Anti-gprc5d antibodies and uses thereof

Abstract

The present application provides an antibody or fragment thereof against G protein-coupled receptor family class C, group 5, member D (GPRC5D). The present application also provides the use of the antibody or fragment thereof as an active ingredient for treating tumors or cancers. Furthermore, the present application also provides a bispecific antibody comprising the antibody or fragment thereof and against GPRC5D and CD3.

Description

[0001] Cross-references to related applications

[0002] This patent application claims priority to Chinese Patent Application No. CN202111663745.2, filed on December 31, 2021, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of biomedicine and relates to a novel anti-GPRC5D antibody or a functional fragment thereof. This invention also relates to the application of said antibody or its functional fragment. Background Technology

[0004] G protein-coupled receptor (GPCR) family C5D member (GPRC5D) is a retinoic acid-induced 40kDa protein with seven transmembrane segments and a short N-terminal extracellular domain. It is expressed in keratinized epithelial cells, pancreas, kidney, small intestine, spleen, and testes. In March 2019, the journal *Science Translational Medicine* published a research article on GPRC5D-targeted CAR-T therapy for multiple myeloma, initially demonstrating the potential of GPRC5D as a novel therapeutic target for multiple myeloma. Tissue expression profiling revealed that GPRC5D is specifically highly expressed in plasma cells of multiple myeloma. Furthermore, diseases associated with GPRC5D have been proposed, including chromosome 13Q14 deletion syndrome.

[0005] Given the significant role of GPRC5D in diseases including multiple myeloma, there is a need for more antibodies to identify GPRC5D. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an antibody or fragment thereof against a member of the G protein-coupled receptor family C class 5D (GPRC5D), and provides its uses based on this antibody or fragment. Furthermore, based on this antibody or fragment, this invention also provides a bispecific antibody against GPRC5D and CD3.

[0007] The present invention provides the following technical solutions.

[0008] On one hand, the present invention provides an antibody or a fragment thereof that is specifically capable of binding to GPRC5D, particularly human GPRC5D.

[0009] Specifically, the antibody or fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) comprise a combination of CDRs selected from the following (HCDR-1, HCDR-2, HCDR-3; LCDR-1, LCDR-2, LCDR-3):

[0010] (1) HCDR-1 containing the amino acid sequence shown in SEQ ID NO:28 (SDYAWN), HCDR-2 containing the amino acid sequence shown in SEQ ID NO:29 (YISYSGSATYNPSLKS), HCDR-3 containing the amino acid sequence shown in SEQ ID NO:30 (GGIAGRGRWGAMDY); and LCDR-1 containing the amino acid sequence shown in SEQ ID NO:31 (KASQNVGTNVA), LCDR-2 containing the amino acid sequence shown in SEQ ID NO:32 (SASYRDS), and LCDR-3 containing the amino acid sequence shown in SEQ ID NO:33 (QQYKSYPLT);

[0011] (2) HCDR-1 comprising the amino acid sequence shown in SEQ ID NO:28 (SDYAWN), HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:34 (YISYSGSATYSPSLKS), HCDR-3 comprising the amino acid sequence shown in SEQ ID NO:30 (GGIAGRGRWGAMDY); and LCDR-1 comprising the amino acid sequence shown in SEQ ID NO:31 (KASQNVGTNVA), LCDR-2 comprising the amino acid sequence shown in SEQ ID NO:32 (SASYRDS), and LCDR-3 comprising the amino acid sequence shown in SEQ ID NO:33 (QQYKSYPLT);

[0012] (3) HCDR-1 containing the amino acid sequence (SYTIQ) shown in SEQ ID NO:35, HCDR-2 containing the amino acid sequence (YIIPSSGYTNYNQKFKD) shown in SEQ ID NO:36, and HCDR-3 containing the amino acid sequence (NYGNWGFTY) shown in SEQ ID NO:37; and LCDR-1 containing the amino acid sequence (KASQNVGSAVT) shown in SEQ ID NO:38, LCDR-2 containing the amino acid sequence (SASNRYT) shown in SEQ ID NO:39, and LCDR-3 containing the amino acid sequence (QQYSNYPLT) shown in SEQ ID NO:40;

[0013] (4) HCDR-1 containing the amino acid sequence (YYVMH) shown in SEQ ID NO:41, HCDR-2 containing the amino acid sequence (YINPYNDGTKYNEKFKG) shown in SEQ ID NO:42, and HCDR-3 containing the amino acid sequence (GGVRRYFDY) shown in SEQ ID NO:43; and LCDR-1 containing the amino acid sequence (RASQDIGSNLN) shown in SEQ ID NO:44, LCDR-2 containing the amino acid sequence (ATSSLDS) shown in SEQ ID NO:45, and LCDR-3 containing the amino acid sequence (LQYATFPYT) shown in SEQ ID NO:46;

[0014] (5) HCDR-1 comprising the amino acid sequence (YYVIH) shown in SEQ ID NO:47, HCDR-2 comprising the amino acid sequence (YINPYNDGTKYNEKFKG) shown in SEQ ID NO:42, and HCDR-3 comprising the amino acid sequence (GGVRRYFDY) shown in SEQ ID NO:43; and LCDR-1 comprising the amino acid sequence (RASQDIGSNLN) shown in SEQ ID NO:44, LCDR-2 comprising the amino acid sequence (ATSSLDS) shown in SEQ ID NO:45, and LCDR-3 comprising the amino acid sequence (LQYATFPYT) shown in SEQ ID NO:46; and

[0015] (6) HCDR-1 containing the amino acid sequence (YYVIH) shown in SEQ ID NO:47, HCDR-2 containing the amino acid sequence (YINPYNAGTKYNEKFKG) shown in SEQ ID NO:48, HCDR-3 containing the amino acid sequence (GGVRRYFDY) shown in SEQ ID NO:43; and LCDR-1 containing the amino acid sequence (RASQDIGSNLN) shown in SEQ ID NO:44, LCDR-2 containing the amino acid sequence (ATSSLDS) shown in SEQ ID NO:45, and LCDR-3 containing the amino acid sequence (LQYATFPYT) shown in SEQ ID NO:46.

[0016] In the context of this invention, the combinations of light and heavy chain CDRs provided are all derived from the antibodies or fragments of this invention. Based on the variable region amino acid sequence contained in a given antibody or fragment, those skilled in the art can conventionally determine the CDRs contained therein. For example, according to a specific embodiment of this invention, the KABAT definition method is used to define the CDRs in the variable region amino acid sequence.

[0017] In the antibody or fragment thereof provided by the present invention, preferably, the heavy chain variable region comprises a sequence selected from the following: an amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:11 or SEQ ID NO:19 or an amino acid sequence having at least 75% identity with said amino acid sequence; and / or, the light chain variable region comprises a sequence selected from the following: an amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:12 or SEQ ID NO:20 or an amino acid sequence having at least 75% identity with said amino acid sequence.

[0018] More preferably, the antibody or its fragment comprises heavy chain variable regions and light chain variable regions comprising sequences selected from combinations of the following:

[0019] (1) The amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:5; and, the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:6;

[0020] (2) The amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:13; and, the amino acid sequence shown in SEQ ID NO:14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:14;

[0021] (3) The amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:7; and, the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:8;

[0022] (4) The amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:15; and, the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:16.

[0023] (5) The amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:9; and, the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:10;

[0024] (6) The amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:17; and, the amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:18.

[0025] (7) The amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:11; and, the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:12; and

[0026] (8) The amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:19; and, the amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:20.

[0027] In the context of this invention, "at least 75% identity" means any percentage of identity between 75% and 100%, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity.

[0028] Specifically, the antibody or fragment of the present invention comprises at least a heavy chain variable region and a light chain variable region, both of which include the aforementioned CDRs and spaced framework regions, with the domains arranged as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Further optionally, the maximum 25% difference in amino acid sequence resulting from the "at least 75% identity" can exist in any framework region within the heavy chain variable region or the light chain variable region, or in any domain or sequence outside the heavy chain variable region and the light chain variable region in the antibody or fragment of the present invention. This difference can be caused by amino acid deletions, additions, or substitutions at any position.

[0029] Regarding the antigen, the antibody or fragment thereof of the present invention is an antibody or fragment thereof against GPRC5D; preferably, the antibody is any form such as mouse anti, rabbit anti, monoclonal antibody, chimeric antibody, partially or fully humanized antibody, or the fragment is a hapten or an antigen-binding fragment of an antibody or hapten, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv; more preferably, the antibody is IgG. According to specific embodiments of the present invention, the present invention provides isolated and structurally characterized human antibodies, such as human monoclonal antibodies, that specifically bind to GPRC5D (e.g., human GPRC5D).

[0030] In addition to the variable region, the antibody or fragment thereof provided by the present invention also includes a constant region, such as a human or mouse constant region, preferably including a human or mouse heavy chain constant region (CH) and / or a light chain constant region (CL); preferably, the antibody or fragment thereof includes a heavy chain and a light chain; more preferably, the antibody or fragment thereof includes a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region.

[0031] According to a specific embodiment of the present invention, the antibody is a monoclonal antibody, preferably a mouse, chimeric, or humanized monoclonal antibody. Preferably, the heavy chain constant region of the monoclonal antibody is an IgG1 or IgG4 subtype, and the light chain constant region is a κ type. Preferably, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:23; the light chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:24.

[0032] According to a specific embodiment of the present invention, the anti-human GPRC5D antibody of the present invention is a monoclonal antibody. Preferably, the anti-human GPRC5D antibody provided by the present invention is an immunoglobulin, for example, the type of the immunoglobulin is human IgA, IgD, IgE, IgG or IgM. More preferably, the antibody is a human IgG1 or IgG4 subtype.

[0033] The antibodies or fragments thereof provided by this invention can also be used to construct bispecific antibodies against GPRC5D×CD3. Therefore, as described below, this invention also provides the following bispecific antibody constructs.

[0034] The present invention provides a bispecific antibody construct comprising a first binding domain that binds to a group of 5D members of the G protein-coupled receptor family C (GPRC5D) and a second binding domain that binds to CD3 on the surface of T cells.

[0035] In the bispecific antibody construct provided by the present invention, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) comprise a combination of CDRs selected from the following (HCDR-1, HCDR-2, HCDR-3; LCDR-1, LCDR-2, LCDR-3):

[0036] (1) HCDR-1 containing the amino acid sequence shown in SEQ ID NO:28 (SDYAWN), HCDR-2 containing the amino acid sequence shown in SEQ ID NO:29 (YISYSGSATYNPSLKS), HCDR-3 containing the amino acid sequence shown in SEQ ID NO:30 (GGIAGRGRWGAMDY); and LCDR-1 containing the amino acid sequence shown in SEQ ID NO:31 (KASQNVGTNVA), LCDR-2 containing the amino acid sequence shown in SEQ ID NO:32 (SASYRDS), and LCDR-3 containing the amino acid sequence shown in SEQ ID NO:33 (QQYKSYPLT);

[0037] (2) HCDR-1 comprising the amino acid sequence shown in SEQ ID NO:28 (SDYAWN), HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:34 (YISYSGSATYSPSLKS), HCDR-3 comprising the amino acid sequence shown in SEQ ID NO:30 (GGIAGRGRWGAMDY); and LCDR-1 comprising the amino acid sequence shown in SEQ ID NO:31 (KASQNVGTNVA), LCDR-2 comprising the amino acid sequence shown in SEQ ID NO:32 (SASYRDS), and LCDR-3 comprising the amino acid sequence shown in SEQ ID NO:33 (QQYKSYPLT);

[0038] (3) HCDR-1 containing the amino acid sequence (SYTIQ) shown in SEQ ID NO:35, HCDR-2 containing the amino acid sequence (YIIPSSGYTNYNQKFKD) shown in SEQ ID NO:36, and HCDR-3 containing the amino acid sequence (NYGNWGFTY) shown in SEQ ID NO:37; and LCDR-1 containing the amino acid sequence (KASQNVGSAVT) shown in SEQ ID NO:38, LCDR-2 containing the amino acid sequence (SASNRYT) shown in SEQ ID NO:39, and LCDR-3 containing the amino acid sequence (QQYSNYPLT) shown in SEQ ID NO:40;

[0039] (4) HCDR-1 containing the amino acid sequence (YYVMH) shown in SEQ ID NO:41, HCDR-2 containing the amino acid sequence (YINPYNDGTKYNEKFKG) shown in SEQ ID NO:42, and HCDR-3 containing the amino acid sequence (GGVRRYFDY) shown in SEQ ID NO:43; and LCDR-1 containing the amino acid sequence (RASQDIGSNLN) shown in SEQ ID NO:44, LCDR-2 containing the amino acid sequence (ATSSLDS) shown in SEQ ID NO:45, and LCDR-3 containing the amino acid sequence (LQYATFPYT) shown in SEQ ID NO:46;

[0040] (5) HCDR-1 comprising the amino acid sequence (YYVIH) shown in SEQ ID NO:47, HCDR-2 comprising the amino acid sequence (YINPYNDGTKYNEKFKG) shown in SEQ ID NO:42, and HCDR-3 comprising the amino acid sequence (GGVRRYFDY) shown in SEQ ID NO:43; and LCDR-1 comprising the amino acid sequence (RASQDIGSNLN) shown in SEQ ID NO:44, LCDR-2 comprising the amino acid sequence (ATSSLDS) shown in SEQ ID NO:45, and LCDR-3 comprising the amino acid sequence (LQYATFPYT) shown in SEQ ID NO:46; and

[0041] (6) HCDR-1 containing the amino acid sequence (YYVIH) shown in SEQ ID NO:47, HCDR-2 containing the amino acid sequence (YINPYNAGTKYNEKFKG) shown in SEQ ID NO:48, HCDR-3 containing the amino acid sequence (GGVRRYFDY) shown in SEQ ID NO:43; and LCDR-1 containing the amino acid sequence (RASQDIGSNLN) shown in SEQ ID NO:44, LCDR-2 containing the amino acid sequence (ATSSLDS) shown in SEQ ID NO:45, and LCDR-3 containing the amino acid sequence (LQYATFPYT) shown in SEQ ID NO:46.

[0042] Preferably, in the first binding domain, the heavy chain variable region comprises a sequence selected from: an amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:11 or SEQ ID NO:19, or an amino acid sequence having at least 75% identity with said amino acid sequence; and / or, the light chain variable region comprises a sequence selected from: an amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:12 or SEQ ID NO:20, or an amino acid sequence having at least 75% identity with said amino acid sequence.

[0043] More preferably, the heavy chain variable region and the light chain variable region included in the first binding structural domain comprise a combination of sequences selected from the following:

[0044] (1) The amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:5; and, the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:6;

[0045] (2) The amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:13; and, the amino acid sequence shown in SEQ ID NO:14 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:14;

[0046] (3) The amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:7; and, the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:8;

[0047] (4) The amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:15; and, the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:16.

[0048] (5) The amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:9; and, the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:10;

[0049] (6) The amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:17; and, the amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:18.

[0050] (7) The amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:11; and, the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:12; or

[0051] (8) The amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:19; and, the amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:20.

[0052] In the bispecific antibody construct provided by the present invention, the second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) comprise a combination of CDRs selected from the following (HCDR-1, HCDR-2, HCDR-3; LCDR-1, LCDR-2, LCDR-3):

[0053] HCDR-1 comprising the amino acid sequence (TYAMN) shown in SEQ ID NO:49, HCDR-2 comprising the amino acid sequence (RIRSKYNNYATYYADSVKD) shown in SEQ ID NO:50, and HCDR-3 comprising the amino acid sequence (HGNFGNSYVSYFAY) shown in SEQ ID NO:51; and LCDR-1 comprising the amino acid sequence (RSSTGAVTTSNYAN) shown in SEQ ID NO:52, LCDR-2 comprising the amino acid sequence (GTNKRAP) shown in SEQ ID NO:53, and LCDR-3 comprising the amino acid sequence (ALWYSNLWV) shown in SEQ ID NO:54.

[0054] Preferably, in the second binding domain, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having at least 75% identity with the amino acid sequence; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:22 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0055] More preferably, the bispecific antibody construct provided by the present invention comprises four polypeptide chains:

[0056] 1) Heavy chain 1, wherein the heavy chain 1 contains structural domains arranged in the order VH-CH1-CH2-CH3 from the N end to the C end;

[0057] 2) Heavy chain 2, which contains structural domains arranged in the order VL-CH1-CH2-CH3 from the N-end to the C-end;

[0058] 3) Light chain 1, wherein the light chain 1 contains structural domains arranged in VL-CL from the N end to the C end;

[0059] 4) Light chain 2, wherein the light chain 2 contains structural domains arranged in VH-CL from the N end to the C end;

[0060] In this invention, VH and VL contained in heavy chain 1 and light chain 1 pair to form the first binding domain for GPRC5D; VL and VH contained in heavy chain 2 and light chain 2 pair to form the second binding domain for CD3. A schematic diagram of the bispecific antibody construct provided by this invention is shown below. Figure 1 .

[0061] According to a specific embodiment of the present invention, in the bispecific antibody construct provided by the present invention, the CH1-CH2-CH3 domain of the heavy chain 1 contains the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having at least 75% identity with said amino acid sequence; the CH1-CH2-CH3 domain of the heavy chain 2 contains the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 75% identity with said amino acid sequence; the CL domain of the light chain 1 contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 75% identity with said amino acid sequence; the CL domain of the light chain 2 contains the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with said amino acid sequence.

[0062] On the other hand, the present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain CDR, a light chain CDR, a light chain variable region, a heavy chain variable region, a heavy chain, or a light chain contained in an antibody or fragment thereof provided by the present invention or a bispecific antibody construct provided by the present invention.

[0063] The nucleic acid molecules of the present invention can be cloned into vectors, and then transformed or transfected into host cells. Therefore, in another aspect, the present invention also provides a vector containing the nucleic acid molecules of the present invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a bacteriophage vector, etc. The vectors or nucleic acid molecules of the present invention can be used to transform or transfect host cells. Therefore, in yet another aspect, the present invention provides a host cell containing the nucleic acid molecules and / or vectors of the present invention, or the host cell is transformed or transfected by the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant, or animal cells.

[0064] The antibodies or fragments thereof provided by this invention, as well as the bispecific antibody constructs, can be obtained using any method known in the art. For example, the host cells provided by this invention can be cultured while allowing the host cells to express both the heavy and light chains of the antibody. Optionally, the method further includes a step of recovering the generated antibody.

[0065] The antibodies or fragments thereof, bispecific antibody constructs, nucleic acid molecules, vectors, host cells, and / or provided by the present invention can be included in compositions, more particularly in pharmaceutical compositions such as pharmaceutical formulations, for use in various purposes as needed. Therefore, in another aspect, the present invention also provides a composition, such as a pharmaceutical composition, comprising the antibodies or fragments thereof described in the present invention, bispecific antibody constructs, nucleic acid molecules, vectors, and / or host cells, and optionally pharmaceutically acceptable excipients.

[0066] In another aspect, the present invention also provides the use of the antibody or fragment thereof, bispecific antibody constructs, nucleic acid molecules, vectors, host cells and / or compositions in the preparation of medicaments for treating diseases, said diseases being tumors or cancers. Preferably, the disease is a tumor or cancer associated with GPRC5D expression (e.g., high expression), such as lymphoma or myeloma, particularly multiple myeloma.

[0067] Accordingly, the present invention also provides a method for treating a disease, the method comprising administering to a subject in need an antibody or fragment thereof, a bispecific antibody construct, a nucleic acid molecule, a vector, a host cell, and / or a composition thereof, wherein the disease is a tumor or cancer. Preferably, the disease is a tumor or cancer associated with GPRC5D expression (e.g., high expression), such as lymphoma or myeloma; the disease is particularly multiple myeloma. Preferably, the subject is a mammal; more preferably, the subject is a human.

[0068] In another aspect, the present invention also provides the use of the antibody or fragment thereof, bispecific antibody constructs, nucleic acid molecules, vectors, host cells, and / or compositions in the preparation of reagents for diagnosing diseases or detecting the presence of antigen GPRC5D, said diseases being tumors or cancers. Preferably, said diseases are tumors or cancers associated with GPRC5D expression (e.g., high expression), such as lymphoma or myeloma, particularly multiple myeloma.

[0069] In another aspect, the present invention provides a kit comprising the antibody or a fragment thereof, a bispecific antibody construct, a nucleic acid molecule, a vector, a host cell, and / or a composition thereof. The kit can be used for the aforementioned treatments, or for diagnostic or antigen detection. Depending on the intended application, the kit may also contain other reagents for treatment, diagnosis, or detection. For example, the kit is for detecting the presence of GPRC5D in any biological sample using an ELISA. Attached Figure Description

[0070] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0071] Figure 1This is a schematic diagram of the structure of the anti-human GPRC5D×CD3 bispecific antibody.

[0072] Figure 2 The results of FACS analysis show the binding of hybridoma cell supernatant to different cells.

[0073] Figure 3 The results of FACS analysis show the binding of hybridoma cell supernatant to different cells.

[0074] Figure 4 The results of FACS analysis show the binding of hybridoma cell supernatant to different cells, where 4-1: MM1R cells; 4-2: Nalm6 cells; 4-3: OPM2 cells; 4-4: NCI-H929 cells.

[0075] Figure 5 The FACS results show the binding of humanized antibodies to human GPRC5D-expressing cells, where 5-1: 9D7 (hz); 5-2: 26D1 (hz); 5-3: 24F5 (hz); 5-4: 6E97 (hz).

[0076] Figure 6 The FACS results show the binding of humanized antibodies to monkey GPRC5D-expressing cells, where 6-1: 9D7 (hz); 6-2: 26D1 (hz); 6-3: 24F5 (hz); 6-4: 6E97 (hz).

[0077] Figure 7 The results of FACS detection show the binding of humanized antibodies to different tumor cell lines, where 7-1: MM.1R cells; 7-2: MOLP8 cells.

[0078] Figure 8 The results show the affinity test results between the humanized antibody and GPRC5D, where 8-1: GPRC5D×CD3 JNJ; 8-2: 9D7 (hz); 8-3: 26D1 (hz); 8-4: 24F5 (hz); 8-5: 6E97 (hz).

[0079] Figure 9 The results of humanized antibody-mediated ADCC detection are shown, where 9-1: wild-type NCI-H929; 9-2: GPRC5D knockout NCI-H929.

[0080] Figure 10 The results of detection of bispecific antibody-mediated T cell killing are shown, where 10⁻¹: co-culture experiment 1; 10⁻²: co-culture experiment 2; 10⁻³: co-culture experiment 3. Detailed Implementation

[0081] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0082] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials, reagents, and other materials used in the following examples are commercially available products. Specifically:

[0083] Human GPRC5D protein: Genbank accession number NM_018654;

[0084] Monkey GPRC5D protein: Genbank accession number XM_005570193.2.

[0085] Example 1 Preparation of hybridoma cells and acquisition of antibody proteins

[0086] Human GPRC5D cells (Kyinno Biotechnology (Beijing) Co., Ltd., catalog number KC-1583) were prepared into a cell concentration of 1×10⁻⁶ cells in PBS containing 2% FBS. 7 Cell suspension of cells per ml.

[0087] The cell suspension was used as an immunogen to immunize mice. Five mice were immunized subcutaneously, and five mice were immunized intramuscularly. Quick Antibody 5W water-soluble adjuvant was used. Two weeks after the booster immunization, the titer was measured. Two mice with high titers were selected for immunization shock. Serum was collected three days later, and spleens were harvested after dissection. The isolated spleen cells were fused with cultured myeloma cells in 96-well plates, and selective medium was added for selection. The medium was changed after 7 days, and cell-based ELISA was performed 10 days later. ELISA was performed using 293T cells expressing human GPRC5D (Kyinno Biotechnology, catalog number KC-1723). Cells with an OD value more than 10 times greater than the negative control (serum from normal mice) were selected for flow cytometry analysis. Flow cytometry also used 293T cells expressing human GPRC5D (Kyinno Biotechnology, catalog number KC-1723).

[0088] Cells that are positive for both ELISA and FACS are selected, and subclonal plating is performed using the limiting dilution method to select monoclonal cells. The culture supernatant from the selected monoclonal cells is then subjected to ELISA and flow cytometry analysis, and cells that are positive for both ELISA and FACS are selected for further culture.

[0089] The culture supernatant of the obtained hybridoma cells was collected, centrifuged, concentrated, and purified using a G protein affinity column. The purified sample was then diluted 10-fold with 20 mM citric acid + 100 mM NaCl, pH 5.5 buffer, concentrated to an appropriate volume using an ultrafiltration concentrator, and aliquoted into 1.5 mL EP tubes (200 μL / tube) and stored at -80°C. Simultaneously, a portion of the sample was subjected to SDS-PAG and FACS analysis to verify protein purity and activity.

[0090] Example 2 ELISA detection of human GPRC5D binding in hybridoma cell culture supernatant

[0091] BXPC-3 cells expressing human GPRC5D (Kyinno Biotechnology, catalog number KC-1717) were prepared into a cell concentration of 1×10⁻⁶ cells in PBS containing 2% FBS. 5 Cell suspension of 100 μl / well was added to each well of a 96-well plate and incubated overnight at 37°C. The liquid in the wells was discarded, and the cells were washed three times with washing buffer. 80 μl of 4% tissue fixative (Beyotime, catalog number P0099) was added to each well, and the plate was fixed at room temperature for 15 min. The cells were washed twice with PBS, air-dried, and 250 μl of blocking buffer containing 2% BSA was added to each well. The plate was incubated at 37°C for 1 h, and washed three times with PBS. 50-100 μl of culture supernatant of the hybridoma cells to be tested was added to each well. Positive controls (containing serum from fusion mice), negative controls (containing serum from normal mice), and blank controls (containing culture medium) were also included. The plates were incubated at 37°C for 1-2 h, washed, and patted dry. Then, add 50-100 μl of enzyme-labeled secondary antibody (1:10000 dilution of horseradish peroxidase-labeled goat anti-mouse IgG (SIGMA, catalog number A9044-2ml)) to each well, incubate at 37°C for 1-2 hours, wash, and blot dry. Add 50-100 μl of freshly prepared substrate development solution (TMB) to each well, and incubate at 37°C for 10-30 minutes.

[0092] The reaction was terminated by adding 2 mol / L H2SO4, and the OD value was read on an enzyme-linked immunosorbent assay (ELISA) reader.

[0093] Result interpretation: A positive result is defined as P / N > 2:1 (P represents the positive value, and N represents the normal mouse serum value). If the negative control well is colorless or nearly colorless, and the positive control well shows clear color, the result can be observed directly with the naked eye. The results are shown in Table 1.

[0094] Table 1. ELISA results of hybridoma cell culture supernatant binding to human GPRC5D

[0095]

[0096]

[0097] Example 3 Construction of anti-human GPRC5D×CD3 bispecific antibody

[0098] A bispecific antibody against human GPRC5D×CD3 was constructed using the GPRC5D-targeting domain and the CD3-targeting domain of Talquetamab (JNJ-64407564) from Johnson & Johnson. A schematic diagram of the structure of this bispecific antibody is shown below. Figure 1 The structural domains contained in each chain, from the N-terminus to the C-terminus, are as follows: Heavy chain 1: VH-CH1-CH2-CH3

[0099] >VH(SEQ ID NO:1)

[0100] QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNS

[0101] DTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTL

[0102] VTVSS

[0103] >CH1-CH2-CH3(SEQ ID NO:25)

[0104] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

[0105] SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPPAA

[0106] GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE

[0107] QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTL

[0108] PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSK

[0109] LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0110] Heavy chain 2: VL-CH1-CH2-CH3

[0111] >VL(SEQ ID NO:2)

[0112] QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL

[0113] >CH1-CH2-CH3(SEQ ID NO:26)

[0114] SSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDDRVEPKSCDKTHTCPPCPAPPAAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0115] Light chain 1: VL-CL

[0116] >VL(SEQ ID NO:3)

[0117] DIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIK

[0118] >CL(SEQ ID NO:24)

[0119] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0120] Light Chain 2: VH-CL

[0121] >VH(SEQ ID NO:4)

[0122] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

[0123] >CL(SEQ ID NO:27)

[0124] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0125] For the four polypeptide chains mentioned above, primers were designed, the corresponding coding genes were synthesized, and they were ligated into eukaryotic expression plasmids. The four recombinant expression plasmids were co-transfected into HEK293F cells. After culturing the cells for 5-7 days, the supernatant of secreted antibodies was harvested. Bispecific antibodies were purified from the cell culture supernatant using MabSelectSure affinity chromatography and SP cation exchange chromatography.

[0126] The obtained bispecific antibody was named "GPRC5D×CD3 JNJ".

[0127] Example 4 FACS detection of human GPRC5D binding in hybridoma cell culture supernatant

[0128] The 293T cells expressing human GPRC5D were prepared in PBS containing 2% FBS to a cell concentration of 1×10⁻⁶ cells / cells. 5 Cell suspension of cells per ml.

[0129] Add 50 μl of cell suspension to each flow cytometry tube (sample tube), then add 50 μl of culture supernatant of the hybridoma cells to be tested and the anti-human GPRC5D×CD3 bispecific antibody GPRC5D×CD3 JNJ as a positive control antibody, and incubate at 4°C for 60 minutes. Add 1 ml of flow cytometry buffer to each flow cytometry tube, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, and repeat the washing three times. Also prepare control tube 1 (without culture supernatant and the secondary antibody mentioned below, only cell suspension) and control tube 2 (without culture supernatant, only cell suspension and the secondary antibody mentioned below).

[0130] Then, add 100 μl of flow cytometry buffer to each flow cytometry tube for resuspending. Add 5 μl of PE-labeled anti-mouse Fc-tagged secondary antibody (Biolegend, catalog number 409304) according to experimental requirements, incubate at 4°C in the dark for 30 minutes, then add 1 ml of flow cytometry buffer, centrifuge at 1200 rpm for 5 minutes at room temperature, discard the supernatant, and repeat the washing three times.

[0131] Add 250 μl of flow cytometry buffer to each tube again, resuspend and mix well, and then perform the analysis. The results are shown in Tables 2-1 to 2-4. Figure 2 2-1 to 2-4 in the middle.

[0132] Table 2-1

[0133] Samples (indicated by hybridoma cell number) 26D1 Positive control antibody EC50 (μg / mL) 0.7881 1.684 MFI.Max 11491 10980

[0134] Table 2-2

[0135] Samples (indicated by hybridoma cell number) 26D1 9D7 EC50 (μg / mL) 0.1688 0.1853 MFI.Max 9755 9785

[0136] Table 2-3

[0137]

[0138]

[0139] Table 2-4

[0140] Samples (indicated by hybridoma cell number) 9D7 57G4 EC50 (μg / mL) 0.2793 0.1918 MFI.Max 9255 9227

[0141] Example 5 FACS detection of hybridoma cell supernatant binding to monkey GPRC5D

[0142] Following the procedure described in Example 4, the human GPRC5D-expressing cells were replaced with 293T cells (Kyinno Biosciences, catalog number KC-1586) expressing monkey GPRC5D, and the binding of the culture supernatant of the hybridoma cells of the present invention to monkey GPRC5D was detected.

[0143] The results of the combined FACS detection are shown in Tables 3-1 to 3-3 and Figure 3The antibody was 3-1 to 3-3; the positive control antibody was GPRC5D×CD3 JNJ.

[0144] Table 3-1

[0145] Samples (indicated by hybridoma cell number) 26D1 Positive control antibody EC50 (μg / mL) 12.31 13.07 MFI.Max 1317 726

[0146] Table 3-2

[0147]

[0148] Table 3-3

[0149]

[0150]

[0151] Example 6 FACS detection of hybridoma cell supernatant binding to different cells

[0152] Following the procedure described in Example 4, the human GPRC5D expressing cells were replaced with other cell types: NALM6 (Kyinno, catalog number KC-0626), NCI-H929 (Kyinno, catalog number KC-0629), MM1R (Kyinno, catalog number KC-0619), and OPM2 (Kyinno, catalog number KC-0631) cells. The binding of the culture supernatant of the hybridoma cells of the present invention to human GPRC5D was then detected.

[0153] The detection results combined with FACS are shown in Table 4 and Figure 4 The antibody was 4-1 to 4-4; the positive control antibody was GPRC5D×CD3JNJ.

[0154] Table 4. FACS results of hybridoma cell supernatant and cell binding.

[0155]

[0156]

[0157] Example 7 Sequence identification of the mouse anti-antibody of the present invention

[0158] Selected hybridoma cell lines were chosen, RNA was extracted from monoclonal cells, reverse transcribed into cDNA, the cDNA was amplified, recovered from the gel, and ligated into a sequencing vector for sequencing analysis.

[0159] The sequence of the variable region of the heavy and light chains of an exemplary mouse antibody obtained from hybridoma cells is shown below, where the bold and underlined portion indicates the CDR (defined according to the KABAT method).

[0160] A1:

[0161] >9D7-H(VH / HCDR-1 / HCDR-2 / HCDR-3:SEQ ID NO:5 / 28 / 29 / 30)

[0162] QVQLKESGPGLVKPSQSLSLTCTVTGYSIT SDYAWN WIRQFPGNKLEWMG YISYSGSATYNPSLKS RISITRDTSKNQFFLQLNSMTTEDTATYCAR GGIAGRGRWGAMDY WGQGTSVTVSS

[0163] >9D7-L(VL / LCDR-1 / LCDR-2 / LCDR-3:SEQ ID NO:6 / 31 / 32 / 33)

[0164] DIVMTQQKFMSTSVGDRVSVTC KASQNVGTNVA WYQNKPGQSPKALIY SASYRDS

[0165] GVPDRFTGRGSGTDFTLTISNVQSEDLAEYFC QQYKSYPLT FGAGTKLELC

[0166] A2:

[0167] >26D1-H(VH / HCDR-1 / HCDR-2 / HCDR-3:SEQ ID NO:7 / 35 / 36 / 37)

[0168] QVQLKQSGAELAGPGASVKMSCCASGYTFT SYTIQ WIKQRPGQGLEWIG YIIPSSGYTNYNQKFKD KATLTADKSSNTAMQLSSLTSEDSAVIYCAN NYGNWGFTY WGQGTLVTVSA

[0169] >26D1-L(VL / LCDR-1 / LCDR-2 / LCDR-3:SEQ ID NO:8 / 38 / 39 / 40)DILMTQSQKFMSTSVRDRVSISC KASQNVGSAVT WYQQKPGQSPRLLIY SASNRYT GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC QQYS NYPLT FGGGTKLEIK

[0170] A3

[0171] >24F5-H(VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NO:9 / 41 / 42 / 43)

[0172] QVQLKESGPELVKPGASVKISCKASGYTFP YYVMH WVKQKPGQGLEWIG YINPYNDGTKYNEKFKG KAALTSDKSSSTAYMDLSSLTSEDSAVYYCAR GGVRRYFDY WGQGTTLTVSS

[0173] >24F5-L(VL / LCDR-1 / LCDR-2 / LCDR-3:SEQ ID NO:10 / 44 / 45 / 46)

[0174] DIQMTQTPSSLSASLGERVSLTC RASQDIGSNLN WLQQEPDGTIKRLIY ATSSLDS GVPKRFSGSRSGSDYSLTISSLESEDFVDYYC LQYATFPYT FGGGTKLEIK

[0175] A4

[0176] >6E97-H(VH / HCDR-1 / HCDR-2 / HCDR-3:SEQ ID NO:11 / 47 / 42 / 43 / )

[0177] QVQLKESGPELVKPGASVKISCKASGYTFP YYVIH WVKQKPGQGLEWIG YINPYNDGTKYNEKFKG KAALTSDKSSSTAYMELSSLTSEDSAVYYCAR GGVRRYFDY WGQGTTLTVSS

[0178] >6E97-L(VL / LCDR-1 / LCDR-2 / LCDR-3:SEQ ID NO:12 / 44 / 45 / 46)

[0179] QIVLTQSPSSLSASLGERVSLTC RASQDIGSNLN WLQQEPDGTIKRLIS ATSSLDS GVPKRFSGSRSGSDYSLTISSLESEDFVDYFC LQYATFPYT FGGGTKLEIK

[0180] Example 8 Preparation of chimeric antibodies and humanized antibodies based on mouse antibodies

[0181] For the A1 antibody (mouse anti-9D7), the following human germline sequences were selected as templates for the heavy and light chains: IGHV4-39 and IGKV1-39, respectively. Homology modeling was performed on the A1 antibody, and the structure of the Fab region was simulated. After homology modeling calculations, the predicted Fab structure of the A1 antibody was finally obtained.

[0182] By comparing the predicted Fab structure and heavy chain with the IGHV4-39 sequence, all mouse amino acids in the VH, except for the CDR region and 2V, 49M, 68I, and 72R which are original mouse amino acids, were replaced with the corresponding human amino acids from the IGHV4-39 template.

[0183] By comparing the predicted Fab structure and light chain with the IGKV1-39 sequence, all mouse amino acids in this VL, except for the CDR region and 42Q, 43S, 46A, and 60D which retained the original mouse amino acids, were replaced with the corresponding human amino acids from the IGKV1-39 template. The resulting humanized sequence is as follows (where the heavy chain and light chain CDRs are shown in bold and underlined, and are obtained according to the KABAT definition):

[0184] The sequence of the humanized antibody is as follows:

[0185] >9D7-H humanized sequence (VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NO:13 / 28 / 34 / 30)

[0186] QVQLQESGPGLVKPSETLSLTCTVSGYSIT SDYAWN WIRQPPGKGLEWMG YISYSGSATYSPSLKS RITISRDTSKNQFSLKLSSVTAADTAVYYCAR GGIAGRGRWGAMDY WGQGTTVTVSS

[0187] >9D7-L humanized sequences (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NO:14 / 31 / 32 / 33)

[0188] DIQMTQSPSSLSASVGDRVTITC KASQNVGTNVA WYQQKPGQSPKALIY SASYRDS GVPDRFSGSGSGTDFTLTISSLQPEDFATYYC QQYKSYPLT FGQGTRLEIK

[0189] For the A2 antibody (mouse anti-26D1), the following human germline sequences were selected as templates for the heavy and light chains: IGHV1-46 and IGKV1-5, respectively. Homology modeling was performed on the A2 antibody, and the structure of the Fab region was simulated. After homology modeling calculations, the predicted Fab structure of the A2 antibody was finally obtained.

[0190] By comparing the predicted Fab structure and heavy chain with the IGHV1-46 sequence, it was found that all the mouse amino acids in the VH, except for the CDR region and 37I, 38K, 48I, 67R, 68A, 70L, 72A, 74K, 76S, 77N, 79A, and 98N which are original mouse amino acids, were replaced with the corresponding human amino acids from the IGHV1-46 template.

[0191] By comparing the predicted Fab structure and light chain with the IGKV1-5 sequence, all mouse amino acids in this VL, except for the CDR region and 45R, 60D, and 87F which retained the original mouse amino acids, were replaced with the corresponding human amino acids from the IGKV1-5 template. The resulting humanized sequence is as follows (where the heavy chain and light chain CDRs are shown in bold and underlined, and are obtained according to the KABAT definition):

[0192] The sequence of the humanized antibody is as follows:

[0193] >26D1-H humanized sequence (VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NO: 15 / 35 / 36 / 37)QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYTIQ WIKQAPGQGLEWIG YIIPSSGYTNYNQKFKD KATLTADKSSNTAYMELSSLTSEDTAVYYCAN NYGNWGFTY WGQGTTVTVSS

[0194] >26D1-L humanized sequence (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NO:16 / 38 / 39 / 40)

[0195] DIQMTQSPSTLSASVGDRVTITC KASQNVGSAVT WYQQKPGKAPRLLIY SASNRYT G

[0196] VPDRFSGSGSGTEFTLTISSLQPDDFATYFC QQYSNYPLT FGQGTRLEIK

[0197] For the A3 antibody (mouse anti-24F5), the following human germline sequences were selected as templates for the heavy and light chains: IGHV1-46 and IGKV1-15, respectively. Homology modeling was performed on the A3 antibody, and the structure of the Fab region was simulated. After homology modeling calculations, the predicted Fab structure of the A3 antibody was finally obtained.

[0198] By comparing the predicted Fab structure and heavy chain with the IGHV1-46 sequence, it was found that all the mouse amino acids in the VH, except for the CDR region and 48I, 68A, 70L, 72S, 74K, and 79A which are original mouse amino acids, were replaced with the corresponding human amino acids from the IGHV1-46 template.

[0199] By comparing the predicted Fab structure and light chain with the IGKV1-15 sequence, all mouse amino acids in this VL, except for the CDR region and 36L, 42G, 43T, 44I, 46R, 66R, 69S, and 71Y which retained the original mouse amino acids, were replaced with the corresponding human amino acids from the IGKV1-15 template. The resulting humanized sequence is as follows (where the heavy and light chain CDRs are shown in bold and underlined, and are obtained according to the KABAT definition):

[0200] The sequence of the humanized antibody is as follows:

[0201] >24F5-H humanized sequence (VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NO: 17 / 41 / 42 / 43)QVQLVQSGAEVKKPGASVKVSCKASGYTFP YYVMH WVRQAPGQGLEWIG YINPYNDGTKYNEKFKG RATLTSDKSTSTAYMELSSLRSEDTAVYYCAR GGVRRYFDY WGQGTTVTVSS

[0202] >24F5-L humanized sequences (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NO:18 / 44 / 45 / 46)

[0203] DIQMTQSPSTLSASVGDRVTITC RASQDIGSNLN WLQQKPGGTIKRLIY ATSSLDS GVPSRFSGSRSGSEYTLTISSLQPDDFATYYC LQYATFPYT FGQGTRLEIK

[0204] For the A4 antibody (mouse anti-6E97), the following human germline sequences were selected as templates for the heavy and light chains: IGHV1-46 and IGKV1-15, respectively. Homology modeling was performed on the A4 antibody, and the structure of the Fab region was simulated. After homology modeling calculations, the predicted Fab structure of the A4 antibody was finally obtained.

[0205] By comparing the predicted Fab structure and heavy chain with the IGHV1-46 sequence, it was found that all the mouse amino acids in the VH, except for the CDR region and 48I, 68A, 70L, 72S, 74K, and 79A which are original mouse amino acids, were replaced with the corresponding human amino acids from the IGHV1-46 template.

[0206] By comparing the predicted Fab structure and light chain with the IGKV1-15 sequence, all mouse amino acids in this VL were replaced with the corresponding human amino acids from the IGKV1-15 template, except for the CDR region and the amino acids 4L, 36L, 42G, 43T, 44I, 46R, 66R, 69S, and 71Y, which retained the original mouse amino acids. The resulting humanized sequence is as follows (where the heavy chain and light chain CDRs are shown in bold and underlined, and are obtained according to the KABAT definition):

[0207] The sequence of the humanized antibody is as follows:

[0208] >6E97-H humanized sequence (VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NO: 19 / 47 / 48 / 43)QVQLVQSGAEVKKPGASVKVSCKASGYTFP YYVIH WVRQAPGQGLEWIG YINPYNAGTKYNEKFKG RATLTSDKSTSTAYMELSSLRSEDTAVYYCAR GGVRRYFDY WGQGTTVTVSS

[0209] >6E97-L humanized sequence (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NO:20 / 44 / 45 / 46)

[0210] DIQLTQSPSTLSASVGDRVTITC RASQDIGSNLN WLQQKPGGTIKRLIS ATSSLDS GVPSRFSGSRSGSEYTLTISSLQPDDFATYYC LQYATFPYT FGQGTRLEIK

[0211] Using the sequence shown in SEQ ID NO:23 as the heavy chain constant region and the sequence shown in SEQ ID NO:24 as the light chain constant region, primers were redesigned for the antibody DNA sequence targeting GRRC5D obtained from sequencing. The genes for the corresponding chimeric antibody and humanized antibody were synthesized, ligated into a eukaryotic expression vector, transformed into DH5alpha competent cells, and cultured overnight at 37°C. Single clones were selected for sequencing identification. Strains with correct sequences were selected, cultured, and plasmids were extracted and transfected into mammalian expression cells 293F. The cells were then incubated at 37°C with 5% CO2 for 7 days.

[0212] Collect the supernatant, centrifuge, filter, and purify using a protein G affinity chromatography column. The purity of the purified antibody is assessed by SDS-PAGE electrophoresis, and the antibody concentration is determined using a BCA protein assay kit. The antibody is then aliquoted and stored at -80°C for later use. The chimeric antibody is named "mouse antibody abbreviation (chi)" and the humanized antibody is named "mouse antibody abbreviation (hz)".

[0213] >CH1-CH3 heavy chain constant region (SEQ ID NO:23)

[0214] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0215] >CL1 light chain constant region (SEQ ID NO:24)

[0216] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0217] Example 9FACS detection of humanized antibody binding to human GPRC5D

[0218] The experimental procedure was as described in Example 4. 293T cells expressing human GPRC5D (Kyinno Biotechnology, catalog number KC-1584) were used. The test antibody, positive control antibody GPRC5D×CD3 JNJ, or negative control antibody (hIgG1) were added. Concentrations are described in [reference needed]. Figure 4 Control tube 1 (without antibodies and secondary antibodies, only cell suspension) and control tube 2 (without antibodies, only cell suspension and secondary antibody) were also set up.

[0219] The results are shown in Table 5 and Figure 5 5-1 to 5-4 in the middle.

[0220] Table 5. FACS detection results of humanized antibody binding to cells

[0221]

[0222]

[0223] Example 10 FACS detection of humanized antibody binding to monkey GPRC5D

[0224] The experimental procedure was as described in Example 4. 293T cells expressing cyno GPRC5D (Kyinno Biosciences, catalog number KC-1586) were used. The test antibody, positive control antibody GPRC5D×CD3 JNJ, or negative control antibody (hIgG1) were added. Concentrations are described in [reference needed]. Figure 6 Control tube 1 (without antibodies and secondary antibodies, only cell suspension) and control tube 2 (without antibodies, only cell suspension and secondary antibody) were also set up.

[0225] The results are shown in Table 6 and Figure 6 6-1 to 6-4 in the middle.

[0226] Table 6. FACS detection results of humanized antibody binding to cells

[0227]

[0228]

[0229] Example 11 FACS detection of humanized antibodies binding to GPRC5A / B / C

[0230] The experimental procedure was as described in Example 4, using five other cell groups expressing members of the G protein-coupled receptor family C: 293T cells expressing human GPRC5A (293T-GPRC5A, Kyinno, catalog number KC-1888), 293T cells expressing human GPRC5B (293T-GPRC5B, Kyinno, catalog number KC-1854), and 293T cells expressing human GPRC5C (293T-GPRC5C, Kyinno, catalog number KC-1889). The test antibody, positive control antibody GPRC5D×CD3 JNJ, or negative control antibody (hIgG1) were added at a concentration of 2 μg / mL. Control tube 1 (cell suspension only, no antibody or secondary antibody) and control tube 2 (cell suspension and secondary antibody only, no antibody) were also included.

[0231] The results are shown in Table 7.

[0232] Table 7. FACS detection results of humanized antibody binding to cells (μg / mL)

[0233]

[0234] Example 12 FACS detection of humanized antibodies binding to tumor cell lines

[0235] The experimental procedure was the same as in Example 4, but the cells used were multiple myeloma cells MM.1R (Kyinno Biosciences, catalog number KC-0619) and MOLP8 (Kyinno Biosciences, catalog number KC-0622). The test antibody, positive control antibody GPRC5D×CD3 JNJ, or negative control antibody (hIgG1) were added. Control tube 1 (no antibody or secondary antibody added, only cell suspension) and control tube 2 (no antibody added, only cell suspension and secondary antibody added) were also set up.

[0236] The results are shown in Table 8 and Figure 7 7-1 and 7-2 in the text.

[0237] Table 8. FACS detection results of humanized antibodies binding to tumor cell lines

[0238]

[0239]

[0240] Example 13 Affinity detection of humanized antibody with GPRC5D protein

[0241] The antibody to be tested was prepared into a 10 μg / mL solution using PBS buffer. Following the Fortebio instructions, the antibody was captured using an AHC chip. Pre-prepared human GPRC5D protein (Kaikai Biotechnology, catalog number GPR-HM05P) (protein diluted 2-fold at a maximum concentration of 200 nM, in 6 gradients) was flowed through the chip under the following conditions: flow rate 30 μl / min; antigen-antibody binding time 200 seconds; dissociation time 500 seconds. The results were fitted using the instrument's dedicated software to analyze the affinity between the antibody and the antigen. The results are shown in Table 9. Figure 8 8-1 to 8-5 in the middle.

[0242] Table 9. Affinity test results between humanized antibodies and GPRC5D protein

[0243] Antibody KD(M) kon(1 / Ms) kdis(1 / s) Positive control antibody 9.47E-10 2.18E+04 2.06E-05 9D7(hz) <1.0E-12 2.26E+04 <1.0E-07 26D1(hz) 1.70E-09 1.85E+04 3.15E-05 24F5(hz) 1.36E-09 3.99E+04 5.42E-05 6E97(hz) 4.58E-10 4.55E+04 2.08E-05

[0244] Example 14 Detection of humanized antibody-mediated ADCC

[0245] 20,000 reporter cells (Jurkat-NFAT-luc-CD16-V158, Kyinno Biotechnology, catalog number KC-1507), 20,000 wild-type NCI-H929 cells (CFSE-H929, Kyinno Biotechnology, catalog number KC-0629) or GPRC5D knockout NCI-H929 cells (CFSE-H929GPRC5DKO, Kyinno Biotechnology, catalog number KC-2203), and different concentrations of antibody (10, 3.16, 1, 0.316, 0.1, 0.0316, 0.01, 0.00316 μg / ml) were added to each well of a U-bottomed 96-well plate. The plates were co-cultured for 6 hours, and then luciferase signal was detected.

[0246] The results are shown in Table 10 and Figure 9 The antibody is 9-1 to 9-2; the positive control antibody is GPRC5D×CD3 JNJ.

[0247] Table 10. Detection results of humanized antibody-mediated ADCC

[0248]

[0249] Example 15 Obtaining anti-CD3 antibodies and detecting target affinity

[0250] The heavy chain variable region sequence SEQ ID NO. 10 and the light chain variable region sequence SEQ ID NO. 5 of the mouse antibody were obtained from patent US10066015B2. Humanization of the antibody sequence was performed, and the resulting humanized sequence is as follows (where the heavy chain and light chain CDRs are shown in bold and underlined, and are obtained according to the KABAT definition):

[0251] >VH humanized sequence (VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NO: 21 / 49 / 50 / 51)QVQLVESGGGLVKPGGSLRLSCAASGFTFS TYAMN WVRQAPGKGLEWVG RIRSKYNNYATYYADSVKD RFTISRDDAKNSLYLQMNSLRAEDTAVYYCAR HGNFGNSYVSYFAY WGQGTTVTVSS

[0252] >VL humanized sequences (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NO:22 / 52 / 53 / 54)

[0253] DIQMTQSPSSLSASVGDRVTITC RSSTGAVTTSNYAN WVQQKPGKAPKALIG GTNKRAP GVPSRFSGSLSGDDATLTISSLQPEDFATYYC ALWYSNLWV FGQGTRLEIK

[0254] As described in Example 8, an antibody containing the above-mentioned humanized sequence was obtained and named SP34V1(hz); similarly, a chimeric antibody was obtained using the heavy and light chain variable region sequences of the above-mentioned murine antibody in patent US10066015B2 and named SP34(chi).

[0255] The humanized antibody and chimeric antibody were prepared into a 10 μg / mL solution using PBS buffer. Following the Fortebio instructions, the antibody was captured using an AHC chip. Pre-prepared human CD3 protein (Kaikai Biotechnology, catalog number GPR-HM05P) (protein diluted 2-fold at a maximum concentration of 200 nM, in 6 gradients) was flowed through the chip under the following conditions: flow rate 30 μl / min; antigen-antibody binding time 200 seconds; dissociation time 500 seconds. The results were fitted using the instrument's dedicated software to analyze the antibody-antigen affinity. The results are shown in Table 11.

[0256] Table 11. Results of antibody-CD3 protein affinity assay

[0257] Antibody KD(M) kon(1 / Ms) kdis(1 / s) SP34V1(hz) 1.80E-08 4.42E+05 7.95E-03 SP34(chi) 5.46E-09 5.01E+05 2.74E-03

[0258] Example 16 Construction of the bispecific antibody of the present invention

[0259] According to Example 3, the bispecific antibody of the present invention was constructed, except that the VH in heavy chain 1 and the VL in light chain 1 were replaced with the VH and VL of the humanized antibodies 9D7(hz), 26D1(hz), 24F5(hz) and 6E97(hz) of the present invention, respectively, and the VL in heavy chain 2 and the VH in light chain 2 were replaced with the humanized CD3-targeting sequence obtained in Example 15.

[0260] The VH and VL containing the humanized antibodies 9D7(hz), 26D1(hz), 24F5(hz) and 6E97(hz) of the present invention, respectively, and the anti-human GPRC5D×CD3 bispecific antibodies with the above sequences are named "GPRC5D×CD3 9D7(hz)", "GPRC5D×CD3 26D1(hz)", "GPRC5D×CD3 24F5(hz)", and "GPRC5D×CD3 26D1(hz)", respectively.

[0261] Example 17 GPRC5D and CD3 dual antibody-mediated T cell killing assay

[0262] PBMCs and CD3 / CD28 magnetic beads were mixed at a ratio of 2:1 in RPMI1640 containing 10 ng / ml IL-2 and 10% FBS and cultured for 7 days to activate T cells.

[0263] Co-culture experiment 1: 293T LDHA-Hibit-GPRC5D cells (Kyinno, catalog number KC-2151) were seeded one day in advance in 96-well plates. 10,000 of these T cells were added to each well, along with different concentrations of the positive control antibody GPRC5D×CD3 JNJ and the bispecific antibody of this invention (1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, 0.000001, 0.000001 μg / ml). The cells were co-cultured for 18 hours in RPMI 1640 containing 10 ng / ml IL-2 and 10% FBS. Nano- The HiBiT Extracellular Reagent (Promega, catalog number N2420) was used to detect LDHA-HIBIT released from dead cells in the supernatant, thereby determining the proportion of cytotoxic cells. Two control groups were set up for each antibody: one group included 293T LDHA-Hibit-GPRC5D cells and the antibody, but without T cells; the other group included 293T LDHA-Hibit cells (Kyinno), T cells, and the antibody to exclude non-antibody-mediated killing and antibody-mediated non-T cell killing. Results are shown below. Figure 10 10-1 in the middle.

[0264] Co-culture experiment 2: NCI-H929 (expressing GPRC5D) and K562 cells (not expressing GPRC5D) were stained with CFSE and named CFSE-H929 and CFSE-K562, respectively. 10,000 activated T cells, 3,000 CFSE-H929 cells, and different concentrations of the positive control antibody GPRC5D×CD3 JNJ and the bispecific antibody of this invention (1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, 0.000001 μg / ml) were added to each well of a U-bottomed 96-well plate and co-cultured for 18 hours in RPMI 1640 containing 10 ng / ml IL-2 and 10% FBS. Then, 7-AAD staining was performed, and flow cytometry was used to count the proportion of CFSE+7-AAD+ cells to CSFE+ cells as the cell killing ratio. This experiment included two control groups for each of the two antibodies: one group included CFSE-H929 cells and the antibody, but without T cells; the other group included CFSE-K562 cells, T cells, and the antibody to exclude non-antibody-mediated killing and antibody-mediated non-T cell killing. Results are shown below. Figure 10 10-2 in the middle.

[0265] Co-culture Experiment 3: Wild-type NCI-H929 cells (Kyinno Biotechnology, catalog number KC-0629) and GPRC5D knockout NCI-H929 cells (Kyinno Biotechnology, catalog number KC-2203) were stained with CFSE and named CFSE-H929 and CFSE-H929GPRC5DKO, respectively. 10,000 activated T cells, 3,000 CFSE-H929 cells, and different concentrations of the positive control antibody GPRC5D×CD3 JNJ and the bispecific antibody of this invention (1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, 0.000001 μg / ml) were added to each well of a U-bottomed 96-well plate and co-cultured for 18 hours in RPMI 1640 containing 10 ng / ml IL-2 and 10% FBS. Then, 7-AAD staining was performed, and flow cytometry was used to count the proportion of CFSE+7-AAD+ cells among CSFE+ cells, which was taken as the cell killing ratio. Two control groups were set up for each of the four antibodies: one group with CFSE-H929 cells and antibody but no T cells; and the other group with CFSE-H929GPRC5DKO, T cells, and antibody, to exclude non-antibody-mediated killing and antibody-mediated non-T cell killing. Results are shown below. Figure 10 10-3 in the middle.

[0266] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to a member of the G protein-coupled receptor family C group 5D (GPRC5D), said antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein said heavy chain variable region (VH) and light chain variable region (VL) comprise: (1) The amino acid sequences shown in SEQ ID NO: 28 for HCDR-1, SEQ ID NO: 29 for HCDR-2, and SEQ ID NO: 30 for HCDR-3; and the amino acid sequences shown in SEQ ID NO: 31 for LCDR-1, SEQ ID NO: 32 for LCDR-2, and SEQ ID NO: 33 for LCDR-3; or (2) The amino acid sequences shown in SEQ ID NO: 28 are HCDR-1, SEQ ID NO: 34 are HCDR-2, and SEQ ID NO: 30 are HCDR-3; and the amino acid sequences shown in SEQ ID NO: 31 are LCDR-1, SEQ ID NO: 32 are LCDR-2, and SEQ ID NO: 33 are LCDR-3.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment contains the following heavy chain variable regions and light chain variable regions: (1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6; or (2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

14.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment is an anti-GPRC5D antibody or its antigen-binding fragment.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a mouse antibody, a monoclonal antibody, a chimeric antibody, or a partially or fully humanized antibody; or the antigen-binding fragment is scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that, The antibody or its antigen-binding fragment also includes a constant region.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The antibody or its antigen-binding fragment further comprises a human or mouse heavy chain constant region (CH) and / or a light chain constant region (CL).

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain and a light chain.

8. The antibody or its antigen-binding fragment according to claim 7, characterized in that, The antibody or its antigen-binding fragment contains a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody is a monoclonal antibody.

10. The antibody or its antigen-binding fragment according to claim 9, characterized in that, The antibody is a mouse, chimeric, or humanized monoclonal antibody.

11. The antibody or its antigen-binding fragment according to claim 9, characterized in that, The heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 subtype, and the light chain constant region is κ type.

12. The antibody or its antigen-binding fragment according to claim 9, characterized in that, The monoclonal antibody comprises a heavy chain constant region, the heavy chain constant region comprising an amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 23; and the monoclonal antibody comprises a light chain constant region, the light chain constant region comprising an amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

24.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody is an immunoglobulin.

14. The antibody according to claim 13, characterized in that, The immunoglobulin type is human IgA, IgD, IgE, IgG, or IgM.

15. The antibody according to claim 13, characterized in that, The antibody is a human IgG1 or IgG4 subtype.

16. A bispecific antibody construct comprising a first binding domain that binds to a group 5D member of the G protein-coupled receptor family C (GPRC5D) and a second binding domain that binds to CD3 on the surface of T cells, wherein the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) and the light chain variable region (VL) comprising: (1) The amino acid sequences shown in SEQ ID NO: 28 for HCDR-1, SEQ ID NO: 29 for HCDR-2, and SEQ ID NO: 30 for HCDR-3; and the amino acid sequences shown in SEQ ID NO: 31 for LCDR-1, SEQ ID NO: 32 for LCDR-2, and SEQ ID NO: 33 for LCDR-3; or (2) The amino acid sequences shown in SEQ ID NO: 28 of HCDR-1, the amino acid sequences shown in SEQ ID NO: 34 of HCDR-2, and the amino acid sequences shown in SEQ ID NO: 30 of HCDR-3; and the amino acid sequences shown in SEQ ID NO: 31 of LCDR-1, the amino acid sequences shown in SEQ ID NO: 32 of LCDR-2, and the amino acid sequences shown in SEQ ID NO: 33 of LCDR-3.

17. The bispecific antibody construct according to claim 16, characterized in that, The first binding structural domain includes the following heavy chain variable region and light chain variable region: (1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6; or (2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

14.

18. The bispecific antibody construct according to claim 16 or 17, characterized in that, The second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) comprise: The amino acid sequences are shown in SEQ ID NO: 49 for HCDR-1, SEQ ID NO: 50 for HCDR-2, and SEQ ID NO: 51 for HCDR-3; and the amino acid sequences are shown in SEQ ID NO: 52 for LCDR-1, SEQ ID NO: 53 for LCDR-2, and SEQ ID NO: 54 for LCDR-3.

19. The bispecific antibody construct according to claim 16 or 17, characterized in that, The second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the amino acid sequence of which is shown in SEQ ID NO:21; and the amino acid sequence of which is shown in SEQ ID NO:

22.

20. The bispecific antibody construct according to claim 16 or 17, characterized in that, The bispecific antibody construct comprises four polypeptide chains: 1) Heavy chain 1, wherein the heavy chain 1 contains structural domains arranged in the order VH-CH1-CH2-CH3 from the N-terminus to the C-terminus; 2) Heavy chain 2, which contains structural domains arranged in the order VL-CH1-CH2-CH3 from the N-terminus to the C-terminus; 3) Light chain 1, wherein the light chain 1 contains structural domains arranged in VL-CL from the N end to the C end; 4) Light chain 2, wherein the light chain 2 contains structural domains arranged in VH-CL from the N end to the C end; In this process, VH and VL contained in heavy chain 1 and light chain 1 pair up to form a first binding domain that binds to GPRC5D; VL and VH contained in heavy chain 2 and light chain 2 pair up to form a second binding domain that binds to CD3.

21. The bispecific antibody construct according to claim 19, characterized in that, The bispecific antibody construct comprises four polypeptide chains: 1) Heavy chain 1, wherein the heavy chain 1 contains structural domains arranged in the order VH-CH1-CH2-CH3 from the N-terminus to the C-terminus; 2) Heavy chain 2, which contains structural domains arranged in the order VL-CH1-CH2-CH3 from the N-terminus to the C-terminus; 3) Light chain 1, wherein the light chain 1 contains structural domains arranged in VL-CL from the N end to the C end; 4) Light chain 2, wherein the light chain 2 contains structural domains arranged in VH-CL from the N end to the C end; In this process, VH and VL contained in heavy chain 1 and light chain 1 pair up to form a first binding domain that binds to GPRC5D; VL and VH contained in heavy chain 2 and light chain 2 pair up to form a second binding domain that binds to CD3.

22. The bispecific antibody construct according to claim 20, characterized in that, The CH1-CH2-CH3 domain in heavy chain 1 contains the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity with said amino acid sequence; the CH1-CH2-CH3 domain in heavy chain 2 contains the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 75% identity with said amino acid sequence; the CL domain in light chain 1 contains the amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 75% identity with said amino acid sequence; the CL domain in light chain 2 contains the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity with said amino acid sequence.

23. The bispecific antibody construct according to claim 21, characterized in that, The CH1-CH2-CH3 domain in heavy chain 1 contains the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity with said amino acid sequence; the CH1-CH2-CH3 domain in heavy chain 2 contains the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 75% identity with said amino acid sequence; the CL domain in light chain 1 contains the amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 75% identity with said amino acid sequence; the CL domain in light chain 2 contains the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity with said amino acid sequence.

24. A nucleic acid molecule comprising an antibody or an antigen-binding fragment thereof encoding any one of claims 1 to 15, or comprising a bispecific antibody construct encoding any one of claims 16 to 23.

25. A carrier comprising the nucleic acid molecule of claim 24.

26. A host cell comprising the nucleic acid molecule of claim 24 or the vector of claim 25.

27. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 15, the bispecific antibody construct of any one of claims 16 to 23, the nucleic acid molecule of claim 24, the vector of claim 25, or the host cell of claim 26.

28. The composition according to claim 27, characterized in that, The composition is a pharmaceutical composition.

29. The composition according to claim 27 or 28, characterized in that, The composition also contains pharmaceutically acceptable excipients.

30. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 15, the bispecific antibody construct of any one of claims 16 to 23, the nucleic acid molecule of claim 24, the vector of claim 25, the host cell of claim 26, or the composition of any one of claims 27 to 29 in the preparation of a medicament for treating a disease, said disease being myeloma.

31. The use according to claim 30, characterized in that, The disease in question is multiple myeloma.

32. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 15, the bispecific antibody construct of any one of claims 16 to 23, the nucleic acid molecule of claim 24, the vector of claim 25, the host cell of claim 26, or the composition of any one of claims 27 to 29 in the preparation of a reagent for detecting the presence of antigen GPRC5D.

33. A kit comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 15, a bispecific antibody construct as claimed in any one of claims 16 to 23, a nucleic acid molecule as claimed in claim 24, a vector as claimed in claim 25, a host cell as claimed in claim 26, or a composition as claimed in any one of claims 27 to 29.

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