Multi-specific antibodies and medical uses thereof

Abstract

The invention relates to an antibody specifically binding to GPRC5D or an antigen binding fragment thereof. The invention also provides a multispecific antibody which comprises a first binding domain capable of specifically binding to GPRC5D and a second binding domain capable of specifically binding to CD3, and a medical application of the multispecific antibody. In addition, the invention also relates to preparation and application of the antibody.

Description

Technical Field

[0001] The present disclosure relates to multispecific antibodies that are capable of simultaneously binding to CD3 and another tumor-associated antigen, such as GPRC5D, binding to tumor cells expressing GPRC5D while simultaneously binding to and activating CD3-positive T cells, thereby promoting T cell-specific killing of tumor cells. Background Art

[0002] GPRC5D (G protein-coupled receptor class C group 5 member D) is an orphan seven-transmembrane receptor belonging to a family of proteins including GPRC5a (RAIG1), GPRC5b, GPRC5c and GPRC5D, which share 31-42% amino acid sequence identity with each other. The endogenous ligands of GPRC5D and its physiological functions are currently unknown.

[0003] GPRC5D is highly and selectively expressed in multiple myeloma cells, approximately 20-fold higher than in normal plasma cells, and has been identified as a promising immunotherapeutic target for multiple myeloma (MMs).

[0004] Cluster of differentiation 3 (CD3) is a multimeric protein complex expressed on T cells that binds to the T cell receptor complex (TCR) and is required for T cell activation. Anti-CD3 antibodies have been shown to aggregate CD3 on T cells, leading to T cell activation. Therefore, anti-CD3 antibodies have been proposed for therapeutic purposes to engage T cell activation. For example, bispecific antibodies capable of binding CD3 and target antigens have been proposed for therapeutic use to engage in targeting T cell immune responses to tissues and cells expressing the target antigen. The recently approved CD19×CD3 bispecific T cell engager (BiTE) Blinatumomab validates this approach.

[0005] Bispecific T cell engagers (BiTEs) are a new class of immunotherapeutic molecules used to treat cancer. These molecules enhance a patient's immune response to tumors by retargeting T cells to tumor cells. BiTEs are composed of two single-chain variable fragments (scFvs) connected in series by a flexible linker. One scFv binds to a T cell-specific molecule (usually CD3), while the other scFv binds to a tumor-associated antigen. This structure and specificity enable BiTEs to connect T cells to tumor cells, ultimately stimulating T cell activation, tumor killing, and cytokine production. BiTEs targeting multiple tumor-associated antigens have been developed for a variety of hematological and solid tumors.

[0006] Talquetamab (US10562968B2, WO2019220368, WO2019220369, WO2023081704) and Forimtamig (WO2021018859) are both GPRC5D x CD3 bispecific antibodies that are in clinical trials for multiple myeloma. Both molecules achieved an ORR of ~70% and can treat patients pretreated with BCMA targeted therapy. In view of the above, although GPRC5D is a promising target for the treatment of multiple myeloma (MMs), there is still a need in the art to develop diversified anti-GPRC5D molecules with different binding potency and / or binding sites or internalization properties, develop diversified BiTEs, and expand and / or improve therapeutic utility and production applicability. Summary of the invention

[0007] In recent years, there has been a growing interest in CD3-targeted BiTEs for cancer immunotherapy. However, challenges related to the efficacy and safety of this therapeutic modality have emerged. In addition to tandem ScFv formats (BiTEs), many bispecific antibody configurations and compositions have been described in the state of the art. Multiple variables can influence the in vivo efficacy of these molecules, including pharmacokinetics, targeted antigenic epitopes, and the relative affinities of the antigen-binding components. However, despite all the previous progress, there is still a need to systematically evaluate the impact of many of these parameters and select the best bispecific binding molecules for cancer therapy.

[0008] The present disclosure meets the above needs by providing novel anti-GPRC5D antibodies, and bispecific antibodies that bind GPRC5D and CD3.

[0009] The inventors of the present application have conducted extensive research to achieve this goal and have achieved the present invention by developing novel anti-GPRC5D antibodies and engineered bispecific antibodies that bind to GPRC5D and CD3.

[0010] Specifically, the present invention includes the following aspects:

[0011] The present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to GPRC5D, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, characterized in that: a) HCDR1 has an amino acid sequence as shown in SEQ ID NO:01 or SEQ ID NO:04; b) HCDR2 has an amino acid sequence as shown in SEQ ID NO:02 or SEQ ID NO:05; c) HCDR3 has an amino acid sequence as shown in SEQ ID NO:03 or SEQ ID NO:06; d) LCDR1 has an amino acid sequence as shown in SEQ ID NO:07 or SEQ ID NO:10; e) LCDR2 has an amino acid sequence as shown in SEQ ID NO:08; f) LCDR3 has an amino acid sequence as shown in SEQ ID NO:09 or SEQ ID NO:11.

[0012] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, characterized in that: a) HCDR1 has an amino acid sequence as shown in SEQ ID NO:32, 39; b) HCDR2 has an amino acid sequence as shown in SEQ ID NO:33, 35, 37, 40; c) HCDR3 has an amino acid sequence as shown in SEQ ID NO:34, 36, 38, 41; d) LCDR1 has an amino acid sequence as shown in SEQ ID NO:42, 45, 47, 49; e) LCDR2 has an amino acid sequence as shown in SEQ ID NO:43; f) LCDR3 has an amino acid sequence as shown in SEQ ID NO:44, 46, 48.

[0013] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:01, SEQ ID NO:02 and SEQ ID NO:03, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:07, SEQ ID NO:08 and SEQ ID NO:09, respectively; or, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:04, SEQ ID NO:05 and SEQ ID NO:06, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:10, SEQ ID NO:08 and SEQ ID NO:11, respectively.

[0014] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44, respectively; or, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:04, SEQ ID NO:35 and SEQ ID NO:36, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:45, SEQ ID NO:43 and SEQ ID NO:46, respectively; or, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:04, SEQ ID NO:37 and SEQ ID NO:38, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:47, SEQ ID NO:08 and SEQ ID NO: NO:48; or, the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41, respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:49, SEQ ID NO:08 and SEQ ID NO:48, respectively.

[0015] In some embodiments, the above-mentioned antibody or antigen-binding fragment thereof is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof.

[0016] In some embodiments, the anti-antibodies or antigen-binding fragments thereof comprise: a) a heavy chain variable region as set forth in SEQ ID NO:18, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and / or, a light chain variable region as set forth in SEQ ID NO:19, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or, b) a heavy chain variable region as set forth in SEQ ID NO:20, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and / or, a light chain variable region as set forth in SEQ ID NO:21, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0017] In some embodiments, the above-mentioned antibodies or antigen-binding fragments include: a) a heavy chain variable region as shown in SEQ ID NO: 18, and / or, a light chain variable region as shown in SEQ ID NO: 19; or, b) a heavy chain variable region as shown in SEQ ID NO: 20, and / or, a light chain variable region as shown in SEQ ID NO: 21.

[0018] In some embodiments, the above-mentioned antibodies or antigen-binding fragments include: a) a heavy chain variable region as shown in SEQ ID NO: 18, and a light chain variable region as shown in SEQ ID NO: 19; or, b) a heavy chain variable region as shown in SEQ ID NO: 20, and a light chain variable region as shown in SEQ ID NO: 21.

[0019] In some embodiments, the anti-antibodies or antigen-binding fragments thereof comprise: a) a heavy chain variable region as set forth in SEQ ID NO:50, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and / or, a light chain variable region as set forth in SEQ ID NO:51, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or, b) a heavy chain variable region as set forth in SEQ ID NO:52, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and / or, a light chain variable region as set forth in SEQ ID NO:53, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or, c) a heavy chain variable region as set forth in SEQ ID NO:54, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and / or, a light chain variable region as set forth in SEQ ID NO: NO:55, or a light chain variable region having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or, d) a heavy chain variable region as shown in SEQ ID NO:56, or a heavy chain variable region having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and / or, a light chain variable region as shown in SEQ ID NO:57, or a light chain variable region having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0020] In some embodiments, the above-mentioned antibodies or antigen-binding fragments include: a) a heavy chain variable region as shown in SEQ ID NO:50, and / or, a light chain variable region as shown in SEQ ID NO:51; or, b) a heavy chain variable region as shown in SEQ ID NO:52, and / or, a light chain variable region as shown in SEQ ID NO:53; or, c) a heavy chain variable region as shown in SEQ ID NO:54, and / or, a light chain variable region as shown in SEQ ID NO:55; or, d) a heavy chain variable region as shown in SEQ ID NO:56, and / or, a light chain variable region as shown in SEQ ID NO:57.

[0021] In some embodiments, the above-mentioned antibodies or antigen-binding fragments include: a) a heavy chain variable region as shown in SEQ ID NO:50, and a light chain variable region as shown in SEQ ID NO:51; or, b) a heavy chain variable region as shown in SEQ ID NO:52, and a light chain variable region as shown in SEQ ID NO:53; or, c) a heavy chain variable region as shown in SEQ ID NO:54, and a light chain variable region as shown in SEQ ID NO:55; or, d) a heavy chain variable region as shown in SEQ ID NO:56, and a light chain variable region as shown in SEQ ID NO:57.

[0022] In some embodiments, the above antibodies also include a human antibody constant region; preferably, the heavy chain constant region of the human antibody constant region is selected from human IgG1, IgG2, IgG3 or IgG4 and variants thereof, and the light chain constant region of the human antibody constant region is selected from human κ, λ chain constant regions and variants thereof.

[0023] In a preferred embodiment, the above-mentioned antibody or antigen-binding fragment comprises the human heavy chain constant region of SEQ ID NO:24 and the human light chain constant region of SEQ ID NO:25.

[0024] In some embodiments, the above-mentioned antibody or antigen-binding fragment comprises: a) a heavy chain as shown in SEQ ID NO:66, and a light chain as shown in SEQ ID NO:67; or, b) a heavy chain as shown in SEQ ID NO:68, and a light chain as shown in SEQ ID NO:69.

[0025] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), dimerization domain V (diabody), disulfide bond-stabilized Fv (dsFv) or a peptide containing CDR.

[0026] In another aspect, the present disclosure provides an isolated nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0027] On the other hand, the present disclosure provides a bispecific antibody, comprising a first binding domain that specifically binds to GPRC5D, and a second binding domain that specifically binds to a second target protein, wherein the first binding domain is the above-mentioned antibody or antigen-binding fragment.

[0028] In a preferred embodiment, the second target protein is CD3.

[0029] In some embodiments, the first binding domain of the bispecific antibodies described above is a complete IgG.

[0030] In some embodiments, the second binding domain of the bispecific antibody is an Fv fragment, preferably a single-chain Fv (scFv) fragment, or a disulfide-bonded Fv fragment.

[0031] In a preferred embodiment, the first binding domain of the bispecific antibody is a complete IgG, and the second binding domain is a scFv.

[0032] In some embodiments, the second binding domain of the above-mentioned bispecific antibody includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively; the light chain variable region includes LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, respectively.

[0033] In some embodiments, the scFv of the above-mentioned second binding domain includes: a heavy chain variable region as shown in SEQ ID NO:22, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%; and / or a light chain variable region as shown in SEQ ID NO:23, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%.

[0034] In some embodiments, the scFv of the second binding domain comprises: a heavy chain variable region as shown in SEQ ID NO:22, and a light chain variable region as shown in SEQ ID NO:23.

[0035] In a preferred embodiment, the scFv of the second binding domain is connected to the complete IgG of the first binding domain via a linker.

[0036] In a preferred embodiment, the N-terminus of the scFv of the second binding domain is connected to the C-terminus of the light chain of the intact IgG of the first binding domain via a linker.

[0037] In some embodiments, the linker is selected from TS(GGGGS)n, (GS)n, (GSGGS)n, (GGGGS)n, (GGGS)n or (GKPGS)n, where n is a positive integer; preferably, n is 1, 2, 3, 4, 5 or 6.

[0038] In some embodiments, the first binding domain of the bispecific antibody comprises a heavy chain constant region and a light chain constant region, and the sequences of the heavy chain constant region and the light chain constant region are shown in SEQ ID NO: 26 and SEQ ID NO: 27, respectively.

[0039] In some embodiments, the bispecific antibody is selected from: a heavy chain as shown in SEQ ID NO:28 and a light chain as shown in SEQ ID NO:29; or, a heavy chain as shown in SEQ ID NO:30 and a light chain as shown in SEQ ID NO:31.

[0040] In another aspect, the present disclosure provides an isolated nucleic acid molecule encoding the above-mentioned bispecific antibody.

[0041] In another aspect, the present disclosure provides an expression vector comprising the isolated nucleic acid molecule described above.

[0042] On the other hand, the present disclosure provides an isolated host cell, comprising the above-mentioned nucleic acid molecule, or the above-mentioned expression vector, characterized in that the host cell is selected from prokaryotic cells or eukaryotic cells; preferably, selected from eukaryotic cells; more preferably, selected from mammalian cells.

[0043] On the other hand, the present disclosure provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned bispecific antibody, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0044] On the other hand, the present disclosure provides the use of the above-mentioned antibody or antigen-binding fragment thereof, or the above-mentioned bispecific antibody, or the pharmaceutical composition in the preparation of a medicament for treating or preventing a GPRC5D-mediated disease or condition, preferably, the GPRC5D-mediated disease or condition is cancer or an autoimmune disease.

[0045] In some embodiments, the cancer is selected from lymphoma, leukemia or myeloma, and the autoimmune disease is selected from systemic lupus erythematosus and rheumatoid arthritis.

[0046] Advantages of the present invention: Bispecific antibodies can specifically bind to the tumor antigen GPRC5D in the subnanomolar or single-digit nM range, while the CD3 binding portion has a moderate or low binding affinity to T cells in the ten-digit nM range. Bispecific antibodies show in vitro efficacy in killing tumor cells and strong target-dependent mediated T cell activation to avoid systemic activation and exhaustion of T cells. In addition, bispecific antibodies are favorable for binding to GPRC5D on tumor cells rather than CD3 on circulating T cells and avoid CD3 sedimentation, thereby increasing drug exposure in tumors. Bispecific antibodies cause extremely low levels of cytokine release, so the therapeutic window will be increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 . Illustration of bispecific antibody configurations for T cell engagement, configuration M "2+2 IgG-scFv". The anti-GPRC5D Fab arms, anti-CD3 scFv and Fc region are shown as dark grey, white and light grey rectangles, respectively.

[0048] Figure 2 Representative binding histograms from flow cytometric analysis of 13A1 hybridoma clones. Solid lines represent binding to Jurkat E6.1 cells (CD3+), and dashed lines represent binding to J.RT3-T3.5 cells (CD3-). F2B was used as a positive control for CD3 binding, and secondary antibody alone was used as a negative control.

[0049] Figure 3 Binding of cynomolgus macaque T cells to 13A1 hybridoma supernatant was analyzed by flow cytometry and is shown as a histogram. SP34 was used as a positive control and clone 27A5 as a negative control.

[0050] Figure 4 .The binding of antibody gene transfected CHO-S cell supernatant to CHO-S-GPRC5D+ cells was detected by flow cytometry analysis.

[0051] Figure 5 Binding curves of anti-GPRC5D recombinant monoclonal antibodies to multiple myeloma cell lines H929 and MM.1S, measured by flow cytometric analysis, and Geomean values ​​at the indicated antibody concentrations are shown. Binding to H929 and MM.1S cells is shown in (A) and (B).

[0052] Figure 6 Anti-GPRC5DxCD3 bispecific antibodies were bound to VLPs displaying human, cynomolgus monkey or mouse GPRC5D protein. Envelope VLP controls served as negative controls.

[0053] Figure 7Binding curves of the anti-GPRC5DxCD3 bispecific antibody to the multiple myeloma cell lines H929 (A) and MM.1S (B), measured by flow cytometric analysis and shown as Geomean values ​​at the indicated antibody concentrations.

[0054] Figure 8 Binding curves of bispecific antibodies to the human T cell line Jurkat E6.1 measured by flow cytometry analysis and shown as Geomean values ​​at the indicated antibody concentrations.

[0055] Fig. 9 Activation of human primary T cells mediated by anti-GPRC5DxCD3 bispecific antibodies. Human PBMCs from a single healthy donor were co-incubated with H929 cell lines at an E:T ratio of 10:1 and serially diluted with the indicated bispecific antibodies for 48 hours. Activated CD4 or CD8 T cells (CD25 + ) as a percentage of the total number of CD4 or CD8 T cells are shown in (A) and (B), respectively.

[0056] Fig.10 Bispecific antibody-mediated PBMC cytotoxicity against the multiple myeloma cell line H929. Human PBMC from a healthy donor were co-cultured with tumor cells at an E:T ratio of 10:1 and serially diluted bispecific antibodies for 48 hours and then analyzed by flow cytometry. The percentage of viable tumor cells is represented by the bispecific antibody dose-response curve on H929 cells.

[0057] Fig.11 . Bispecific antibodies induce expanded human T cells to kill multiple myeloma cell lines. 14-day expanded T cells from PBMCs of a single healthy donor were co-cultured with H929 (E:T=1:1), MM.1S (E:T=1:5), or RPMI8226 (E:T=1:1) for 48 hours, and then cytotoxicity was measured using flow cytometry. Dose-response curves of bispecific antibodies on MM.1S (A), H929 (B), and RPMI8226 (C) represent the percentage of viable tumor cells.

[0058] Fig.12 . Bispecific antibodies induce PBMC to release inflammatory cytokines when co-cultured with H929 cells. Fresh PBMC were incubated with H929 (E:T=10:1) in the presence of different bispecific antibodies at the indicated concentrations for 48 h. TM The Human Essential Immune Response Kit detects the levels of inflammatory cytokines in the culture supernatant. The levels of IFNγ (A) and IL-6 (B) are shown.

[0059] Fig.13Efficacy of bispecific antibodies in the humanized H929CDX model. Each NSG mouse was inoculated with 5 million H929 cells by subcutaneous injection on day 0, and then intravenously injected three days later. Injected with 10 million expanded human T cells, the designated bispecific antibodies were 2μg / mouse, twice a week, and each group contained 5 mice.

[0060] Fig.14 Efficacy of bispecific antibodies in the humanized MM.1S CDX model. Each NSG mouse was inoculated with 10 million MM.1S cells by subcutaneous injection on day 0 and 8, followed by intravenous injection of 10 million expanded human T cells and the indicated bispecific antibodies twice a week at 2 μg / mouse, with 5 mice per group.

[0061] Detailed description of the invention

[0062] In the present invention, unless otherwise defined, the scientific and technical terms used herein have the meanings generally understood by those skilled in the art. In addition, the laboratory operations of cell culture, molecular genetics, nucleic acid chemistry and immunology used herein are routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0063] definition

[0064] Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, because these methods, protocols and reagents may be different. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which will only be limited by the appended claims.

[0065] For the interpretation of the specification, the following definitions shall apply, and where appropriate, terms used in the singular may also include the plural, and vice versa. It should be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0066] "Bispecific antibodies" are antibodies that have binding activity to two different antigens (or different epitopes of the same antigen). The bispecific antibodies disclosed herein are specific to two different antigens, i.e., GPRC5D as the first antigen and CD3 as the second antigen. The bispecific antibodies disclosed herein include a first binding domain, referred to as B1, which is an anti-GPRC5D antibody or an antigen-binding fragment thereof, and a second binding domain, B2, which is an anti-CD3 antibody or an antigen-binding fragment thereof.

[0067] One homodimer, bispecific antibody that finds particular use in the present invention is an IgG-scFv configuration, such as Figure 1In this embodiment, the configuration relies on the use of a scFv attached to the C-terminus of an IgG light chain, thereby forming a second antigen binding domain, wherein the Fab portion of the IgG binds the GPRC5D antigen and two "additional" scFv domains bind the CD3 antigen.

[0068] The term "antibody" as used herein refers to a monoclonal antibody or a fragment thereof. Antibodies consist of two pairs of "light chains" (LC) and "heavy chains" (HC) (such light chain / heavy chain pairs are abbreviated herein as LC / HC). The light and heavy chains of such antibodies are polypeptides consisting of several domains. Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. Each light chain includes a light chain variable domain VL and a light chain constant domain CL. The variable domains VH and VL can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The "constant domains" of the heavy and light chains are not directly involved in the binding of the antibody to the target, but exhibit various effector functions.

[0069] The term "antibody" includes mouse antibodies, fully human antibodies, chimeric antibodies, humanized antibodies and genetically engineered antibodies (variant or mutant antibodies), as long as their characteristic properties are retained. Particularly preferred are fully human antibodies or humanized antibodies, especially recombinant fully human antibodies or humanized antibodies.

[0070] As used herein, the term "antigen binding portion" or "antigen binding fragment" of an antibody refers to one or more parts or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human GPRC5D or CD3, or a portion thereof). It has been shown that certain fragments of a full-length antibody can perform the antigen binding function of an antibody. Examples of binding fragments included in the term "antigen binding portion" include (i) Fab fragments: monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments: bivalent fragments consisting of two Fab fragments linked by a disulfide bond in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments consisting of a VH domain; (vi) isolated complementary determining regions (CDRs) capable of specifically binding to an antigen. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected by synthetic linkers using recombinant methods, enabling them to be made into a single protein chain, in which the VL and VH domains are paired to form a monovalent molecule (called a single-chain variable fragment (scFv)). Useful linkers include glycine-serine polymers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is at least one (usually 3 to 4) integer, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. In some embodiments, scFv may also include a positively charged scFv linker, including (GKPGS)4. Antigen binding molecules containing VH and / or VL are also included within the scope of the present disclosure. In the case of VH, the molecule may also include one or more of the CH1, hinge, CH2, or CH3 regions. Such single-chain antibodies are also intended to be included in the term "antigen binding portion" of antibodies. Other forms of single-chain antibodies, such as dimers, are also included. Dimers are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites.

[0071] As used herein, the term "comprising" with respect to the bispecific antibodies used herein means that the bispecific antibodies only contain those binding agents that are GPRC5D and CD3 binding agents. Therefore, according to the disclosed bispecific antibodies, including a bivalent anti-GPRC5D antibody that specifically binds to GPRC5D, and a bivalent antibody that specifically binds to CD3, with respect to GPRC5D and CD3, only two binding valencies of GPRC5D are bound, while CD3 only binds two valencies, and is therefore tetravalent. As used herein, the term "peptide linker" used in the present invention may mainly include the following amino acid residues: glycine, serine, alanine or threonine. The length of the connecting peptide should be sufficient to connect the two molecules so that they have the correct conformation relative to each other, thereby maintaining the desired activity. Useful linkers include glycine-serine polymers, including, for example, TS (GGGGS) n, (GS) n, (GSGGS) n, (GGGGS) n and (GGGS) n, wherein n is at least one (usually 3 to 4) integer), glycine-alanine polymers, alanine-serine polymers and other flexible linkers. In some embodiments, the scFv may further comprise a positively charged scFv linker, including (GKPGS)4.

[0072] "Fab fragment" as used herein refers to an antibody fragment, including a light chain fragment containing the VL domain and the constant domain (CL) of the light chain, and the VH domain and the first constant domain (CH1) of the heavy chain. Two different chain compositions of the cross Fab molecule are possible and are included in the bispecific antibodies of the present invention: on the one hand, the variable regions of the Fab heavy chain and the light chain are exchanged, i.e., the cross Fab molecule includes a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). This cross Fab molecule is also referred to as crossFab (VLVH). On the other hand, when the constant regions of the Fab heavy chain and the light chain are exchanged, the cross Fab molecule includes a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL), and a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1). This cross Fab molecule is also referred to as crossFab (CLCH1). The term "Fab fragment" also includes part or all of the hinge region, such as a Fab fragment. As used herein, "F(ab)2 fragment" refers to a bivalent monospecific antibody fragment without an Fc portion. Preferably, the F(ab)2 fragment is linked at the C-terminus via a disulfide bond in the hinge region, and typically such a "F(ab)2 fragment" is a F(ab')2 fragment.

[0073] As used herein, "Fc portion of an antibody" is a term well known to those skilled in the art and is defined based on papain cleavage of an antibody. The antibody of the present invention, which includes an Fc portion, comprises as an Fc portion, preferably an Fc portion derived from human origin, and preferably all other parts of a human constant region.

[0074] In some embodiments, the bispecific binding molecules of the present disclosure are homodimers. In some embodiments, the bispecific binding molecules of the present disclosure are heterotrimers (light chain combined with heavy chain heterodimers), wherein the heavy chain heterodimers are in a configuration such as described in Brinkmann and Kontermann, MABS 9: 182-212 (2017). For example, in the structure of the bispecific binding molecules of the present disclosure, "knob-in-hole", HA-TF, ZW1, CH3 charge pair, EW-RVT, LUZ-Y, chain exchange engineered domain body (SEEDbody), DuoBody, BEAT, Triomab / Quadroma or CrossMAb strategies are used to promote heterodimerization (e.g., excessive homodimerization) of polypeptides comprising an immunoglobulin Fc region. In certain embodiments, a "knob-in-hole" approach can be used, wherein a prominent "knob-in-hole" structure of a domain is obtained by replacing another amino acid with a larger side chain (e.g., arginine, phenylalanine, tyrosine or tryptophan) with an amino acid with a small side chain (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine or valine). A recessed "hole" structure of a domain can be obtained by replacing an amino acid with a large side chain (e.g., arginine, phenylalanine, tyrosine or tryptophan) with another amino acid with a smaller side chain (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine or valine). In certain embodiments, the "knob-in-hole" mutation is located in the CH3 domain.

[0075] As used herein, the "variable domain" used herein (variable domain of light chain (VL), variable region of heavy chain (VH)) refers to each of a pair of light and heavy chains that are directly involved in binding an antibody to a target. The domains of variable human light and heavy chains have the same general structure, each domain comprising four framework (FR) regions, whose sequences are widely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a beta sheet conformation, and the CDRs can form loops connecting the beta sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and, together with the CDRs from the other chain, form a target binding site. The heavy and light chain CDR3 regions of the antibodies according to the present invention play a particularly important role in the binding specificity / affinity of the antibody, and therefore provide a further object of the present invention.

[0076] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody, which primarily contributes to antigen binding. Kabat EA et al. (1991) Sequences of proteins of immunological interest, NIH publication 91-3242 (one of the most commonly used definitions of the six CDRs) provides the most commonly used definitions of the six CDRs. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 of light chain variable domains (LCDR1, LCDR2, LCDR3, or L1, L2, L3), and CDR1, CDR2, and CDR3 of heavy chain variable domains (HCDR1, HCDR2, HCDR3, or H1, H2, H3).

[0077] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within a given HCVR and / or LCVR amino acid sequence disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, Chothia (Chothia et al., 1989) Nature 342: 877-883), based on the three-dimensional structure of antibodies and the topology of CDR loops, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, U.S. Department of Health and Human Services, National Institutes of Health (1987), AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics Database (IMGT) (imgt.cines.fr / World Wide Web), and North CDR definitions based on association propagation clustering using a large number of crystal structures. One skilled in the art can readily determine the CDRs defined by each numbering system.

[0078] The comparison of CDR numbers in different systems is as follows:

[0079]

[0080] Note 1: Some of these definitions (particularly for the Chothia loop) vary depending on the individual publications studied; Note 2: Any numbering scheme can be used for these CDR definitions, with the exception of the contact definitions which use the Chothia or Martin (Enhanced Chothia) definitions; Note 3: When numbering using the Kabat numbering convention, the ends of the Chothia HCDR1 loop vary between H32 and H34, depending on the length of the loop. This is because the Kabat numbering scheme places the insert at H35A and H35B.

[0081] As used herein, the term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the prior art. Human antibodies can also be produced in transgenic animals (e.g., mice) that can produce complete repertoires or selected human antibodies in the absence of endogenous immunoglobulin production after immunization. Transferring human germline immunoglobulin gene arrays in such germline mutant mice will result in the production of human antibodies when the target is attacked. Human antibodies can also be produced in phage display libraries.

[0082] As used herein, the term "recombinant human antibody" as used herein is intended to include all human antibodies prepared, expressed, manufactured or isolated by recombinant means, such as antibodies isolated from host cells (e.g., NSO or CHO cells) or from animals (e.g., mice), which have transgenic human immunoglobulin genes, or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant human antibodies have variable and constant regions in rearranged form. The recombinant human antibodies of the present invention have been subjected to in vivo somatic hypermutation. Therefore, although the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, they may not naturally exist in the human antibody germline repertoire in vivo.

[0083] As used herein, the term "epitope" includes any polypeptide determinant that is capable of specific binding to an antibody. In certain embodiments, epitope determinants include chemically active molecular surface groups, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics. An epitope is the region where a target binds to an antibody.

[0084] As used herein, the term "nucleic acid molecule" as used herein refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. When a nucleic acid is in a functional relationship with another nucleic acid sequence, it is "operably linked". For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to a coding sequence.

[0085] The method for preparing the nucleic acid is a conventional method in the art. Preferably, it comprises the following steps: obtaining the nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining the nucleic acid molecule encoding the above protein by artificial full-length sequence synthesis.

[0086] Those skilled in the art know that the base sequence encoding the amino acid sequence of the protein can be appropriately replaced, deleted, changed, inserted or added to provide a polynucleotide homologue. The homologue of the polynucleotide of the present invention can be prepared by replacing, deleting or adding one or more bases of the protein encoding sequence to maintain the activity of the antibody.

[0087] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be attached. In another embodiment, the vector is a viral vector, wherein additional DNA segments can be attached to the viral genome. The vectors disclosed herein are capable of self-replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial replication origin), or can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating together with the host genome (e.g., non-episomal mammalian vectors).

[0088] The recombinant expression vector can be obtained by conventional methods in the art, that is, by connecting the nucleic acid molecule of the present invention to various expression vectors to construct. The expression vector is one of a variety of conventional vectors in the art, as long as it can carry the above-mentioned nucleic acid molecule. The vector preferably includes: various plasmids, cosmids, phages or viral vectors, etc.

[0089] As used herein, the term "transfected cells" as used herein includes recombinant eukaryotic host cells expressing antibodies, such as CHO cells, NS / 0 cells, HEK293 cells, plant cells or fungi, including yeast cells.

[0090] The DNA molecule sequence of the antibody or its fragment according to the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the sequences encoding the light chain and the heavy chain can be fused together to form a single-chain antibody.

[0091] Once the relevant sequence is obtained, it can be obtained in bulk using recombinant methods. This is usually achieved by cloning the sequence into a vector, transforming cells with the vector, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0092] In addition, related sequences can be synthesized artificially, especially when the fragment length is short. Usually, several small fragments are synthesized first and then connected together to obtain a long sequence fragment.

[0093] At present, the DNA sequence encoding the antibody of the present invention (or its fragment or derivative thereof) can be completely obtained by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or, for example, vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0094] Generally, the resulting host cells are cultured under conditions suitable for expressing the antibodies of the invention. The antibodies of the invention are then purified using conventional immunoglobulin purification procedures, for example, conventional separation and purification methods well known to those skilled in the art, such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.

[0095] The obtained antibodies can be identified by conventional methods. For example, the binding specificity of the monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). For example, the binding affinity of the monoclonal antibody can be determined by Scatchard analysis (Munson et al., Anal. Biochem., 107: 220 (1980)).

[0096] The antibodies of the present invention can be expressed on cells or cell membranes, or secreted outside the cells. If necessary, various separation methods can be used for separation and purification according to the physical and chemical properties of the recombinant protein. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, protein precipitant treatment (such as salt precipitation), centrifugation, osmotic cell lysis, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel chromatography), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and any other liquid chromatography, and combinations thereof.

[0097] As used herein, the term "variant" of a polypeptide, for example, an antigen binding fragment, a protein or an antibody is a polypeptide in which one or more amino acid residues are inserted, deleted, added and / or substituted, compared to another polypeptide sequence, and includes fusion polypeptides. In addition, protein variants include protein variants modified by protease cleavage, phosphorylation or other post-translational modifications, but retain the biological activity of the antibodies disclosed herein, such as binding and specificity to CD3. The variant may be identical to the sequence of an antibody or antigen binding fragment thereof disclosed herein, or about 99%, 98%, 97%, 96%, 95%, 94%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% identical.

[0098] As used herein, the term "K D"(M) refers to the dissociation equilibrium constant for a specific antibody-antigen interaction. "K D " refers to the dissociation constant, which is obtained by the ratio of Kd to Ka (i.e., Kd / Ka), expressed in molar concentration (M). In view of the present disclosure, the K of an antibody can be determined using methods in the art. D For example, the K D It can be determined by using surface plasmon resonance, for example by using a biosensor system, such as the AUTO® system, or by using bio-layer interferometry, such as the Octet RED96 system.

[0099] As used herein, the term "affinity" is the strength of the interaction between an antibody or antigen-binding fragment thereof and an antigen, which is determined by the properties of the antigen, such as the size, shape and / or charge of the antigen, and the CDR sequence of the antibody or antigen-binding fragment. Methods for determining affinity are known in the art, and the following methods may be referred to.

[0100] As used herein, the term “EC 50 ” refers to the concentration that is 50% of the maximum effect, that is, the concentration that can cause 50% of the maximum effect.

[0101] As used herein, the term "pharmaceutical composition" used herein is intended to refer to a mixture containing one or more compounds or physiologically / pharmaceutically acceptable salts or prodrugs thereof and other chemical components described herein, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, which is beneficial to the absorption of the active ingredient and the exertion of biological activity.

[0102] As used herein, the terms "administration" and "treatment", when applied to an animal, human, subject, cell, tissue, organ or biological fluid, refer to contacting an animal, human, subject, cell, tissue, organ or biological fluid with an exogenous drug, therapeutic, diagnostic agent or composition. For example, "administration" and "treatment" can refer to treatment, pharmacokinetics, diagnosis, research and experimental methods. Treatment of cells includes contacting cells with an agent, and contacting a fluid with an agent, wherein the fluid is in contact with the cell. "Administration" and "treatment" also refer to in vitro and ex vivo treatment, for example, treatment of a cell by an agent, a diagnostic, a combination composition or by another cell. "Treatment" when applied to humans, veterinary medicine or research subjects refers to treatment, prevention or preventive measures, research and diagnostic applications. DETAILED DESCRIPTION

[0103] The present invention is further illustrated by the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental methods without detailed conditions in the following examples generally meet conventional conditions, such as the conditions described by Sambrook, Joseph F.Sambrook et al. "Guide to Molecular Cloning Laboratory" (Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer (e.g., product manual). Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise stated, the experimental materials and reagents used in the following examples are all commercially available materials and reagents.

[0104] The room temperature described in the examples is conventional room temperature in the art, generally 18-27°C.

[0105] Example 1. Immunization and screening of antibodies

[0106] Anti-GPRC5D antibody

[0107] Immunization and screening were performed using full-length human GPRC5D. RenMab GPRC5D-KO mice (purchased from Biocytogen) aged 6-8 weeks were immunized once every 14 days for 4 rounds using conventional DNA immunization methods. Mice were sacrificed five days after the last injection. Tissues were isolated under sterile conditions and washed with fresh medium. Lymphocytes were released from lymph nodes or spleen. The platform screened plasma cells that secreted GPRC5D-specific antibodies, and the V region sequences of the antibody light and heavy chains were obtained by reverse transcription and PCR amplification sequencing.

[0108] Anti-CD3 antibodies

[0109] Antibodies were obtained by immunizing transgenic mice encoding human immunoglobulin heavy chain and kappa light chain variable regions with a combination of human CD3 antigen, cynomolgus CD3 antigen, human CD3 DNA naturally expressing human CD3, or Jurkat E6.1 cells (Sigma-Aldrich, catalog number 88042803). Antibody immune responses were monitored by CD3-specific immunoassays. When the desired immune response was achieved, splenocytes were harvested from each mouse, fused with mouse myeloma cells to maintain their viability, formed hybridoma cells, and screened for CD3 specificity.

[0110] Splenic lymphocytes and myeloma cells Sp2 / 0 CRL-158) and hybridoma cells were obtained by electrofusion or PEG fusion. TMPEG fusion was performed by HY technology (STEMCELL Technologies). The ratio of primary cells to mouse myeloma cells was 1:1 for electrofusion and 10:1 for PEG fusion.

[0111] Screening of hybridoma clones specifically binding to human / cynomolgus monkey CD3 protein by ELISA

[0112] ELISA was performed using the DuoSet ELISA Auxiliary Kit (R&D System, DY008). ELISA plates were coated with 5ug / ml human CD3δε (AcroBio CDD-H52W1) and cynomolgus CD3δε (AcroBio, CDD-C52W4) overnight. The plates were washed three times with wash buffer to wash off excess unbound protein. The plates were then blocked for 1 hour at room temperature. 50ul CD3 hybridoma supernatant was added to each replicate well and incubated for 1 hour. Excess unbound antibodies were washed away, and 50ul 1:1000 diluted secondary antibody goat anti-mouse IgG Fc-HRP (Jackson ImmunoResearch, 115-035-071) was added for another 1 hour. The plates were washed before adding chemiluminescent agents (color A and color B) according to the manufacturer's protocol. The optical density of the 450nm samples was measured by a microplate reader (PerkinElmer). The results are shown in Table 1.

[0113] Table 1. Binding activity results of ELISA assay

[0114]

[0115] The results showed that the supernatant of the hybridoma clone 13A1 antibody disclosed in the present invention had a high affinity for human and cynomolgus monkey CD3.

[0116] Jurkat binding screening

[0117] Human T cell line (CD3 + Jurkat cell line clones E6.1 and CD3 - The hybridoma supernatants were tested for binding using flow cytometry (clone J.RT3-T3.5 (ATCC)). Undiluted hybridoma supernatants were used for primary staining, followed by fluorescently labeled secondary antibodies (PE goat anti-mouse IgG, BioLegend Cat#405307). Figure 2 Examples of selected flow cytometry histograms are shown, with the solid line representing the expression of 100% sigmoid protein in Jurkat E6.1 cells (CD3 + ), the dotted line indicates the binding with J.RT3-T3.5 cells (CD3 -The binding spectrum of clone 13A1 to Jurkat E6.1 cells was enhanced compared to that to J.RT3-T3.5 cells.

[0118] Cyno-T cells (HSC-F) combined

[0119] The clones selected for Jurkat binding were further tested for binding to cynomolgus monkey T cells (HSC-F) using flow cytometry. Anti-CD3 antibody SP34 was used as a positive control (Conrad, ML, Davis, WC and Koop, BF (2007). The cynomolgus monkey T cell line (HSC-F) was obtained from the Non-Human Primate Reagent Resource (NHPRR). The staining was performed similarly to the above, and representative results are shown in Figure 3 shown.

[0120] Example 2. Sequencing of positive clones

[0121] Sequencing of GPRC5D-resistant clones

[0122] The V region sequences of the antibody light chain and heavy chain were obtained by reverse transcription and PCR amplification sequencing.

[0123] Table 2. KABAT CDR sequences of the heavy chain variable region of GPRC5D clones

[0124]

[0125] Table 3. KABAT CDR sequences of the light chain variable region of GPRC5D clones

[0126]

[0127] Table 4. Heavy and light chain variable region sequences of GPRC5D clones

[0128]

[0129] Sequencing of anti-CD3 clones

[0130] The process of cloning sequences from positive hybridomas is as follows. Hybridoma cells in the logarithmic growth phase are collected, RNA is extracted, reverse transcribed, and then the VDJ region is amplified. The cDNA library amplified by each clone is subjected to second-generation sequencing. The amino acid sequences of the heavy chain and light chain variable region DNA sequences corresponding to antibody 13A1 are obtained. In the manufacturability assessment, the framework region was mutated several times, and the amino acid sequences and CDR sequences of the heavy chain variable region and light chain variable region of each antibody are shown in the following table. The amino acid residues of the CDR in VH / VL are numbered and annotated according to the Kabat&Wu definition system.

[0131] Table 5. CDR sequences of the heavy chain variable regions of CD3 hybridoma clones

[0132]

[0133]

[0134] Table 6. CDR sequences of light chain variable regions of CD3 hybridoma clones

[0135]

[0136] Table 7. Heavy and light chain variable region sequences of CD3 hybridoma clones

[0137]

[0138] CD3ScFv engineered mutation

[0139] The mutagenesis was designed to obtain a panel of anti-CD3 antibodies with different affinities and T-cell activation activities, as well as potentially lower immunogenicity, by converting specific residues to the germline sequence.

[0140] Template selection and mutation design of hybridoma clone 13A1-1

[0141] Table 8.13A1-1 Mutations in the light and heavy chain variable regions

[0142]

[0143] Note: For example, according to the Kabat numbering system, I14T indicates a mutation from I to T at position 14. WT indicates that the sequence does not contain an amino acid mutation.

[0144] Table 9. KABAT CDR sequences of the heavy chain variable domain of 13A1-1_mut36

[0145]

[0146] Table 10. KABAT CDR sequences of the light chain variable domain of 13A1-1_mut36

[0147]

[0148] Table 11. Heavy chain variable domain and light chain variable domain sequences of 13A1-1_mut36

[0149]

[0150] Example 3 Screening of anti-GPRC5D antibodies

[0151] Using the ExpiCHO transient expression system, anti-GPRC5D antibody light and heavy chains were expressed and supernatants were tested by binding to CHO-S cells overexpressing human GPRC5D. The parent anti-GPRC5D antibody from Talquetamab (clone: ​​GC5B596 from US10562968B2) was expressed as IgG1 and used as a positive control. Clones Ab04 and Ab17 showed binding to GPRC5D. + Strong binding of cells ( Figure 4 ) and were selected for further study.

[0152] The endocytosis of MM.1S cells by antibody supernatants expressed by ExpiCHO was evaluated. Cells were labeled with antibodies and pHAb (Promega, catalog number G9845). The internalized antibody-pHAb fluoresces in a low pH environment. Endocytosis was shown by comparing the mean fluorescence intensity (MFI) after 3 or 6 hours of incubation. Compared to the negative control, clones 04 and 17 showed internalization after 3 hours and 6 hours of incubation. The data are summarized in Table 12.

[0153] Table 12. Antibody internalization efficiency

[0154] clone Internalization 3h MFI Internalization 6h MFI 04 6206 8591 17 5018 8238 GC5B596 3793 5284 Negative control 2951 3087

[0155] Example 4 Construction, expression and purification of recombinant antibodies

[0156] Molecular cloning of recombinant antibodies:

[0157] The cDNA sequences encoding the VH and VL regions of the monoclonal antibodies or designed bispecific antibodies were directly synthesized as DNA fragments with a 5' in-frame leader sequence (MGWSCIILFLVATATGVHS). These DNA fragments were cloned into mammalian expression vectors (backbone sequences similar to pcDNA3.1) using the NEBuilder DNA assembly cloning kit (New England Biolabs).

[0158] The constant region of the monospecific antibody here is hIgG1.

[0159] Table 13. Monoclonal antibody heavy chain constant region and light chain constant region sequence

[0160]

[0161] Table 14. Sequences of monoclonal antibodies

[0162]

[0163] The bispecific antibody of the present invention is of IgG-scFv configuration, such as Figure 1As shown. This configuration relies on the use of scFv attached to the C-terminus of the IgG light chain, thereby forming a second antigen-binding domain, the Fab portion of the IgG binding to GPRC5D, and two "extra" scFv domains binding to CD3. The constant region of the bispecific antibody adopts the hIgG4 sequence with engineered mutations (S228P, F234A, L235A).

[0164] Table 15. Bispecific antibody heavy chain constant region and light chain constant region sequences

[0165]

[0166] Table 16. Sequences of peptide linkers

[0167] Linkers in anti-CD3 ScFv (GGGGS)6 Linker between anti-GPRC5D CL and anti-CD3 VH TS(GGGGS)3

[0168] Table 17. Sequences of bispecific antibodies

[0169]

[0170]

[0171] Expression and purification of recombinant antibodies:

[0172] The heavy chain expression plasmid and the light chain plasmid were co-transfected into ExpiCHO-S cells (ThermoFisher #A29127) using the ExpiFectamine CHO transfection kit (ThermoFisher, A29129). The plasmid DNA concentration reached 1.0 ug per ml of suspended cells according to the manufacturer's instructions. The transfected cells were cultured on a shaker at 37°C, 8% CO2 for 5 to 7 days. The culture medium was collected and the antibodies were purified using a HiTrap MabSelect SuRe column (Cytiva, #17549112) on an AKTA Pure 25 machine (Cytiva). The eluted antibodies were neutralized with Tris buffer (pH 9.0) and PBS buffer exchange was performed. The concentrated protein was further purified using a SEC-superdex200 column (Cytiva, #28989335) on an AKTA Pure 25 machine. The components of each peak were concentrated. The final product was quantified by UV absorption, and its purity was determined by SDS-PAGE and HPLC.

[0173] Example 5 Basic antigen binding properties of recombinant anti-GPRC5D monoclonal antibodies

[0174] The binding affinity of recombinant anti-GPRC5D monoclonal antibodies to cell surface GPRC5D antigens was tested in a flow cytometry-based cell binding assay. Multiple myeloma cell lines were blocked in ice culture medium for 30 minutes and then incubated with different concentrations of primary antibodies in culture medium on ice for 1 hour. The cells were then washed with PBS and labeled with secondary antibody (R-phycoerythrin AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment specific, Jackson ImmunoResearch 109-116-170), diluted 1:200 in culture medium and incubated on ice for 1 hour. The cells were washed with PBS and then analyzed by flow cytometry. Binding curves were generated and analyzed by Flowjo and GraphPad Prism software. The binding curves of recombinant antibodies to H929 and MM.1S are shown in Figure 2. Figure 5 As shown, K D The values ​​are summarized in Table 18. Anti-GPRC5D antibody GC5B596, the parent antibody of Talquetamab (from patent WO2019220368, see SEQ ID NO: 51, 52, 41, 42) was used as a control. HBMAB-0088 and HBMAB-0097 showed strong binding to H929 and MM.1S cells with higher affinity than the control antibody.

[0175] Table 18. Binding affinity of recombinant anti-GPRC5D monoclonal antibodies to multiple myeloma cell lines

[0176]

[0177] Example 6 Antigen Binding Properties of T Cell Engaging Bispecific Antibodies (TCEs)

[0178] The selected GPRC5DxCD3 bispecific antibodies were tested for binding to cynomolgus monkey GPRC5D and mouse GPRC5D. VLPs displaying human GPRC5D (Kactusbio Cat. No. GPR-HM05P), cynomolgus monkey GPRC5D (Kactusbio Cat. No. GPR-CM05P) or mouse GPRC54D (Kactusbio Cat. No. GPR-MM05P) were coated on MSD plates (MSD Cat. No. L15XA-3) at a concentration of 5 μg / ml in PBS at 4°C overnight. Envelope VLPs (Kactusbio Cat. No. VLP-HM00C) were coated in parallel and served as negative controls. After coating, the plates were washed 3 times with PBS 0.05% Tween20 and then blocked with 3% BSA in PBS for 1 hour at room temperature. After washing, the plates were incubated with different concentrations of bispecific antibodies for 1 hour at room temperature. The plates were then washed and incubated for 1 hour at room temperature with 1 μg / ml Sulfo-tagged goat anti-human Fc generated by adding a Sulfo-tag to goat anti-human Fc (ImmunoResearch Cat. No. 109-005-170) and MSD GOLD SULFO-TAG NHS-Ester coupling kit (MSD Cat. No. R31AA-1) according to the manufacturer's instructions. After washing, MSD GOLD read buffer B was added to the plate at 150 μl / well and the plate was read using a MESO Quickplex SQ 120MM reader. Data are shown in Figure 6 and as shown in Table 19. Both M44-mut36 and M53-mut36 bound to human, cynomolgus monkey or mouse GPRC5D and did not bind to the VLP control.

[0179] Table 19. Binding affinity of GPRC5D x CD3TCE on VLPs displaying human GPRC5D, cynomolgus monkey GPRC5D or mouse GPRC5D.

[0180]

[0181] Each arm of the GPRC5DxCD3 bispecific antibody was functionally tested for binding to cell surface GPRC5D or CD3 in a cell-based binding assay as described in Example 5. TCE was performed with the GPRC5D+ multiple myeloma cell lines H929 and MM.1S and human CD3 + Binding curves of T cell line Jurkat E6.1 cells are shown in Figure 7 and Figure 8 As shown, K DThe values ​​are summarized in Table 20. M44-Mut36 and M53-Mut36 bound more strongly to GPRC5D-positive cells than the control TCE Talquetamab and were positive for binding to T cells.

[0182] Table 20. Binding affinity of GPRC5D x CD3TCE to multiple myeloma cell lines H929 and MM.1S and human T cell line Jurkat E6.1

[0183] H929 MM.1S Jurkat E6.1 BsAb <![CDATA[K D (nM)]]> <![CDATA[K D (nM)]]> <![CDATA[K D (nM)]]> M44-mut36 1.7 1.5 6 M53-mut36 3.8 2.9 26 Talquetamab 25.0 15.0 4

[0184] Example 7 Effect of bispecific antibodies on human PBMC activation

[0185] The ability of the GPRC5DxCD3 bispecific antibody to activate human primary T cells was tested. CD25 expression is induced during T cell activation, and surface CD25 levels can be used as an indicator of T cell activation. The expression of CD25 on human primary T cells after bispecific antibody treatment of target tumor cells was evaluated based on a flow cytometric assay. The multiple myeloma cell line H929 was pre-labeled with CellTrace Violet (ThermoFisher Cat. No.: C34557). Human PBMCs from healthy donors were co-cultured with pre-labeled H929 cells at an effector to tumor ratio (E-to-T) of 10:1 for 48 hours in the presence of different concentrations of different bispecific antibodies. The cells were washed with 1x PBS and anti-CD4-BV605 (BioLegend Cat#317438, clone OKT4), anti-CD8-FITC (Invitrogen Cat#11-0088-42, clone RPA-T8), and anti-CD25-APC (BioLegend Cat#302610, clone BC96) antibodies. + CD25 + and CD8 + CD25 + T cell populations were stained. Flow cytometry data were acquired on a BD Fortessa X20. Data were analyzed using FlowJo software. GraphPad Prism was used to plot CD25 + The percentage of the population as a percentage of total CD4 or CD8 T cells. Calculate EC 50 See Table 21 for values.

[0186] Table 21. GPRC5D x CD3TCE induces T cell activation EC 50 (nM)

[0187] <![CDATA[EC 50 (pM)]]> CD4T activation CD8 T activation M44-mut36 5.3 2.3 M53-mut36 11 4.2 Talquetamab 40.5 39.5

[0188] In the presence of H929 cells, strong activation of primary human CD4 and CD8 T cells by the bispecific antibodies was observed in PBMCs treated with M44-Mut36 and M53-Mut36, which was superior to the control Talquetamab. 50 The value is better than the control ( Fig. 9 ).

[0189] Example 8 Cytotoxicity of human PBMC or expanded T cells redirected by bispecific antibodies to GPRC5D+ multiple myeloma cell lines

[0190] The GPRC5DxCD3 bispecific antibody was tested for its ability to inhibit GPRC5D in co-culture experiments with primary human immune cells. + Cytotoxicity in tumor cell lines.

[0191] PBMC co-culture cytotoxicity assay:

[0192] Multiple myeloma cell line H929 was labeled with 5 μM CellTrace Violet dye (ThermoFisher Cat#C34557) according to the manufacturer's instructions. Human PBMCs from healthy donors were isolated from LRS chambers using lymphocyte separation medium (Corning Cat#25-072-CV) according to the manufacturer's instructions. Viable PBMCs and tumor cells were counted and mixed at an ET ratio of 10:1. Bispecific antibodies were serially diluted with a starting concentration of 10 nM. Cells were incubated with antibodies at 37°C for 48 hours. Then, cells were incubated with Invitrogen ELISA according to the manufacturer's instructions. TM LIVE / DEAD TM Cells were stained with Fixable Near-Infrared Dead Cell Stain Kit (ThermoFisher Scientific Cat#L10119) and analyzed using a BD FortessaX20 flow cytometer.

[0193] The number of viable tumor cells in each well was normalized to the control well containing tumor cells alone in the absence of effector cells. M44-Mut36 and M53-Mut36 can effectively redirect PBMCs and kill H929 tumor cells ( Fig.10 ), with better in vitro cytotoxicity than Talquetamab. EC 50 The values ​​are summarized in the following table (Table 22).

[0194] Table 22. EC of GPRC5D x CD3TCE induced cytotoxicity in H929 cells 50 value

[0195] <![CDATA[EC 50 (pM)]]> M44-mut36 0.64 M53-mut36 1.03 Talquetamab 15.45

[0196] Expanded Human T Cell Co-culture Cytotoxicity Assay:

[0197] T cell co-culture cytotoxicity assays were performed similarly to those described above, using expanded T cells as effector cells. Briefly, cells were isolated from 100% T cells using the EasySep Human T Cell Isolation Kit (STEMCELL Technologies Cat. No. 17951) according to the manufacturer’s protocol in the presence of recombinant IL2 and Dynabeads. TM T cells were expanded in RPMI1640 10% FBS, 1% penicillin / streptomycin medium for 14 days in the presence of human T activator CD3 / CD28 (ThermoFisher Cat#11161D), and then cultured for another day without T cell activation beads. Multiple myeloma cell lines H929, MM.1S or RPMI8226 were labeled with CellTrace Violet (ThermoFisher Cat.No.C34557) and incubated with expanded T cells in a 37°C incubator for 48 hours in the presence of different concentrations of bispecific antibodies. The E:T ratios of T cells to MM.1S, H929 and RPMI8226 were 1:5, 1:1 and 1:1, respectively. Cells were collected and stained with Live / Dead stain kit (ThermoFisher Cat.No.C34557) for 633 or 635 nm excitation and analyzed by flow cytometry. The number of viable tumor cells per well was normalized to wells with tumor cells only.

[0198] M44-Mut36 and M53-Mut36 induced strong killing activity on MM.1S and H929 cells expressing high GPRC5D by expanding T cells ( Fig.11 AB). The killing activity against RPMI8226 cells is weaker ( Fig.11 C) EC 50 The values ​​are summarized in Table 23.

[0199] Table 23. GPRC5D x CD3TCE induces cytotoxicity in multiple myeloma cells EC 50 value

[0200]

[0201]

[0202] Example 9 Quantification of cytokine release from PBMCs when bispecific antibodies mediate target cell binding

[0203] The potential of the GPRC5DxCD3 bispecific antibody to induce T cell-mediated cytokine production was analyzed. Freshly isolated human PBMC cells were co-cultured with the multiple myeloma cell line H929 (E to T = 10:1) in a U-bottom 96-well plate at 37°C and 5% CO2 for 48 hours. After centrifugation of the cell pellet, the supernatant of each well was transferred to a V-bottom 96-well plate. Cytokines (including IFNγ, IL6) released from PBMCs were measured using the LEGENDplex multiplex detection kit (BioLegend #740930) according to the manufacturer's instructions. Data were collected and analyzed on iQue3 (Sartorius). Individual cytokine levels for each sample were interpolated from the standard curve using GraphPad Prism software.

[0204] The results showed that compared with the effective concentration of TCE, the levels of inflammatory factors (FNγ, IL-6) released by bispecific antibody-mediated T cell activation were extremely low. Fig.12 As shown, M44-Mut36 and M53-Mut36 induced cytokine levels comparable to those of Talquetamab.

[0205] Example 10 Evaluation of the therapeutic efficacy of bispecific antibodies in the T cell humanized multiple myeloma CDX mouse model.

[0206] Expanded T cell humanized H929CDX model:

[0207] Human T cells were isolated from human PBMCs as described above and cultured in the presence of recombinant IL2 and Dynabeads. TM The cells were expanded for 7-10 days in the presence of human T activator CD3 / CD28 (ThermoFisher). NSG mice (6-8 weeks old) were implanted with multiple myeloma cell line H929 (prepared in PBS 20% matrix gel) at a volume of 5 million cells / mouse. Three days later, bispecific antibodies and expanded human T cells were injected intravenously. The mice were injected into the retro-orbital vein at the specified dose. Tumor volume was measured three times a week.

[0208] On day 0, 6- to 8-week-old NOD-scid-IL2Rgnull (NSG) mice (Jackson Laboratories) were injected subcutaneously in the flank with 5 × 10 6 H929 tumor cells and 20% Matrigel. On day 3, the animals were randomly divided into 5 treatment groups, with 5 mice in each group; each mouse was injected intravenously (retro-orbital vein) with 10x 10 6Expanded human T cells were mixed with bispecific antibodies (2 μg / mouse) or PBS. Mice received 3 doses of expanded primary T cells every 3 or 4 days and 5 doses of bispecific antibodies or PBS solution alone every 3 or 4 days. Body weight and tumor volume were measured three times a week. Tumor volume was measured using a caliper and calculated using the following formula: Volume = (length x width x width) / 2.

[0209] The M44-Mut36 and M53-mut36 treatment groups showed complete tumor regression at a dose of 2ug / animal (~0.1mpk), whereas Talquetamb only partially inhibited tumor growth ( Fig.13 ). These results indicate that M44-Mut36 and M53-Mut36 have strong anti-tumor effects.

[0210] Expanding T Cell Humanized MM.1S CDX Model

[0211] The antitumor activity of M44-Mut36 and M53-Mut36 was also evaluated in the MM.1S CDX model in a similar manner as described above. Briefly, 10 7 MM.1S cells were inoculated into NSG mice. On day 8, the mice were randomly divided into 5 groups, with 5 mice in each group. Then, the tumor-bearing mice were injected with 10 7 The mice were given 4 doses of expanded human T cells and 2 μg of bispecific antibody per mouse. All mice received a total of 4 doses of expanded T cells and bispecific antibody every 3 or 4 days. Tumor volume was measured three times a week.

[0212] M44-Mut36 and M53-Mut36 exhibited potent antitumor activity against MM.1S in vivo, whereas Talquetamab caused minimal tumor inhibition compared with the control group ( Fig.14 ). These results suggest that M44-Mut36 and M53-Mut36 can effectively target multiple myeloma in vivo.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to GPRC5D, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, characterized in that: a) HCDR1 has the amino acid sequence shown in SEQ ID NO: 01 or SEQ ID NO: 04; b) HCDR2 has the amino acid sequence shown in SEQ ID NO: 02 or SEQ ID NO: 05; c) HCDR3 has the amino acid sequence shown in SEQ ID NO: 03 or SEQ ID NO: 06; d) LCDR1 has the amino acid sequence shown in SEQ ID NO: 07 or SEQ ID NO: 10; e) LCDR2 has the amino acid sequence shown in SEQ ID NO:08; f) LCDR3 has the amino acid sequence shown in SEQ ID NO:09 or SEQ ID NO:

11.

2. The antibody or antigen-binding fragment thereof that specifically binds to GPRC5D according to claim 1, characterized in that: a) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:01, SEQ ID NO:02 and SEQ ID NO:03, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:07, SEQ ID NO:08 and SEQ ID NO:09, respectively; or b) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:04, SEQ ID NO:05 and SEQ ID NO:06, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:10, SEQ ID NO:08 and SEQ ID NO:11, respectively.

3. The antibody or antigen-binding fragment thereof that specifically binds to GPRC5D according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof.

4. The antibody or antigen-binding fragment thereof that specifically binds to GPRC5D according to claim 3, characterized in that: The antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region as set forth in SEQ ID NO:18, or a heavy chain variable region having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and / or a light chain variable region as set forth in SEQ ID NO:19, or a light chain variable region having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or, b) a heavy chain variable region as set forth in SEQ ID NO:20, or a heavy chain variable region having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, and / or a light chain variable region as set forth in SEQ ID NO:21, or a light chain variable region having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; Preferably, the antibody or antigen-binding fragment comprises: a) a heavy chain variable region as shown in SEQ ID NO: 18, and / or, a light chain variable region as shown in SEQ ID NO: 19; or, b) a heavy chain variable region as shown in SEQ ID NO: 20, and / or, a light chain variable region as shown in SEQ ID NO: 21; More preferably, the antibody or antigen-binding fragment comprises: a) a heavy chain variable region as shown in SEQ ID NO: 18, and a light chain variable region as shown in SEQ ID NO: 19; or, b) a heavy chain variable region as shown in SEQ ID NO: 20, and a light chain variable region as shown in SEQ ID NO:

21.

5. The antibody or antigen-binding fragment thereof that specifically binds to GPRC5D according to claim 4, characterized in that: The antibody also includes a human antibody constant region; preferably, the heavy chain constant region of the human antibody constant region is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 and variants thereof, and the light chain constant region of the human antibody constant region is selected from human κ, λ chain constant regions and variants thereof; more preferably, the antibody includes the human heavy chain constant region of SEQ ID NO: 24 and the human light chain constant region of SEQ ID NO:

25.

6. The antibody or antigen-binding fragment thereof that specifically binds to GPRC5D according to claim 5, characterized in that: The antibody or antigen-binding fragment comprises: a) a heavy chain as shown in SEQ ID NO:66, and a light chain as shown in SEQ ID NO:67; Or, b) a heavy chain as shown in SEQ ID NO:68, and a light chain as shown in SEQ ID NO:

69.

7. The antibody or antigen-binding fragment thereof that specifically binds to GPRC5D according to any one of claims 1 to 6, characterized in that: The antigen binding fragment is selected from Fab, Fab', F(ab')2, variable fragment (Fv), single chain variable fragment (scFv), dimerization domain V (diabody), disulfide bond stabilized Fv (dsFv) or a peptide containing CDR.

8. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. A bispecific antibody comprising a first binding domain that specifically binds to GPRC5D, and a second binding domain that specifically binds to a second target protein, wherein: The first binding domain is the antibody or antigen-binding fragment of any one of claims 1-7.

10. The bispecific antibody according to claim 9, characterized in that The second target protein is CD3.

11. The bispecific antibody according to claim 9 or 10, characterized in that: The first binding domain is an intact IgG.

12. The bispecific antibody according to any one of claims 9 to 11, characterized in that The second binding domain comprises a heavy chain variable region and a light chain variable region, Wherein, the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14 respectively; the light chain variable region includes LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17 respectively.

13. The bispecific antibody according to claim 12, characterized in that: The second binding domain is an Fv fragment, preferably a single-chain Fv (scFv) fragment, or a disulfide-bonded Fv fragment.

14. The bispecific antibody according to any one of claims 9 to 13, characterized in that The first binding domain is a complete IgG and the second binding domain is a scFv.

15. The bispecific antibody according to claim 14, characterized in that The scFv of the second binding domain comprises: a heavy chain variable region as set forth in SEQ ID NO:22, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as set forth in SEQ ID NO:23, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; Preferably, the scFv of the second binding domain comprises: a heavy chain variable region as shown in SEQ ID NO:22, and a light chain variable region as shown in SEQ ID NO:

23.

16. The bispecific antibody according to any one of claims 13 to 15, characterized in that The scFv of the second binding domain is connected to the intact IgG of the first binding domain via a linker.

17. The bispecific antibody according to claim 16, characterized in that The N-terminus of the scFv of the second binding domain is connected to the C-terminus of the light chain of the intact IgG of the first binding domain via a linker.

18. The bispecific antibody according to claim 16 or 17, characterized in that: The linker is selected from TS(GGGGS)n, (GS)n, (GSGGS)n, (GGGGS)n, (GGGS)n or (GKPGS)n, where n is a positive integer; preferably, n is 1, 2, 3, 4, 5 or 6.

19. The bispecific antibody according to any one of claims 9 to 18, characterized in that The first binding domain includes a heavy chain constant region and a light chain constant region, and the sequences of the heavy chain constant region and the light chain constant region are shown in SEQ ID NO: 26 and SEQ ID NO: 27, respectively.

20. The bispecific antibody according to claim 19, characterized in that The bispecific antibody is selected from: The heavy chain shown in SEQ ID NO:28 and the light chain shown in SEQ ID NO:29; Or, the heavy chain as shown in SEQ ID NO:30 and the light chain as shown in SEQ ID NO:

31.

21. An isolated nucleic acid molecule encoding the bispecific antibody of any one of claims 9-20.

22. An expression vector comprising the isolated nucleic acid molecule of claim 21.

23. An isolated host cell comprising the nucleic acid molecule of claim 21, or the expression vector of claim 22, wherein: The host cell is selected from prokaryotic cells or eukaryotic cells; preferably, selected from eukaryotic cells; more preferably, selected from mammalian cells.

24. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or the bispecific antibody according to any one of claims 9 to 20, and one or more pharmaceutically acceptable carriers, diluents or excipients.

25. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or the bispecific antibody according to any one of claims 9 to 20, or the pharmaceutical composition according to claim 24, in the preparation of a medicament for treating or preventing a disease or condition mediated by GPRC5D; preferably, the disease or condition mediated by GPRC5D is cancer or an autoimmune disease; more preferably, the cancer is selected from lymphoma, leukemia or myeloma, and the autoimmune disease is selected from systemic lupus erythematosus and rheumatoid arthritis.

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