivo antitumor ability of GPRC5D CAR-T cells 3×10 6 Human multiple myeloma cells MM.1S were subcutaneously inoculated into the right axilla of NPG female mice, and the mice were divided into three groups (AB3-BBZ CAR T group, 18-BBZ CAR T group, and UTD group), with 8 mice in each group. The day of tumor cell inoculation was recorded as D0. The average tumor volume at 16 days after inoculation was approximately 200 mm 3 AB3-BBZ CAR T, 18-BBZ CAR T, and UTD cells were injected into the tail vein. The injection volume was 3 × 10 6 100 cells / mouse. Twenty days after CAR T cell injection (36 days after tumor inoculation), the tumor volume of mice in the UTD group was 2000 mm 3 The experiment was terminated because

[0309] The results are shown in Figure 12. The mice in the AB3-BBZ CAR-T group showed tumor regression on the 9th day after CAR-T injection (25th day after tumor inoculation), and the tumors of all 8 mice in the group disappeared on the 13th day after CAR-T injection (29th day after tumor inoculation). As a control, the mice in the 18-BBZ CAR-T group did not show tumor regression on the 20th day after CAR-T injection (36th day after tumor inoculation), and its tumor inhibition rate was only 46.02% compared with the UTD group. This result indicates that AB3-BBZ CAR-T has a better in vivo antitumor effect than 18-BBZ CAR-T (P<0.0001). At the same time, the changes in the body weight of the mice in each treatment group were detected, and the results are shown in Figure 13.

[0310] Example 15. Modified antibodies based on AB3 The light or heavy chain sequences of AB3 were randomly mutated, and a phage library was constructed and screened using human GPRC5D antigen (Kactus) to obtain AB6, AB7, and AB8 antibodies. ELISA tests showed that AB6, AB7, and AB8 significantly bound to human GPRC5D (Figure 14). As a result of sequencing, the heavy chain variable region of AB6 is shown in SEQ ID NO: 76, the heavy chain variable region of AB7 is shown in SEQ ID NO: 23, the heavy chain variable region of AB8 is shown in SEQ ID NO: 80, the light chain variable regions of AB6 and AB8 are shown in SEQ ID NO: 25, the light chain variable region of AB7 is shown in SEQ ID NO: 78, the HCDR1 of AB6 is shown in SEQ ID NO: 70, the HCDR2 is shown in SEQ ID NO: 72, the HCDR1 of AB7 is shown in SEQ ID NO: 1, the HCDR2 is shown in SEQ ID NO: 2, the HCDR1 of AB8 is shown in SEQ ID NO: 71, the HCDR2 is shown in SEQ ID NO: 73, the HCDR3 of AB6, AB7, and AB8 is shown in SEQ ID NO: 3, the LCDR1 of AB6 and AB8 is shown in SEQ ID NO: 4, the LCDR2 is shown in SEQ ID NO: 5, the LCDR1 of AB7 is shown in SEQ ID NO: 74, the LCDR2 is shown in SEQ ID NO: 75, and the LCDR3 of AB6, AB7, and AB8 is shown in SEQ ID NO: 6.

[0311] Example 16. Binding of modified antibodies to GPRC5D cell lines of different species The three clones selected in Example 15 were prokaryotically expressed and affinity purified using a nickel column to obtain purified single-chain antibodies (scFv) (shown in SEQ ID NO: 82, SEQ ID NO: 84, and SEQ ID NO: 86, respectively). The C-terminus of this single-chain antibody contains a his tag and a flag tag. Stably transfected cell lines of CHO-K1-huGPRC5D and CHO-K1-muGPRC5D were constructed according to the method of Example 1. The above purified scFv (10ug / ml) was incubated with CHO-K1-huGPRC5D cells and CHO-K1-muGPRC5D cells, as well as non-transfected CHO-K1 cells, and the secondary antibody anti-Flag-488 (R&D, used at a dilution of 1:200) was used to fluorescently label the scFv bound to the cells, and the fluorescence intensity was detected by a flow cytometer, and the mean fluorescence intensity (MFI) was calculated for the experimental data using FlowJo analysis software. The detection results are shown in Figure 15. The binding ability of AB6, AB7, and AB8 to human GPRC5D-expressing cell lines and mouse GPRC5D cell lines is significantly improved compared to AB3, but they do not bind to CHO-K1 cells that do not express GPRC5D, indicating that antibodies AB6, AB7, and AB8 have good species binding properties and cell binding specificity.

[0312] Example 17. EC50 assay of anti-GPRC5D antibodies binding to GPRC5D-expressing cell lines Using 293T-huGPRC5D cells and MM.1S cell line endogenously expressing GPRC5D as described in Example 1, the EC50 values ​​of the antibodies described in Example 15 were measured according to the method of Example 10. Primary antibodies AB3 (Fab), AB6 (scFv), AB7 (scFv) and AB8 (scFv): starting with 3-fold gradient dilution at 30 μg / mL, diluted 12-fold gradient, and co-incubated with secondary antibody (Anti-Flag-488: 1:200, R&D). The results are shown in Figure 16. All of the AB3-based modified antibodies have good cell binding activity, among which the EC50 of AB6 and AB7 on both cells is lower than AB3.

[0313] Example 18 Construction of anti-GPRC5D antibody scFv-Fc format and monomer rate assay Anti-GPRC5D antibodies (scFv) AB3, AB6, AB7 and AB8 were fused and expressed with the Fc (huFc shown in SEQ ID NO: 91) segment of human IgG, and transfected into 293F cells using 293Fectin transfection reagent. The supernatant was collected on the 7th day and affinity purified using Protein A packing. The purified protein was passed through a GE XK16 / 40 empty chromatography column, the monomer peak was collected, and monomer rate detection was performed. The results showed that the antibody monomer rate was high and there was no obvious difference. The antibody protein was concentrated using a Millipore ultrafiltration tube with a cutoff capacity of 10KD, and the concentration was measured by OD280 / extinction coefficient. The specific results are shown in Table 3.

[0314] [Table 3]

[0315] Example 19 Affinity Assay of Anti-GPRC5D Antibodies Bio-GPRC5D (Acro, Cat. No. GPD-H82D6) 741.4Ru was captured using a SA chip provided by CYTIVA (Cat. No.: BR100531). A 3-fold gradient dilution of the target antibodies (AB3-huFc, AB7-huFc) starting from 150 nM was used as the mobile phase, and Gly.Hcl pH 1.7 was regenerated for 180 seconds. After the detection was completed, the affinity results were fitted using evaluation software. The affinity KD of AB3-huFc to Bio-GPRC5D was 4.68 nM, and the affinity KD of AB7-huFc to Bio-GPRC5D was 2.14 nM (Figure 17).

[0316] Example 20 Construction of Chimeric Antigen Receptor (CAR) Plasmids Second-generation chimeric antigen receptor lentiviral plasmids expressing AB6, AB7, and AB8 were constructed according to the method of Example 12. The amino acid sequence of AB6-BBZ is shown in SEQ ID NO:88, the amino acid sequence of AB7-BBZ is shown in SEQ ID NO:89, and the amino acid sequence of AB8-BBZ is shown in SEQ ID NO:90.

[0317] Example 21 Preparation of CAR-T cells and assay of their killing effect in vitro Using the CAR expression plasmids prepared in Examples 12 and 20, AB3-BBZ CAR-T cells, AB6-BBZ CAR-T cells, AB7-BBZ CAR-T cells, and AB8-BBZ CAR-T cells were obtained according to the method of Example 12, and the T cells not transfected with the virus were used as UTD. The cell killing experiment was carried out according to the method of Example 13. The results are shown in Figure 18. AB3 CAR-T, AB6 CAR-T, AB7 CAR-T, and AB8 CAR-T had no killing effect on 293T cells that did not express GPRC5D, and 293T-huGPRC5D cells all showed obvious killing effect on MM.1S that expressed GPRC5D, and the killing effect was positively correlated with the effector / target ratio. This indicates that AB3 CAR-T, AB6 CAR-T, AB7 CAR-T, and AB8 CAR-T have specific killing effects on cells expressing human GPRC5D.

[0318] All documents mentioned in this application are incorporated herein by reference as if each document was individually incorporated by reference. Furthermore, after reading the above teachings of this application, those skilled in the art will be able to make various changes or modifications to this application, and these equivalents will also fall within the scope defined by the appended claims of this application.

[0319] The sequences according to the present application are shown in the table below. TIFF2025508343000004.tif244170TIFF2025508343000005.tif244170TIFF2025508343000006.tif245170TIFF2025508343000007.tif244170TIFF2025508343000008.tif243170TIFF2025508343000009.tif244170TIFF2025508343000010.tif245170TIFF2025508343000011.tif245170TIFF2025508343000012.tif244170TIFF2025508343000013.tif244170TIFF2025508343000014.tif93170

Claims

1. An antibody that recognizes GPRC5D, wherein the antibody is selected from any one of the following groups: (1) An antibody comprising a heavy chain variable region comprising HCDR1 set forth in any one of SEQ ID NOs: 1, 11, 17, 70, or 71, and / or HCDR2 set forth in any one of SEQ ID NOs: 2, 12, 18, 72, or 73, and / or HCDR3 set forth in any one of SEQ ID NOs: 3, 7, 9, 13, or 19; (2) An antibody comprising a light chain variable region comprising an LCDR1 set forth in any of SEQ ID NOs: 4, 14, 20, or 74, and / or an LCDR2 set forth in any of SEQ ID NOs: 5, 15, 21, or 75, and / or an LCDR3 set forth in any of SEQ ID NOs: 6, 8, 10, 16, or 22; (3) An antibody comprising the heavy chain variable region of the antibody according to (1) and the light chain variable region of the antibody according to (2); (4) An antibody that is a mutant of the antibody according to any one of (1) to (3), which has the same or similar activity as the antibody according to any one of (1) to (3). is selected from Preferably, the antibody comprises at least one CDR of a heavy chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, 35, 39, 76 or 80 or a variant thereof, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with any one of said sequences; and / or at least one CDR of a light chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, 41 or 78 or a variant thereof, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with any one of said sequences, More preferably, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise the amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 31, 35, 39, 76, or 80, or a variant thereof, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the aforementioned sequences; and / or the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which comprise the amino acid sequence set forth in any one of SEQ ID NOs: 25, 29, 33, 37, 41, or 78, or a variant thereof, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the aforementioned sequences. An antibody characterized by:

2. The CDR regions of the heavy chain variable region and / or the CDR regions of the light chain variable region of the antibody have a sequence selected from the following groups: (1) An antibody comprising HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, and HCDR3 as set forth in SEQ ID NO: 3; LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, and LCDR3 as set forth in SEQ ID NO: 6; or (2) an antibody comprising HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, and HCDR3 as set forth in SEQ ID NO: 7; LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, and LCDR3 as set forth in SEQ ID NO: 8; or (3) An antibody comprising HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, and HCDR3 as set forth in SEQ ID NO: 9; LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, and LCDR3 as set forth in SEQ ID NO: 10; or (4) An antibody comprising HCDR1 as set forth in SEQ ID NO: 11, HCDR2 as set forth in SEQ ID NO: 12, and HCDR3 as set forth in SEQ ID NO: 13; LCDR1 as set forth in SEQ ID NO: 14, LCDR2 as set forth in SEQ ID NO: 15, and LCDR3 as set forth in SEQ ID NO: 16; or (5) An antibody comprising HCDR1 as set forth in SEQ ID NO: 17, HCDR2 as set forth in SEQ ID NO: 18, and HCDR3 as set forth in SEQ ID NO: 19; LCDR1 as set forth in SEQ ID NO: 20, LCDR2 as set forth in SEQ ID NO: 21, and LCDR3 as set forth in SEQ ID NO: 22; or (6) An antibody comprising HCDR1 as set forth in SEQ ID NO: 70, HCDR2 as set forth in SEQ ID NO: 72, and HCDR3 as set forth in SEQ ID NO: 3; LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, and LCDR3 as set forth in SEQ ID NO: 6; or (7) An antibody comprising HCDR1 as set forth in SEQ ID NO: 71, HCDR2 as set forth in SEQ ID NO: 73, and HCDR3 as set forth in SEQ ID NO: 3; LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, and LCDR3 as set forth in SEQ ID NO: 6; or (8) An antibody comprising HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, and HCDR3 as set forth in SEQ ID NO: 3; LCDR1 as set forth in SEQ ID NO: 74, LCDR2 as set forth in SEQ ID NO: 75, and LCDR3 as set forth in SEQ ID NO: 6; (9) An antibody that is a mutant of the antibody according to any one of (1) to (8), having the same or similar activity as the antibody according to any one of (1) to (8). The antibody of claim 1, wherein the antibody is selected from the group consisting of:

3. The antibody has a sequence from one of the following groups: (1) An antibody in which the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:23 or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:23, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:25 or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:25; (2) an antibody in which the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:27 or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:27, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:29 or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:29; (3) An antibody in which the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:31 or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:31, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:33 or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:33; (4) An antibody in which the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 35 or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 35, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 37 or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 37; (5) An antibody in which the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:39, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:39, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:41, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:41; (6) An antibody wherein the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:76, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:76, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:25, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:25; (7) An antibody wherein the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 80, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 80, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 25, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 25; (8) An antibody wherein the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:23 or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:23, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:78 or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:78; (9) An antibody that is a mutant of the antibody according to any one of (1) to (8), having the same or similar activity as the antibody according to any one of (1) to (8). Selected from: The antibody described in claim 1.

4. The antibody may be a whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab') ２ 2. The antibody of claim 1, which is selected from the group consisting of an Fd fragment, an Fd fragment, a dAb fragment, a multifunctional antibody or an scFv-Fc antibody, a hybridoma antibody, a chimeric antibody, a humanized antibody, a fully human antibody, and a monoclonal antibody.

5. The antibody (1) An antibody wherein the heavy chain has the amino acid sequence set forth in SEQ ID NO:45, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:45, and the light chain has the amino acid sequence set forth in SEQ ID NO:47, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:47; (2) an antibody wherein the heavy chain has the amino acid sequence set forth in SEQ ID NO:49, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:49, and the light chain has the amino acid sequence set forth in SEQ ID NO:51, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:51; (3) An antibody wherein the heavy chain has the amino acid sequence set forth in SEQ ID NO:53, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:53, and the light chain has the amino acid sequence set forth in SEQ ID NO:55, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:55; (4) An antibody that is a mutant of the antibody according to any one of (1) to (3), which has the same or similar activity as the antibody according to any one of (1) to (3). The antibody of claim 1, wherein the antibody is selected from the group consisting of:

6. The antibody of claim 1, characterized in that the antibody comprises an amino acid sequence set forth in SEQ ID NO: 43, 82, 84 or 86, or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to said sequence.

7. An antibody that recognizes GPRC5D, the antibody comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 25, 29, 33 or 78, or 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; The amino acid sequences of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region are (i) SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; (ii) SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:7; (iii) SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:9; (iv) SEQ ID NO: 70, SEQ ID NO: 72, and SEQ ID NO: 3; (v) SEQ ID NO: 71, SEQ ID NO: 73, and SEQ ID NO: 3 An antibody characterized by:

8. The antibody of claim 7, wherein the heavy chain variable region is selected from SEQ ID NOs: 23, 27, 31, 76, or 80.

9. An antibody that recognizes GPRC5D, the antibody comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 23, 27, 31, 76 or 80, or an amino acid sequence having 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; The amino acid sequences of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region are (i) SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; (ii) SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:8; (iii) SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:10; (ii) SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 6 An antibody characterized by:

10. The antibody of claim 9, wherein the light chain variable region is selected from SEQ ID NO: 25, 29, 33 or 78.

11. An antibody that recognizes GPRC5D, comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises SEQ ID NO: 25, 29, 33, or 78, or an amino acid sequence having 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the heavy chain variable region comprises SEQ ID NO: 23, 27, 31, 76, or 80, or an amino acid sequence having 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

12. An immune complex comprising the antibody of claim 1 and a functional molecule linked thereto.

13. The extracellular domain of a chimeric receptor comprises the antibody of claim 1, wherein the chimeric receptor comprises a chimeric antigen receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC), or a combination thereof. A chimeric receptor characterized by:

14. The chimeric receptor is a chimeric antigen receptor (CAR) comprising the antibody of claim 1, a transmembrane domain, and an intracellular signaling domain; Preferably, the CAR comprises, from the N-terminus to the C-terminus, the antibody of claim 1, a transmembrane domain, and an intracellular signaling domain; Preferably, the antibody is linked to the transmembrane region via a hinge domain. The chimeric receptor of claim 13.

15. the intracellular signaling region comprises one or more costimulatory signaling domains and / or primary signaling domains; Preferably, the primary signal domain is selected from TCRξ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, ICOS, CD66d, CD3ζ, and / or the costimulatory signaling domain is selected from CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1BBL, MyD88, and 4-1BB; and / or the transmembrane region comprises the transmembrane region of CD8 or CD28; Preferably, The intracellular signaling region is selected from the group consisting of CD3ζ, FcεRIγ, CD27, CD28, CD137, CD134, MyD88, and CD40 intracellular signaling region sequences, and combinations thereof. The chimeric receptor of claim 14.

16. the chimeric receptor The antibody of claim 1, the transmembrane domain of CD8 / CD28, and CD3ζ; or The antibody of claim 1, the transmembrane domain of CD8 / CD28, the intracellular signaling domain of CD137, and CD3ζ; or The antibody of claim 1, the transmembrane domain of CD8 / CD28, the intracellular signaling domain of CD28, and CD3ζ; or The antibody according to claim 1, the transmembrane domain of CD8 / CD28, the intracellular signaling domain of CD28, CD137, and CD3ζ is one selected from Preferably, the amino acid sequences of the CD8 transmembrane domain, the CD137 intracellular signaling domain, and the CD3ζ are set forth in SEQ ID NOs: 60, 61, and 62, respectively, or are amino acid sequences having 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; More preferably, the amino acid sequence of the chimeric receptor is set forth in any one of SEQ ID NOs: 63, 88, 89, and 90, or an amino acid sequence having 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. The chimeric receptor of claim 15.

17. 1) a nucleic acid encoding the antibody of claim 1, the immune complex of claim 12, or the chimeric receptor of claim 13; 2) A vector containing the nucleic acid according to 1); 3) A virus containing the nucleic acid according to 1) or the vector according to 2) A biomaterial, which is any one of

18. The antibody according to claim 1, the immune complex according to claim 12, the chimeric receptor according to claim 13, the nucleic acid according to claim 17-1, and / or the vector according to claim 17-2, Preferably, the cells include T cells, natural killer cells, natural killer T cells, NK92 cells, stem cell-derived immune effector cells, or combinations thereof; More preferably, the T cells comprise natural T cells and / or T cells induced by pluripotent stem cells; Preferably, the T cells comprise autologous and / or allogeneic T cells; Preferably, the T cells are primary T cells; Preferably, said T cells comprise human autologous T cells.

19. and / or further comprising a coding sequence for an exogenous cytokine. also expressing chimeric receptors that do not target GPRC5D, and / or chemokines are also expressed, and / or also express chemokine receptors, and / or It also represents the safety switch, The cell of claim 18.

20. A pharmaceutical composition comprising an antibody described in claim 1, an immune complex described in claim 12, a chimeric receptor described in claim 13, a nucleic acid described in claim 17-1, or a vector described in claim 17-2, and a pharmaceutically acceptable adjuvant.

21. Use of an antibody described in claim 1, an immune complex described in claim 12, a chimeric receptor described in claim 13, a nucleic acid described in claim 17-1, or a vector described in claim 17-2) in the preparation of a drug for treating / diagnosing a disease, wherein the disease involves expression of GPRC5D, preferably the disease is selected from inflammatory diseases, infectious diseases, autoimmune diseases and tumors, preferably the tumor is multiple myeloma.

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