Anti-GPRC5D antibody and its applications

Abstract

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

Description

Technical Field

[0001] Cross - reference to related applications This application claims the priority of a Chinese patent application filed on December 31, 2021, with the application number CN202111663745.2, and incorporates the entire content thereof by reference herein. The present invention belongs to the field of biopharmaceuticals and relates to a novel anti - GPRC5D antibody or its functional fragment. The present invention further relates to the application of the antibody or its functional fragment.

Background Art

[0002] G - protein - coupled receptor family C group 5 member D (GPRC5D) is a retinoic acid - induced 40 kDa protein, which has seven transmembrane regions and one short N - terminal extracellular region, and is expressed in keratinized epithelial cells, pancreas, kidney, small intestine, spleen, testis, etc. In March 2019, a research paper on treating multiple myeloma targeting GPRC5D CAR - T was published in the journal Science Translational Medicine, initially showing the potential of GPRC5D as a new target for treating multiple myeloma. Through research on tissue expression profiles, it was found that GPRC5D is specifically highly expressed in myeloma plasma cells. It has also been proposed that diseases related to GPRC5D include chromosome 13Q14 deletion syndrome, etc. In view of the significant role of GPRC5D in diseases including multiple myeloma, more antibodies that recognize GPRC5D are needed.

Summary of the Invention

[0003] Regarding the above technical problems, the present invention provides an antibody or its fragment against G - protein - coupled receptor family C group 5 member D (GPRC5D), and also provides the use of the antibody or its fragment. Here, the present invention further provides a bispecific antibody against GPRC5D and CD3 by the antibody or its fragment. The present invention provides the following. According to one embodiment, the present invention provides an antibody or fragment thereof that can specifically bind to GPRC5D, particularly human GPRC5D. 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) include a combination of complementary determining regions (CDR-1, HCDR-2, HCDR-3; LCDR-1, LCDR-2, LCDR-3) selected from the following: (1) HCDR-1 containing the amino acid sequence (SDYAWN) shown in SEQ ID NO: 28, HCDR-2 containing the amino acid sequence (YISYSGSATYNPSLKS) shown in SEQ ID NO: 29, HCDR-3 containing the amino acid sequence (GGIAGRGRWGAMDY) shown in SEQ ID NO: 30; and LCDR-1 containing the amino acid sequence (KASQNVGTNVA) shown in SEQ ID NO: 31, LCDR-2 containing the amino acid sequence (SASYRDS) shown in SEQ ID NO: 32, and LCDR-3 containing the amino acid sequence (QQYKSYPLT) shown in SEQ ID NO: 33; (2) HCDR-1 containing the amino acid sequence (SDYAWN) shown in SEQ ID NO: 28, HCDR-2 containing the amino acid sequence (YISYSGSATYSPSLKS) shown in SEQ ID NO: 34, HCDR-3 containing the amino acid sequence (GGIAGRGRWGAMDY) shown in SEQ ID NO: 30; and LCDR-1 containing the amino acid sequence (KASQNVGTNVA) shown in SEQ ID NO: 31, LCDR-2 containing the amino acid sequence (SASYRDS) shown in SEQ ID NO: 32, and LCDR-3 containing the amino acid sequence (QQYKSYPLT) shown in SEQ ID NO: 33; (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, 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; (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, 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; (5) HCDR-1 containing the amino acid sequence (YYVIH) shown in SEQ ID NO: 47, HCDR-2 containing the amino acid sequence (YINPYNDGTKYNEKFKG) shown in SEQ ID NO: 42, 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, LCDR-3 containing the amino acid sequence (LQYATFPYT) shown in SEQ ID NO: 46; and (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. In the context of the present invention, the combinations of light-chain and heavy-chain CDRs provided are all derived from the antibody or fragment thereof of the present invention, and those skilled in the art can generally determine the CDRs contained therein based on the amino acid sequence of the variable region contained in a given antibody or fragment thereof. For example, according to a specific embodiment of the present invention, the CDRs in the amino acid sequence of the variable region are limited using the KABAT definition method. In the antibody or fragment thereof provided by the present invention, preferably, the heavy chain variable region includes a sequence selected from the amino acid sequences 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 the said amino acid sequence; and / or, the light chain variable region includes a sequence selected from the amino acid sequences 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 the said amino acid sequence. More preferably, the heavy chain variable region and light chain variable region contained in the antibody or fragment thereof include a combination of sequences selected from the following: (1) The amino acid sequence shown in Sequence ID No. 5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 5; and the amino acid sequence shown in Sequence ID No. 6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 6; (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; (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; (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; (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; (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; (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 (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. In the context of the present invention, "at least 75% identity" means any percentage between 75% and 100%, for example, 75%, 80%, 85%, 90%, and even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In particular, the antibody or fragment thereof of the present invention comprises at least a heavy chain variable region and a light chain variable region, both comprising the above-mentioned CDR and spacing framework regions, with the sequence scheme of each domain being FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Furthermore, selectively, the difference of up to 25% in the amino acid sequence due to the "at least 75% identity" may be present in any framework region within the heavy chain variable region or the light chain variable region, or in any domain or sequence other than the heavy chain variable region and the light chain variable region of the antibody or fragment thereof of the present invention. The difference can be caused by the deletion, addition, or substitution of amino acids at any position. With respect to the antigen, the antibody or fragment thereof of the present invention is an antibody or fragment thereof for anti-GPRC5D; preferably, the antibody is any form such as a mouse antibody, rabbit antibody, monoclonal antibody, chimeric antibody, partially or fully humanized antibody, or the fragment is a half-antibody or an antigen-binding fragment of an antibody or half-antibody, e.g., scFv, BsFv, dsFv, (dsFv)2, Fab, Fab, F(ab')2, or Fv; more preferably, the antibody is IgG. According to a specific embodiment of the present invention, the present invention provides a separated and structurally characterized human antibody, e.g., a human monoclonal antibody, that specifically binds to GPRC5D (e.g., human GPRC5D). The antibody or fragment thereof provided by the present invention includes, in addition to a variable region, a constant region, such as a human or mouse constant region, preferably a human or mouse heavy chain constant region (CH) and / or 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 and / or a κ or λ type light chain constant region of IgG, IgA, IgM, IgD, or IgE. 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 of the IgG1 or IgG4 subtype, and the light chain constant region is of the κ type. Preferably, the heavy chain constant region of the monoclonal antibody includes 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; and the light chain constant region of the monoclonal antibody includes 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. According to specific embodiments 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. The antibodies or fragments thereof provided by the present invention may also be used to construct bispecific antibodies against GPRC5D×CD3. Therefore, as described below, the present invention further provides the following bispecific antibody constructs. The present invention provides a bispecific antibody construct comprising a first binding domain that binds to member D of the C family of G protein-coupled receptors (GPRC5D), and a second binding domain that binds to CD3 on the surface of T cells. 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): (1) HCDR-1 containing the amino acid sequence (SDYAWN) shown in SEQ ID NO: 28, HCDR-2 containing the amino acid sequence (YISYSGSATYNPSLKS) shown in SEQ ID NO: 29, HCDR-3 containing the amino acid sequence (GGIAGRGRWGAMDY) shown in SEQ ID NO: 30; and LCDR-1 containing the amino acid sequence (KASQNVGTNVA) shown in SEQ ID NO: 31, LCDR-2 containing the amino acid sequence (SASYRDS) shown in SEQ ID NO: 32, and LCDR-3 containing the amino acid sequence (QQYKSYPLT) shown in SEQ ID NO: 33; (2) HCDR-1 containing the amino acid sequence (SDYAWN) shown in SEQ ID NO: 28, HCDR-2 containing the amino acid sequence (YISYSGSATYSPSLKS) shown in SEQ ID NO: 34, HCDR-3 containing the amino acid sequence (GGIAGRGRWGAMDY) shown in SEQ ID NO: 30; and LCDR-1 containing the amino acid sequence (KASQNVGTNVA) shown in SEQ ID NO: 31, LCDR-2 containing the amino acid sequence (SASYRDS) shown in SEQ ID NO: 32, and LCDR-3 containing the amino acid sequence (QQYKSYPLT) shown in SEQ ID NO: 33; (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, 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; (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, 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; (5) HCDR-1 containing the amino acid sequence (YYVIH) shown in SEQ ID NO: 47, HCDR-2 containing the amino acid sequence (YINPYNDGTKYNEKFKG) shown in SEQ ID NO: 42, 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, LCDR-3 containing the amino acid sequence (LQYATFPYT) shown in SEQ ID NO: 46; and (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. Preferably, in the first binding domain, the heavy chain variable region includes a sequence selected from the amino acid sequences 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 the aforementioned amino acid sequences; and / or, the light chain variable region includes a sequence selected from the amino acid sequences 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 the aforementioned amino acid sequences. More preferably, the heavy chain variable region and light chain variable region included in the first binding domain include a combination of sequences selected from the following: (1) The amino acid sequence shown in Sequence ID No. 5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 5; and the amino acid sequence shown in Sequence ID No. 6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 6; (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; (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; (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; (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; (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; (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 (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. 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): HCDR-1 containing the amino acid sequence (TYAMN) shown in SEQ ID NO: 49; HCDR-2 containing the amino acid sequence (RIRSKYNNYATYYADSVKD) shown in SEQ ID NO: 50; HCDR-3 containing the amino acid sequence (HGNFGNSYVSYFAY) shown in SEQ ID NO: 51; and LCDR-1 containing the amino acid sequence (RSSTGAVTTSNYAN) shown in SEQ ID NO: 52; LCDR-2 containing the amino acid sequence (GTNKRAP) shown in SEQ ID NO: 53; and LCDR-3 containing the amino acid sequence (ALWYSNLWV) shown in SEQ ID NO: 54. Preferably, in the second binding domain, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 75% identity with the aforementioned amino acid sequence; and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 75% identity with the aforementioned amino acid sequence. More preferably, the bispecific antibody construct provided by the present invention comprises four polypeptide chains: 1) Heavy chain 1 containing domains arranged according to VH-CH1-CH2-CH3 from the N end to the C end; 2) Heavy chain 2 containing domains arranged according to VL-CH1-CH2-CH3 from the N end to the C end; 3) Light chain 1 containing domains arranged according to VL-CL from the N end to the C end; 4) Light chain 2 containing domains arranged according to VH-CL from the N end to the C end; Here, VH and VL contained in heavy chain 1 and light chain 1 pair up to form a first binding domain that binds to GPRC5D; and VL and VH contained in heavy chain 2 and light chain 2 pair up to form a second binding domain that binds to CD3. A schematic diagram of the structure of the bispecific antibody construct provided by the present invention is shown in Figure 1. According to a specific embodiment of the present invention, in the bispecific antibody construct provided by the present invention, the CH1-CH2-CH3 domain in heavy chain 1 comprises 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 comprises 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 comprises 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; and the CL domain in light chain 2 comprises 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. In another embodiment, the present invention further provides nucleic acid molecules comprising a heavy chain CDR, a light chain CDR, a light chain variable region, a heavy chain variable region, a nucleotide sequence encoding a heavy chain or a light chain, contained in an antibody or fragment thereof provided by the present invention or in a bispecific antibody construct provided by the present invention. The nucleic acid molecules of the present invention may be cloned into a vector and further transformed or transfected into host cells. Therefore, according to yet another embodiment, the present invention further provides a vector comprising the nucleic acid molecules of the present invention. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a phagemide vector. The vector or nucleic acid molecules of the present invention may be used for transformation or transfection into host cells. Therefore, according to yet another embodiment, the present invention provides a host cell comprising the nucleic acid molecules and / or vector of the present invention, or the host cell is transformed or transfected with the nucleic acid molecules and / or vector of the present invention. The host cell may be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant, or animal cell. The antibodies or fragments thereof and bispecific antibody constructs provided by the present invention may be obtained by any method known in the art. For example, if it is permissible for the heavy and light chains of the antibody to be expressed in the host cells provided by the present invention, the host cells are cultured. Optionally, the method further includes the step of recovering the antibody produced. The antibodies or fragments thereof, bispecific antibody constructs, nucleic acid molecules, vectors and / or host cells provided by the present invention may be included in a composition, and more particularly in a pharmaceutical composition, such as a drug formulation, thereby being used for a variety of purposes as required by the actual circumstances. For this reason, according to yet another embodiment, the present invention further provides a composition, such as a pharmaceutical composition, which comprises the antibodies or fragments thereof, bispecific antibody constructs, nucleic acid molecules, vectors and / or host cells described in the present invention, and any pharmaceutically acceptable adjuvants. In yet another embodiment, the present invention further provides the use of the antibody or fragment thereof, bispecific antibody constructs, nucleic acid molecules, vectors, host cells and / or compositions in the manufacture of a drug for treating a disease, 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, and in particular multiple myeloma. Accordingly, the present invention further provides a method for treating a disease, the method comprising the step of administering to a subject requiring the antibody or fragment thereof, a bispecific antibody construct, a nucleic acid molecule, a vector, a host cell and / or composition of the present invention, 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. According to yet another aspect, the present invention further provides the use of the antibody or its fragment, bispecific antibody construct, nucleic acid molecule, vector, host cell and / or composition in the manufacture of a reagent for diagnosing a disease or detecting the presence of antigen GPRC5D, 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, particularly multiple myeloma. According to yet another aspect, the present invention provides a kit comprising the antibody or its fragment, bispecific antibody construct, nucleic acid molecule, vector, host cell and / or composition of the present invention. The kit may be used for the above-mentioned treatment, or may be used for diagnosis or detection of an antigen. Depending on the intended application mode, the kit may further comprise other reagents used for treatment, diagnosis or detection. For example, the kit is a kit for detecting the presence of GPRC5D in any biological sample by ELISA.

Brief Description of the Drawings

[0004] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. [Figure 1] It is a schematic diagram of the structure of an anti-human GPRC5D×CD3 bispecific antibody. [Figure 2] The FACS detection results of the binding of the supernatant of hybridoma cells to different cells are shown. [Figure 3] The FACS detection results of the binding of the supernatant of hybridoma cells to different cells are shown. [Figure 4] The FACS detection results of the binding of the supernatant of hybridoma cells to different cells are shown, in which 4-1: MM1R cells; 4-2: Nalm6 cells; 4-3: OPM2 cells; 4-4: NCI-H929 cells. [Figure 5] The FACS detection results of the binding of the humanized antibody to cells expressing human GPRC5D are shown, in which 5-1: 9D7 (hz); 5-2: 26D1 (hz); 5-3: 24F5 (hz); 5-4: 6E97 (hz). [Figure 6]The FACS detection results for the binding of humanized antibodies to cells expressing monkey GPRC5D are shown, including 6-1:9D7(hz); 6-2:26D1(hz); 6-3:24F5(hz); 6-4:6E97(hz). [Figure 7] The FACS detection results for the binding of humanized antibodies to different tumor cell lines are shown, including 7-1: MM.1R cells; 7-2: MOLP8 cells. [Figure 8] The results of detecting the affinity between humanized antibodies and GPRC5D are shown, including 8-1:GPRC5D×CD3 JNJ; 8-2:9D7(hz); 8-3:26D1(hz); 8-4:24F5(hz); 8-5:6E97(hz). [Figure 9] The results of detecting humanized antibody-mediated ADCC are shown, including 9-1: wild-type NCI-H929; 9-2: GPRC5D knockout NCI-H929. [Figure 10] The results of detecting bispecific antibody-mediated T cell injury are shown, including 10-1: co-culture experiment 1; 10-2: co-culture experiment 2; 10-3: co-culture experiment 3. [Modes for carrying out the invention]

[0005] The present invention will be described below with reference to specific examples. Those skilled in the art should understand that these examples are merely for illustrative purposes and do not limit the scope of the invention in any way. The experimental methods in the following examples are all standard methods unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available unless otherwise specified. Among them, Human GPRC5D protein: Genbank Accession No. NM_018654; Monkey GPRC5D protein: Genbank Accession No. XM_005570193.2. Example 1 Production of hybridoma cells and acquisition of antibody proteins Cells expressing human GPRC5D (kyinno, catalog number KC-1583) were subjected to a cell concentration of 1 × 10⁶ in PBS containing 2% FBS. 7 It was prepared as a cell suspension at a concentration of cells / mL. This 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 as the adjuvant. Antibody titers were measured two weeks after booster immunization. Two mice with high antibody titers were selected for immunoimpact, and serum was collected three days later. After dissection, the spleen was collected. The isolated spleen cells and cultured myeloma cells were fused in a 96-well plate, and selective medium was added simultaneously for screening. The culture medium was changed after seven days, and ELISA detection was performed on the cells after ten days. For ELISA detection, human GPRC5D-expressing 293T cells (kyinno, catalog number KC-1723) were used. Cells with an OD value greater than 10 times that of the negative control (serum of a normal mouse) were selected, and further detection was performed by flow cytometry. For flow cytometry, human GPRC5D-expressing 293T cells (kyinno, catalog number KC-1723) were used. Cells that tested positive by both ELISA and FACS were selected, and monoclonal cells were selected by subcloning and plating using the limiting dilution method. The culture supernatant was taken from the selected monoclonal cells and detected by ELISA and flow cytometry. Double-positive cells were selected and cultured on an expanded scale. 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 buffer solution (20 mM citrate + 100 mM NaCl, pH 5.5), concentrated to the appropriate volume using an ultrafiltration concentrator, and dispensed at 200 μL / tube into 1.5 mL EP tubes, stored at -80°C. Simultaneously, some samples were taken and identified using SDS-PAG and FACS to verify protein purity and activity. Example 2 ELISA detection of the binding of hybridoma cell culture supernatant to human GPRC5D. BXPC-3 cells expressing human GPRC5D (kyinno, catalog number KC-1717) were subjected to a cell concentration of 1 × 10⁶ in PBS containing 2% FBS. 5 Cell suspensions were prepared at a concentration of cells / mL and added to a 96-well plate at 100 μL / well. The plates were incubated overnight at 37°C. The liquid in the wells was discarded, and the plates were simultaneously washed three times with washing solution. 80 μL of 4% tissue fixative (Beyotime, catalog number P0099) was added to each well, and the plates were fixed at room temperature for 15 minutes. The plates were then washed twice with PBS and dried. 250 μL of blocking solution containing 2% BSA was added to each well, and the plates were incubated at 37°C for 1 hour. The plates were then washed three times with PBS. 50–100 μL of the culture supernatant of the test hybridoma cells was added to each well, and a positive control (with serum from fused mice), a negative control (with serum from normal mice), and a blank control (with culture medium) were simultaneously placed. The plates were incubated at 37°C for 1–2 hours, washed, and dried. Subsequently, 50–100 μL of the enzyme-labeled secondary antibody, sheep anti-mouse IgG (SIGMA, catalog number A9044-2 mL) labeled with horseradish peroxidase diluted at 1:10000, was added to each well. The mixture was incubated at 37°C for 1–2 hours, washed, and dried. 50–100 μL of freshly prepared substrate chromogenic solution TMB was added to each well and incubated at 37°C for 10–30 minutes. The reaction was stopped by adding 2 mol / L H2SO4, and the OD value was read using an ELISA (enzyme-linked immunosorbent assay) reader. Result interpretation: A P / N ratio of >2:1 (where P represents a positive value and N represents the value in normal mouse serum) was considered positive. If the negative control well was colorless or nearly colorless and the positive control well was clearly colored, the results could be directly observed visually. The results are shown in Table 1.

[0006] [Table 1] Example 3 Construction of a bispecific anti-human GPRC5D x CD3 antibody A bispecific anti-human GPRC5D × CD3 antibody was constructed using the GPRC5D-targeting domain and the CD3-targeting domain of Johnson & Johnson's Talquetamab (JNJ-64407564). A schematic diagram of the structure of this bispecific antibody is shown in Figure 1, where the domains contained in each chain, from the N-terminus to the C-terminus, are as follows. Heavy chain 1 is VH-CH1-CH2-CH3. >VH(Sequence ID 1) QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSS >CH1-CH2-CH3 (Sequence ID 25) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPPAAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy chain 2: VL-CH1-CH2-CH3 >VL(Sequence ID 2) QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL >CH1-CH2-CH3 (Sequence ID 26) SSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDDRVEPKSCDKTHTCPPCPAPPAAGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light chain 1: VL-CL >VL(Sequence ID 3) DIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIK >CL(Sequence ID 24) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Light chain 2: VH-CL >VH(Sequence ID 4) EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS >CL(Sequence ID 27) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Primers were designed for the four polypeptide chains described above, the corresponding coding genes were synthesized, and introduced into eukaryotic expression plasmids. The resulting four recombinant expression plasmids were co-transfected into HEK293F cells, and the cells were cultured for 5-7 days to obtain the supernatant of secreted antibodies. Bispecific antibodies were purified from the cell culture supernatant using MabSelectSure affinity chromatography and SP cation exchange chromatography. The resulting bispecific antibody was named "GPRC5D×CD3 JNJ". Example 4 FACS detection of binding between hybridoma cell culture supernatant and human GPRC5D 293T cells expressing human GPRC5D as described above were placed in PBS containing 2% FBS at a cell concentration of 1 × 10⁶ 5 It was prepared as a cell suspension at a concentration of cells / mL. 50 μL of cell suspension was placed in each flow tube (sample tube), and then 50 μL each of the culture supernatant of the test hybridoma cells and the anti-human GPRC5D×CD3 bispecific antibody GPRC5D×CD3 JNJ as a positive control antibody were added, and the tubes were incubated at 4°C for 60 minutes. 1 mL of flow buffer was added to each flow tube, and the tubes were centrifuged at 1200 rpm for 5 minutes. The supernatant was discarded, and the tubes were washed three times. Simultaneously, control tube 1 (containing only the cell suspension, without the culture supernatant and the secondary antibody listed below) and control tube 2 (containing only the cell suspension and the secondary antibody listed below, without the culture supernatant) were set up. Subsequently, 100 μL of flow buffer was added to each flow tube for resuspending, and 5 μL of PE-labeled anti-mouse Fc tagged secondary antibody (Biolegend, catalog number 409304) was added as required by the experiment. The mixture was incubated at 4°C in the dark for 30 minutes, then 1 mL of flow buffer was added, and the mixture was centrifuged at room temperature at 1200 rpm for 5 minutes. The supernatant was discarded, and the mixture was washed three times. 250 μL of flow buffer was added to each flow tube again, the mixture was resuspended and uniformly mixed, and then detected using an instrument. The detection results are shown in Tables 2-1 to 2-4 and Figures 2-1 to 2-4.

[0007] [Table 2-1]

[0008] [Table 2-2]

[0009] [Table 2-3]

[0010] [Table 2-4] Example 5 FACS detection of binding between hybridoma cell supernatant and monkey GPRC5D. Following the procedure described in Example 4, cells expressing human GPRC5D were replaced with 293T cells expressing monkey GPRC5D (kyinno, catalog number KC-1586), and the binding of the culture supernatant of the hybridoma cells of the present invention to monkey GPRC5D was detected. The detection results for FACS binding are shown in Tables 3-1 to 3-3 and Figures 3-1 to 3-3, and the positive control antibody is GPRC5D×CD3 JNJ.

[0011] [Table 3-1]

[0012] [Table 3-2]

[0013] [Table 3-3] Example 6 FACS detection of binding between hybridoma cell supernatant and different cell types. Following the procedure described in Example 4, cells expressing human GPRC5D were replaced with other cells, namely 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), and the binding of the culture supernatant of the hybridoma cells of the present invention to human GPRC5D was detected. The detection results for FACS binding are shown in Table 4 and Figures 4-1 to 4-4 in Figure 4. The positive control antibody is GPRC5D×CD3 JNJ.

[0014] [Table 4] Example 7 Identification of the sequence of the mouse antibody of the present invention We selected hybridoma cell lines from the screening process, extracted RNA from monoclonal cells, reverse transcribed it into cDNA, amplified the cDNA, recovered it from the gel, and then introduced it into a sequencing vector for sequencing analysis. The sequences of the heavy and light chain variable regions of exemplary mouse antibodies obtained from hybridoma cells are as follows, where the bolded and underlined portion indicates the CDR (as defined by the KABAT method). A1: >9D7-H(VH / HCDR-1 / HCDR-2 / HCDR-3:Sequence IDs 5 / 28 / 29 / 30)

[0015] [ka] >9D7-L(VL / LCDR-1 / LCDR-2 / LCDR-3:Sequence IDs 6 / 31 / 32 / 33)

[0016] [ka] A2: >26D1-H(VH / HCDR-1 / HCDR-2 / HCDR-3:Sequence IDs 7 / 35 / 36 / 37)

[0017] [ka] >26D1-L(VL / LCDR-1 / LCDR-2 / LCDR-3:Sequence IDs 8 / 38 / 39 / 40)

[0018] [ka] A3 >24F5-H(VH / HCDR-1 / HCDR-2 / HCDR-3:Sequence IDs 9 / 41 / 42 / 43)

[0019] [ka] TLTVSS >24F5-L(VL / LCDR-1 / LCDR-2 / LCDR-3:Sequence IDs 10 / 44 / 45 / 46)

[0020] [ka] A4 >6E97-H(VH / HCDR-1 / HCDR-2 / HCDR-3:Sequence IDs 11 / 47 / 42 / 43 / )

[0021] [ka] >6E97-L(VL / LCDR-1 / LCDR-2 / LCDR-3:Sequence IDs 12 / 44 / 45 / 46)

[0022] [ka] Example 8 Production of chimeric antibodies and humanized antibodies using mouse antibodies For the A1 antibody (mouse antibody 9D7), the following human germline sequences were selected as templates for the heavy and light chains, respectively: IGHV4-39 and IGKV1-39. Homology modeling was performed on the A1 antibody, and structural simulations of the Fab region were conducted. The Fab structure of the A1 antibody was finally predicted by the homology modeling calculations. By comparing and analyzing the predicted Fab structure and heavy chain with the IGHV4-39 sequence, we found that in this VH, all other mouse amino acids were replaced with corresponding human amino acids using IGHV4-39 as a template, except for the CDR region and 2V, 49M, 68I, and 72R, which were the original mouse amino acids. By comparing and analyzing the predicted Fab structure and light chain with the IGKV1-39 sequence, it was found that all other mouse amino acids were replaced with corresponding human amino acids using IGKV1-39 as a template, except for the CDR region and 42Q, 43S, 46A, and 60D in this VL, where the original mouse amino acids were retained. The resulting humanized sequence is as follows (wherein the heavy chain and light chain CDR are shown in bold and underlined, and obtained according to the KABAT definition method). The sequence of the humanized antibody is as follows: >9D7-H humanized sequence (VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NOs. 13 / 28 / 34 / 30)

[0023] [ka] [ka] >9D7-L humanized sequence (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NOs. 14 / 31 / 32 / 33)

[0024] [ka] For the A2 antibody (mouse antibody 26D1), the following human germline sequences were selected as templates for the heavy and light chains, respectively: IGHV1-46 and IGKV1-5. Homology modeling was performed on the A2 antibody, and structural simulations of the Fab region were conducted. The Fab structure of the A2 antibody was finally predicted by the homology modeling calculations. By comparing and analyzing the predicted Fab structure and heavy chain with the IGHV1-46 sequence, we found that in this VH, all other mouse amino acids were replaced with corresponding human amino acids using IGHV1-46 as a template, except for the CDR region and 37I, 38K, 48I, 67R, 68A, 70L, 72A, 74K, 76S, 77N, 79A, and 98N, which were the original mouse amino acids. By comparing and analyzing the predicted Fab structure and light chain with the IGKV1-5 sequence, it was found that all other mouse amino acids were replaced with corresponding human amino acids using IGKV1-5 as a template, except for the CDR region and 45R, 60D, and 87F in this VL, where the original mouse amino acids were retained. The resulting humanized sequence is as follows (wherein the heavy chain and light chain CDR are shown in bold and underlined, and obtained according to the KABAT definition method). The sequence of the humanized antibody is as follows: >26D1-H humanized sequence (VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NOs. 15 / 35 / 36 / 37)

[0025] [ka] >26D1-L humanized sequence (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NOs. 16 / 38 / 39 / 40)

[0026] [ka] For the A3 antibody (mouse antibody 24F5), the following human germline sequences were selected as templates for the heavy and light chains, respectively: IGHV1-46 and IGKV1-15. Homology modeling was performed on the A3 antibody, and structural simulations of the Fab region were conducted. The Fab structure of the A3 antibody was finally predicted by the homology modeling calculations. By comparing and analyzing the predicted Fab structure and heavy chain with the IGHV1-46 sequence, we found that in this VH, all other mouse amino acids were replaced with corresponding human amino acids using IGHV1-46 as a template, except for the CDR region and 48I, 68A, 70L, 72S, 74K, and 79A, which were the original mouse amino acids. By comparing and analyzing the predicted Fab structure and light chain with the IGKV1-15 sequence, it was found that in this VL, all mouse amino acids were replaced with corresponding human amino acids using IGKV1-15 as a template, except for the CDR region and 36L, 42G, 43T, 44I, 46R, 66R, 69S, and 71Y, where the original mouse amino acids were retained. The resulting humanized sequence is as follows (wherein the heavy chain and light chain CDR are shown in bold and underlined, and obtained according to the KABAT definition method). The sequence of the humanized antibody is as follows: >24F5-H humanized sequence (VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NOs. 17 / 41 / 42 / 43)

[0027] [ka] >24F5-L humanized sequence (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NOs. 18 / 44 / 45 / 46)

[0028] [ka] For the A4 antibody (mouse antibody 6E97), the following human germline sequences were selected as templates for the heavy and light chains, respectively: IGHV1-46 and IGKV1-15. Homology modeling was performed on the A4 antibody, and structural simulations of the Fab region were conducted. The Fab structure of the A4 antibody was finally predicted by the homology modeling calculations. By comparing and analyzing the predicted Fab structure and heavy chain with the IGHV1-46 sequence, we found that in this VH, all other mouse amino acids were replaced with corresponding human amino acids using IGHV1-46 as a template, except for the CDR region and 48I, 68A, 70L, 72S, 74K, and 79A, which were the original mouse amino acids. By comparing and analyzing the predicted Fab structure and light chain with the IGKV1-15 sequence, it was found that in this VL, all mouse amino acids were replaced with corresponding human amino acids using IGKV1-15 as a template, except for the CDR region and 4L, 36L, 42G, 43T, 44I, 46R, 66R, 69S, and 71Y, where the original mouse amino acids were retained. The resulting humanized sequence is as follows (wherein the heavy chain and light chain CDR are shown in bold and underlined, and obtained according to the KABAT definition method). The sequence of the humanized antibody is as follows: >6E97-H humanized sequence (VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NOs. 19 / 47 / 48 / 43)

[0029] [ka] >6E97-L humanized sequence (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NOs. 20 / 44 / 45 / 46)

[0030] [ka] The sequence shown in SEQ ID NO: 23 was used as the heavy chain constant region, and the sequence shown in SEQ ID NO: 24 was used as the light chain constant region. Primers were redesigned for the antibody DNA sequence against the GRRC5D target obtained by sequencing, and the genes for the corresponding chimeric and humanized antibodies were synthesized and introduced into eukaryotic expression vectors. DH5alpha-competent cells were transformed, incubated overnight in a 37°C constant temperature incubator, and monoclonal strains were selected and identified by sequencing. Strains with correct sequences were selected, the strains were shaken, and large quantities of plasmid were extracted. These plasmids were transfected into mammalian expression cells 293F and cultured in a 37°C, 5% CO2 incubator for 7 days. The supernatant was collected, centrifuged, filtered, and purified using a protein G affinity chromatography column. The purity of the purified antibody was detected by SDS-PAGE electrophoresis, and the antibody concentration was detected using a BCA protein detection kit. The antibodies were then aliquoted and stored in a refrigerator at -80°C. Here, the chimeric antibody was named "mouse antibody abbreviation (chi)" and the humanized antibody was named "mouse antibody abbreviation (hz)". >CH1-CH3 heavy chain constant region (SEQ ID NO: 23) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >CL1 light chain steady region (SEQ ID NO: 24) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Example 9 FACS detection of binding of humanized antibodies to human GPRC5D The experimental procedure followed the instructions in Example 4. Human GPRC5D-expressing 293T cells (kyinno, catalog number KC-1584) were used, and the test antibody, the positive control antibody GPRC5D×CD3 JNJ, or the negative control antibody (hIgG1) was added. The concentrations were as shown in Figure 4. Simultaneously, control tube 1 (containing only the cell suspension, without the antibody or secondary antibody) and control tube 2 (containing only the cell suspension and secondary antibody, without the antibody) were set up. The results are shown in Table 5 and Figures 5-1 to 5-4.

[0031] [Table 5] Example 10 FACS detection of binding of humanized antibodies to monkey GPRC5D The experimental procedure followed the instructions in Example 4. 293T cells expressing monkey (cyno)GPRC5D (kyinno, catalog number KC-1586) were used. The test antibody, the positive control antibody GPRC5D×CD3 JNJ, or the negative control antibody (hIgG1) was added. The concentrations are shown in Figure 6. Simultaneously, control tube 1 (containing only the cell suspension, without the antibody or secondary antibody) and control tube 2 (containing only the cell suspension and secondary antibody, without the antibody) were set up. The results are shown in Table 6 and Figures 6-1 to 6-4 in Figure 6.

[0032] [Table 6] Example 11 FACS detection of binding of humanized antibodies to GPRC5A / B / C The experimental procedure followed the instructions in Example 4, using cells expressing other members of the G protein-coupled receptor family C group 5: 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, the positive control antibody GPRC5D×CD3 JNJ, or the negative control antibody (hIgG1) were added at a concentration of 2 μg / mL. Simultaneously, control tube 1 (containing only the cell suspension, without antibody or secondary antibody) and control tube 2 (containing only the cell suspension and secondary antibody, without antibody) were set up. The results are shown in Table 7.

[0033] [Table 7] Example 12 FACS detection of binding of humanized antibodies to tumor cell lines The experimental procedure followed the instructions in Example 4, replacing the cells used with multiple myeloma cells MM.1R (kyinno, catalog number KC-0619) and MOLP8 (kyinno, catalog number KC-0622), and adding the test antibody, the positive control antibody GPRC5D×CD3 JNJ, or the negative control antibody (hIgG1). Simultaneously, control tube 1 (containing only the cell suspension, without the antibody or secondary antibody) and control tube 2 (containing only the cell suspension and secondary antibody, without the antibody) were set up. The results are shown in Table 8 and Figures 7-1 and 7-2.

[0034] [Table 8] Example 13 Detection of affinity between humanized antibodies and GPRC5D protein The test antibody was prepared as a 10 μg / mL solution using PBS buffer, and the antibody was captured using an AHC chip according to the Fortebio instructions. Pre-prepared human GPRC5D protein (Kactus Biosystems, catalog number GPR-HM05P) (diluted 2-fold in 6 steps to a maximum concentration of 200 nM) was flowed through the chip under specific conditions: flow rate of 30 μL / min, antigen-antibody binding time of 200 seconds, and dissociation time of 500 seconds. The affinity between the antibody and antigen was analyzed by fitting the measured results using the instrument's dedicated software. The results are shown in Table 9 and Figures 8-1 to 8-5.

[0035] [Table 9] Example 14 Detection of humanized antibody-mediated ADCC In a 96-well plate with a U-bottom, 20,000 reporter cells Jurkat-NFAT-luc-CD16-V158 (kyinno, catalog number KC-1507), 20,000 wild-type NCI-H929 cells (CFSE-H929, kyinno, catalog number KC-0629), or GPRC5D knockout NCI-H929 cells (CFSE-H929GPRC5DKO, kyinno, catalog number KC-2203), and different concentrations of antibody (10 μg / mL, 3.16 μg / mL, 1 μg / mL, 0.316 μg / mL, 0.1 μg / mL, 0.0316 μg / mL, 0.01 μg / mL, 0.00316 μg / mL) were added to each well. After co-culturing for 6 hours, the luciferase signal was detected. The results are shown in Table 10 and Figures 9-1 to 9-2, and the positive control antibody is GPRC5D×CD3 JNJ.

[0036] [Table 10] Example 15 Acquisition of anti-CD3 antibodies and detection of affinity with targets From patent US10066015B2, we obtained the heavy chain variable region sequence (SEQ ID NO. 10) and the light chain variable region sequence (SEQ ID NO. 5) of a mouse-derived antibody. Humanization of the antibody sequence was performed, and the resulting humanized sequence is as follows (wherein the heavy chain and light chain CDRs are shown in bold and underlined, respectively, and were obtained according to the KABAT definition method). >VH humanized sequences (VH / HCDR-1 / HCDR-2 / HCDR-3: SEQ ID NOs. 21 / 49 / 50 / 51)

[0037] [ka] >VL humanized sequences (VL / LCDR-1 / LCDR-2 / LCDR-3: SEQ ID NOs. 22 / 52 / 53 / 54)

[0038] [ka] As described in Example 8, an antibody containing the above humanized sequence was obtained and named SP34V1(hz). Similarly, a chimeric antibody was obtained using the heavy chain and light chain variable region sequences of the above mouse-derived antibody described in Patent US10066015B2 and named SP34(chi). The humanized antibody and chimeric antibody were prepared as a 10 μg / mL solution using PBS buffer, and the antibodies were captured using an AHC tip according to the Fortebio instructions. Pre-prepared human CD3 protein (Kactus Biosystems, catalog number GPR-HM05P) (diluted 2-fold in 6 steps to a maximum concentration of 200 nM) was flowed through the tip under specific conditions: flow rate 30 μL / min, antigen-antibody binding time 200 seconds, and dissociation time 500 seconds. The affinity between the antibody and antigen was analyzed by fitting the measured results using the instrument's dedicated software. The results are shown in Table 1.

[0039] [Table 11] Example 16 Construction of the bispecific antibody of the present invention As described in Example 3, the bispecific antibody of the present invention was constructed, with the differences being that VH in heavy chain 1 and VL in light chain 1 were replaced with VH and VL of the humanized antibodies 9D7(hz), 26D1(hz), 24F5(hz), and 6E97(hz) of the present invention, respectively, and VL in heavy chain 2 and VH in light chain 2 were replaced with the humanized sequence for CD3 obtained in Example 15, respectively. The humanized antibodies of the present invention, VH and VL, each containing 9D7(hz), 26D1(hz), 24F5(hz), and 6E97(hz), respectively, and the anti-human GPRC5D×CD3 bispecific antibody with the above sequence were named "GPRC5D×CD3 9D7(hz)", "GPRC5D×CD3 26D1(hz)", "GPRC5D×CD3 24F5(hz)", and "GPRC5D×CD3 26D1(hz)", respectively. Example 17 GPRC5D-CD3 double antibody-mediated T cell toxicity experiment PBMCs and CD3 / CD28 magnetic beads were mixed in a 2:1 ratio in RPMI1640 containing 10 ng / mL of IL-2 and 10% FBS, and cultured for 7 days to amplify and activate T cells. Co-culture experiment 1: 293T LDHA-Hibit-GPRC5D cells (kyinno, catalog number KC-2151) were plated one day in advance (96-well plate), and then 10,000 of the above T cells were added to each well. In addition, different concentrations of the positive control antibody GPRC5D×CD3 JNJ and the bispecific antibodies of the present invention (1 μg / mL, 0.1 μg / mL, 0.01 μg / mL, 0.001 μg / mL, 0.0001 μg / mL, 0.00001 μg / mL, 0.000001 μg / mL, 0.000001 μg / mL, 0.0000001 μg / mL) were added, and the cells were co-cultured for 18 hours in RPMI1640 containing 10 ng / mL of IL-2 and 10% FBS. The proportion of injured cells was detected by detecting LDHA-HIBIT released from dead cells in the supernatant using Nano-Glo® HiBiT Extracellular Reagent (Promega, catalog number N2420). In this experiment, two sets of controls were set up for each of the two antibodies: one with 293T LDHA-Hibit-GPRC5D cells and antibody added, but without T cells; and another with 293T LDHA-Hibit cells (kyinno), T cells, and antibody added to eliminate non-antibody-mediated injury and antibody-mediated non-T cell injury. The results are shown in 10-1 in Figure 10. 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. In a U-bottom 96-well plate, 10,000 activated T cells and 3,000 CFSE-H929 cells were added to each well, and different concentrations of the positive control antibody GPRC5D×CD3 JNJ and the bispecific antibodies of the present invention (1 μg / mL, 0.1 μg / mL, 0.01 μg / mL, 0.001 μg / mL, 0.0001 μg / mL, 0.00001 μg / mL, 0.000001 μg / mL, 0.000001 μg / mL) were added to each well, and the cells were co-cultured for 18 hours in RPMI1640 containing 10 ng / mL of IL-2 and 10% FBS. Subsequently, the cells were stained with 7-AAD and detected by flow cytometry. The proportion of CFSE+7-AAD+ cells among CSFE+ cells was counted as the cytotoxicity rate. In this experiment, two sets of controls were set up for each of the two antibodies: one with CFSE-H929 cells and antibody added, but without T cells; and another with CFSE-K562, T cells, and antibody added to eliminate non-antibody-mediated injury and antibody-mediated non-T cell injury. The results are shown in Figure 10, 10-2. Co-culture experiment 3: Wild-type NCI-H929 (kyinno, catalog number KC-0629) and GPRC5D knockout NCI-H929 cells (kyinno, catalog number KC-2203) were stained with CFSE and named CFSE-H929 and CFSE-H929GPRC5DKO, respectively. In a U-bottom 96-well plate, 10,000 activated T cells and 3,000 CFSE-H929 cells were added to each well. Different concentrations of the positive control antibody GPRC5D×CD3 JNJ and the bispecific antibodies of the present invention (1 μg / mL, 0.1 μg / mL, 0.01 μg / mL, 0.001 μg / mL, 0.0001 μg / mL, 0.00001 μg / mL, 0.000001 μg / mL, 0.0000001 μg / mL) were added to each well, and the cells were co-cultured for 18 hours in RPMI1640 containing 10 ng / mL of IL-2 and 10% FBS. Subsequently, the cells were stained with 7-AAD and detected by flow cytometry. The proportion of CFSE+7-AAD+ cells among CSFE+ cells was counted as the cytotoxicity rate. In this experiment, two control groups were established for each of the four antibodies: one with CFSE-H929 cells and antibody added, but without T cells; and another with CFSE-H929GPRC5DKO cells, T cells, and antibody added to eliminate non-antibody-mediated injury and antibody-mediated non-T cell injury. The results are shown in Figure 10, 10-3. Although specific embodiments of the present invention have been described above, this does not limit the present invention. Those skilled in the art may make various changes and modifications based on the present invention, provided they do not deviate from the spirit of the invention, but all such corresponding changes and modifications should fall within the scope of protection of the claims appended to the present invention.

Claims

1. An antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL) and binding to member D of family group C of G protein-coupled receptors (GPRC5D), The heavy chain variable region (VH) and light chain variable region (VL) include combinations of CDRs selected from the following (HCDR-1, HCDR-2, HCDR-3; LCDR-1, LCDR-2, LCDR-3): Antibodies or their antigen-binding fragments: (1) HCDR-1 containing the amino acid sequence shown in SEQ ID NO: 28, HCDR-2 containing the amino acid sequence shown in SEQ ID NO: 29, HCDR-3 containing the amino acid sequence shown in SEQ ID NO: 30; and LCDR-1 containing the amino acid sequence shown in SEQ ID NO: 31, LCDR-2 containing the amino acid sequence shown in SEQ ID NO: 32, and LCDR-3 containing the amino acid sequence shown in SEQ ID NO: 33; (2) HCDR-1 containing the amino acid sequence shown in SEQ ID NO: 28, HCDR-2 containing the amino acid sequence shown in SEQ ID NO: 34, HCDR-3 containing the amino acid sequence shown in SEQ ID NO: 30; and LCDR-1 containing the amino acid sequence shown in SEQ ID NO: 31, LCDR-2 containing the amino acid sequence shown in SEQ ID NO: 32, and LCDR-3 containing the amino acid sequence shown in SEQ ID NO: 33; (3) HCDR-1 containing the amino acid sequence shown in SEQ ID NO: 35, HCDR-2 containing the amino acid sequence shown in SEQ ID NO: 36, HCDR-3 containing the amino acid sequence shown in SEQ ID NO: 37; and LCDR-1 containing the amino acid sequence shown in SEQ ID NO: 38, LCDR-2 containing the amino acid sequence shown in SEQ ID NO: 39, and LCDR-3 containing the amino acid sequence shown in SEQ ID NO: 40; (4) HCDR-1 containing the amino acid sequence shown in SEQ ID NO: 41, HCDR-2 containing the amino acid sequence shown in SEQ ID NO: 42, HCDR-3 containing the amino acid sequence shown in SEQ ID NO: 43; and LCDR-1 containing the amino acid sequence shown in SEQ ID NO: 44, LCDR-2 containing the amino acid sequence shown in SEQ ID NO: 45, and LCDR-3 containing the amino acid sequence shown in SEQ ID NO: 46; (5) HCDR-1 containing the amino acid sequence shown in SEQ ID NO: 47, HCDR-2 containing the amino acid sequence shown in SEQ ID NO: 42, HCDR-3 containing the amino acid sequence shown in SEQ ID NO: 43; and LCDR-1 containing the amino acid sequence shown in SEQ ID NO: 44, LCDR-2 containing the amino acid sequence shown in SEQ ID NO: 45, LCDR-3 containing the amino acid sequence shown in SEQ ID NO: 46; and (6) HCDR-1 containing the amino acid sequence shown in SEQ ID NO: 47, HCDR-2 containing the amino acid sequence shown in SEQ ID NO: 48, HCDR-3 containing the amino acid sequence shown in SEQ ID NO: 43; and LCDR-1 containing the amino acid sequence shown in SEQ ID NO: 44, LCDR-2 containing the amino acid sequence shown in SEQ ID NO: 45, and LCDR-3 containing the amino acid sequence shown in SEQ ID NO:

46.

2. The heavy chain variable region includes a sequence selected from the amino acid sequences 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 the said amino acid sequence; and / or The light chain variable region includes a sequence selected from the amino acid sequences 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 the aforementioned amino acid sequences. The antibody or antigen-binding fragment thereof according to claim 1.

3. The heavy chain variable region and light chain variable region contained in the antibody or its antigen-binding fragment include a combination of sequences selected from the following: The antibody or antigen-binding fragment thereof according to claim 1: (1) The amino acid sequence shown in Sequence ID No. 5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 5; and the amino acid sequence shown in Sequence ID No. 6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 6; (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; (3) The amino acid sequence shown in Sequence ID No. 7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 7; and the amino acid sequence shown in Sequence ID No. 8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 8; (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; (5) The amino acid sequence shown in Sequence ID No. 9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 9; and the amino acid sequence shown in Sequence ID No. 10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 10; (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; (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 (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.

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

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is IgG.

6. The antibody or antigen-binding fragment according to claim 1, further comprising a constant region.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain.

8. The antibody or antigen-binding fragment according to claim 1, further comprising a heavy chain constant region and / or a κ or λ-type light chain constant region of IgG, IgA, IgM, IgD, or IgE.

9. It comprises a first binding domain that binds to member D of the C family of G protein-coupled receptors (GPRC5D), and a second binding domain that binds to CD3 on the surface of T cells. The first binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL), The heavy chain variable region (VH) and light chain variable region (VL) include combinations of CDRs selected from the following (HCDR-1, HCDR-2, HCDR-3; LCDR-1, LCDR-2, LCDR-3): Bispecific antibody construct: (1) HCDR-1 containing the amino acid sequence shown in SEQ ID NO: 28, HCDR-2 containing the amino acid sequence shown in SEQ ID NO: 29, HCDR-3 containing the amino acid sequence shown in SEQ ID NO: 30; and LCDR-1 containing the amino acid sequence shown in SEQ ID NO: 31, LCDR-2 containing the amino acid sequence shown in SEQ ID NO: 32, and LCDR-3 containing the amino acid sequence shown in SEQ ID NO: 33; (2) HCDR-1 containing the amino acid sequence shown in SEQ ID NO: 28, HCDR-2 containing the amino acid sequence shown in SEQ ID NO: 34, HCDR-3 containing the amino acid sequence shown in SEQ ID NO: 30; and LCDR-1 containing the amino acid sequence shown in SEQ ID NO: 31, LCDR-2 containing the amino acid sequence shown in SEQ ID NO: 32, and LCDR-3 containing the amino acid sequence shown in SEQ ID NO: 33; (3) HCDR-1 containing the amino acid sequence shown in SEQ ID NO: 35, HCDR-2 containing the amino acid sequence shown in SEQ ID NO: 36, HCDR-3 containing the amino acid sequence shown in SEQ ID NO: 37; and LCDR-1 containing the amino acid sequence shown in SEQ ID NO: 38, LCDR-2 containing the amino acid sequence shown in SEQ ID NO: 39, and LCDR-3 containing the amino acid sequence shown in SEQ ID NO: 40; (4) HCDR-1 containing the amino acid sequence shown in SEQ ID NO: 41, HCDR-2 containing the amino acid sequence shown in SEQ ID NO: 42, HCDR-3 containing the amino acid sequence shown in SEQ ID NO: 43; and LCDR-1 containing the amino acid sequence shown in SEQ ID NO: 44, LCDR-2 containing the amino acid sequence shown in SEQ ID NO: 45, and LCDR-3 containing the amino acid sequence shown in SEQ ID NO: 46; (5) HCDR-1 containing the amino acid sequence shown in SEQ ID NO: 47, HCDR-2 containing the amino acid sequence shown in SEQ ID NO: 42, HCDR-3 containing the amino acid sequence shown in SEQ ID NO: 43; and LCDR-1 containing the amino acid sequence shown in SEQ ID NO: 44, LCDR-2 containing the amino acid sequence shown in SEQ ID NO: 45, LCDR-3 containing the amino acid sequence shown in SEQ ID NO: 46; and (6) HCDR-1 containing the amino acid sequence shown in SEQ ID NO: 47, HCDR-2 containing the amino acid sequence shown in SEQ ID NO: 48, HCDR-3 containing the amino acid sequence shown in SEQ ID NO: 43; and LCDR-1 containing the amino acid sequence shown in SEQ ID NO: 44, LCDR-2 containing the amino acid sequence shown in SEQ ID NO: 45, and LCDR-3 containing the amino acid sequence shown in SEQ ID NO:

46.

10. In the first binding domain, the heavy chain variable region includes a sequence selected from the amino acid sequences 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 the aforementioned amino acid sequences; and / or the light chain variable region includes a sequence selected from the amino acid sequences 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 the aforementioned amino acid sequences. The bispecific antibody construct according to claim 9.

11. The heavy chain variable region and light chain variable region included in the first binding domain include a combination of sequences selected from the following: The bispecific antibody construct according to claim 9: (1) The amino acid sequence shown in Sequence ID No. 5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 5; and the amino acid sequence shown in Sequence ID No. 6 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 6; (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; (3) The amino acid sequence shown in Sequence ID No. 7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 7; and the amino acid sequence shown in Sequence ID No. 8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 8; (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; (5) The amino acid sequence shown in Sequence ID No. 9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 9; and the amino acid sequence shown in Sequence ID No. 10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in Sequence ID No. 10; (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; (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 (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.

12. The second binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL), and the heavy chain variable region (VH) and the light chain variable region (VL) include a combination of CDRs selected from the following (HCDR-1, HCDR-2, HCDR-3; LCDR-1, LCDR-2, LCDR-3): The bispecific antibody construct according to claim 9: HCDR-1 containing the amino acid sequence shown in SEQ ID NO: 49, HCDR-2 containing the amino acid sequence shown in SEQ ID NO: 50, HCDR-3 containing the amino acid sequence shown in SEQ ID NO: 51; and LCDR-1 containing the amino acid sequence shown in SEQ ID NO: 52, LCDR-2 containing the amino acid sequence shown in SEQ ID NO: 53, and LCDR-3 containing the amino acid sequence shown in SEQ ID NO:

54.

13. In the second binding domain, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 75% identity with the aforementioned amino acid sequence; and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 75% identity with the aforementioned amino acid sequence. The bispecific antibody construct according to claim 12.

14. The aforementioned bispecific antibody construct is as follows: ： 1) Heavy chain 1 containing domains arranged according to VH-CH1-CH2-CH3 from the N end to the C end; 2) Heavy chain 2 containing domains arranged according to VL-CH1-CH2-CH3 from the N end to the C end; 3) Light chain 1 containing domains arranged according to VL-CL from the N end to the C end; 4) Light chain 2 containing domains arranged according to VH-CL from the N end to the C end; Including, here, The VH and VL contained in the heavy chain 1 and the light chain 1 pair up to form the first binding domain that binds to GPRC5D; the VL and VH contained in the heavy chain 2 and the light chain 2 pair up to form the second binding domain that binds to CD3. The bispecific antibody construct according to claim 9.

15. A nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, or a nucleotide sequence encoding a bispecific antibody construct according to any one of claims 9 to 14.

16. A vector comprising the nucleic acid molecule described in claim 15.

17. A host cell comprising the nucleic acid molecule described in claim 15.

18. A host cell comprising the vector according to claim 16.

19. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a bispecific antibody construct according to any one of claims 9 to 14.

20. Use of the pharmaceutical composition according to claim 19 in the manufacture of a drug for treating a disease, wherein the disease is a tumor or cancer.

21. The use according to claim 20, wherein the disease is a tumor or cancer associated with GPRC5D expression.

22. The use according to claim 20, wherein the disease is lymphoma or myeloma.

23. The use according to claim 20, wherein the disease is multiple myeloma.

24. Use of the pharmaceutical composition according to claim 19 in the manufacture of a reagent used for diagnosing a disease or detecting the presence of the antigen GPRC5D, wherein the disease is a tumor or cancer.

25. The use according to claim 24, wherein the disease is a tumor or cancer associated with GPRC5D expression.

26. The use according to claim 24, wherein the disease is lymphoma or myeloma.

27. The use according to claim 24, wherein the disease is multiple myeloma.

28. A kit comprising the pharmaceutical composition described in claim 19.

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