GPRC5D antibody and its applications

Abstract

The present invention relates to an antibody related to G protein-coupled receptor class C group 5 member D (GPRC5D) and its application. Specifically, the present invention discloses an antibody or an antigen-binding fragment thereof that specifically binds to GPRC5D, a nucleic acid encoding the antibody, an expression vector and an expression cell, a manufacturing method, a pharmaceutical composition, and their use in the manufacture of a pharmaceutical composition for treating a disease, for example, in the treatment of a tumor. This is of great significance for the development of GPRC5D antibody therapeutic agents and detection reagents.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to a Chinese patent application bearing patent application number 202111668463.1 and title "GPRC5D antibody and its application" filed with the State Intellectual Property Office of the People's Republic of China on December 31, 2021. The above-mentioned prior application is incorporated by reference in its entirety into this application.

[0002] <Technical field> The present application relates to the biomedicine field, specifically to GPRC5D antibodies and their applications. [Background technology]

[0003] Multiple myeloma (MM) is a plasma cell malignant tumor characterized by unlimited proliferation of plasma cells in the bone marrow like tumor cells, accompanied by the secretion of monoclonal immunoglobulin, which causes a series of clinical symptoms such as multiple osteolytic lesions, hypercalcemia, anemia, kidney damage, and repeated infections. Hematopoietic stem cell transplantation and combination therapy with immunomodulators and protease inhibitors have significantly improved the prognosis. In recent years, CAR T therapy has made great progress in the treatment of multiple myeloma, and in particular, CAR T targeting B cell maturation antigen (BCMA) has shown positive clinical efficacy in the treatment of multiple myeloma. However, for multiple myeloma patients with BCMA-negative or low BCMA expression, relapse still occurs after receiving CAR-T cell therapy targeting BCMA, so the treatment of multiple myeloma needs to find other novel targets with more potential.

[0004] GPRC5D is a G protein-coupled receptor class C group 5 member D, which belongs to orphan receptors and is a seven-transmembrane protein. GPRC5D is a new multiple myeloma target following BCMA. Tissue expression profiling studies have found that GPRC5D is specifically highly expressed in plasma cells of multiple myeloma, but is low expressed in normal tissues and is restricted to the immune-tolerant hair follicle region. And the expression of GPRC5D and BCMA does not overlap. In tumor recurrence models where BCMA has been lost, GPRC5D CAR-T still has therapeutic efficacy and overcomes tumor evasion. In theory, dual targeting of GPRC5D and BCMA can target more people and also result in better clinical responses. Therefore, there is a huge potential clinical need for the development of GPRC5D-specific antibodies and related biological products. Summary of the Invention [Means for solving the problem]

[0005] The present application discloses antibodies or antigen-binding fragments thereof that specifically bind to G protein-coupled receptor class C group 5 member D (GPRC5D), multispecific antigen-binding molecules, nucleic acid fragments, vectors, host cells, immune effector cells, methods of manufacture, pharmaceutical compositions, pharmaceutical uses and methods of treating tumors or cancer (e.g., B-cell lymphoma or multiple myeloma).

[0006] In one aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: (1) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 has any one of the LCDR1 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to said sequence, the LCDR2 has any one of the LCDR2 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to said sequence, and the LCDR3 has any one of the LCDR3 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to said sequence,

[0007] [Table 1] and, (2) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 has any one of the HCDR1 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to the sequence; the HCDR2 has any one of the HCDR2 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to the sequence; and the HCDR3 has any one of the HCDR3 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to the sequence.

[0008] [Table 2]

[0009] In another aspect, the present application provides a multispecific antigen-binding molecule, wherein the multispecific antigen-binding molecule comprises the above-mentioned antibody or antigen-binding fragment thereof and another antigen-binding molecule that binds to an antigen other than GPRC5D or binds to a GPRC5D epitope different from the above-mentioned antibody or antigen-binding fragment thereof.

[0010] In another aspect, the present application provides a chimeric antigen receptor (CAR), wherein said chimeric antigen receptor comprises at least a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane domain and an intracellular signaling domain, and said extracellular antigen binding domain comprises said GPRC5D antibody or antigen-binding fragment thereof, or said multispecific antigen binding molecule.

[0011] In another aspect, the present application provides an immune effector cell, wherein said immune effector cell expresses said chimeric antigen receptor or comprises a nucleic acid fragment encoding said chimeric antigen receptor.

[0012] In another aspect, the present application provides an isolated nucleic acid fragment, said nucleic acid fragment encoding said antibody or antigen-binding fragment thereof, said multispecific antigen-binding molecule, or said chimeric antigen receptor.

[0013] In another aspect, the present application provides a vector, wherein said vector comprises the nucleic acid fragment described above.

[0014] In another aspect, the present application provides a host cell, wherein said host cell comprises the aforementioned vector.

[0015] In another aspect, the present application provides a method of producing said antibody or antigen-binding fragment thereof, or said multispecific antigen-binding molecule, wherein said method comprises culturing said cell and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by said cell.

[0016] In another aspect, the present application provides a method of producing the immune effector cell, wherein the method comprises introducing a nucleic acid fragment encoding the CAR into an immune effector cell.

[0017] In another aspect, the present application provides a pharmaceutical composition, wherein said pharmaceutical composition comprises said antibody or antigen-binding fragment thereof, said multispecific antigen-binding molecule, said immune effector cell, said nucleic acid fragment, said vector, or a product produced according to said method.

[0018] In another aspect, the present application provides a method of treating a tumor or cancer, wherein the method comprises administering to a subject an effective amount of the above-described antibody or antigen-binding fragment thereof, the above-described multispecific antigen-binding molecule, the above-described immune effector cell, the above-described nucleic acid fragment, the above-described vector, or a product produced according to the above-described method, or the above-described pharmaceutical composition, wherein the tumor or cancer is a tumor or cancer that expresses GPRC5D.

[0019] In another aspect, the present application provides the use of said antibody or antigen-binding fragment thereof, said multispecific antigen-binding molecule, said immune effector cell, said nucleic acid fragment, said vector or a product produced according to said method or said pharmaceutical composition in the manufacture of a medicament for treating a tumor or cancer, wherein said tumor or cancer expresses GPRC5D.

[0020] In another aspect, the present application provides the above antibody or antigen-binding fragment thereof, the above multispecific antigen-binding molecule, the above immune effector cell, the above nucleic acid fragment, the above vector or a product produced according to the above method or the above pharmaceutical composition for treating a tumor or cancer, wherein the tumor or cancer is a tumor or cancer that expresses GPRC5D.

[0021] In another aspect, the present application provides a kit, wherein said kit comprises said antibody or antigen-binding fragment thereof, said multispecific antigen-binding molecule, said immune effector cell, said nucleic acid fragment, said vector, or a product produced according to said method, or said pharmaceutical composition.

[0022] In another aspect, the present application provides a method for detecting GPRC5D expression in a biological sample, the method comprising contacting the biological sample with the antibody or antigen-binding fragment thereof under conditions allowing the formation of a complex between the antibody or antigen-binding fragment thereof and GPRC5D.

[0023] In another aspect, the present application provides the use of the above-mentioned antibody or antigen-binding fragment thereof in the manufacture of a reagent for detecting GPRC5D.

[0024] BCMA negativity or low expression is a common cause of relapse or refractory multiple myeloma, and the present application focuses on another target GPRC5D for relapsed / refractory multiple myeloma, and provides an antibody or antigen-binding fragment thereof targeting human GPRC5D, which binds with high affinity to human or monkey GPRC5D, but does not or binds with low affinity to GPRC5A, GPRC5B or GPRC5C proteins, which has important implications for the further development of antibodies (monospecific or multispecific antibodies) against GPRC5D target, cell therapy products. [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 shows the expression level of endogenous cellular GPRC5D detected by FACS. [Figure 2A] FIG. 1 shows the results of FACS detection of CHOK1 stably transfected cell lines expressing human GPRC5A protein. [Figure 2B] FIG. 1 shows the results of FACS detection of CHOK1 stably transfected cell lines expressing human GPRC5B protein. [Figure 2C] FIG. 1 shows the results of FACS detection of CHOK1 stably transfected cell lines expressing human GPRC5C protein. [Figure 2D] FIG. 1 shows the results of FACS detection of a CHOK1 stably transfected cell line expressing human GPRC5D protein. [Diagram 3]FIG. 1 shows the results of FACS detection of a stably transfected HEK293T cell line expressing human GPRC5D protein. [Figure 4A] FIG. 1 shows the results of FACS detection of a CHOK1 stably transfected cell line expressing monkey GPRC5D protein. [Figure 4B] FIG. 1 shows the results of FACS detection of a stably transfected HEK293T cell line expressing monkey GPRC5D protein. [Figure 5A] FIG. 1 shows the binding reaction between a control antibody and NCI-H929 tumor cells detected by FACS. [Figure 5B] FIG. 1 shows the binding reaction between a control antibody and 293T-human-GPRC5D recombinant cells detected by FACS. [Figure 5C] FIG. 1 shows the binding reaction between a control antibody and 293T-monkey-GPRC5D recombinant cells detected by FACS. [Figure 6A] FIG. 1 shows detection of the binding reaction between a chimeric antibody and CHOK1-hGPRC5D by cell-based ELISA. [Figure 6B] FIG. 1 shows detection of the binding reaction between a chimeric antibody and CHOK1-hGPRC5D by cell-based ELISA. [Figure 6C] FIG. 1 shows detection of the binding reaction between a chimeric antibody and CHOK1-hGPRC5D by cell-based ELISA. [Figure 7A] FIG. 1 shows detection of the binding reaction between a chimeric antibody and CHOK1-cynoGPRC5D by cell-based ELISA. [Figure 7B] FIG. 1 shows detection of the binding reaction between a chimeric antibody and CHOK1-cynoGPRC5D by cell-based ELISA. [Figure 7C] FIG. 1 shows detection of the binding reaction between a chimeric antibody and CHOK1-cynoGPRC5D by cell-based ELISA. [Figure 8A]FIG. 1 shows detection of binding reaction between chimeric antibody and CHOK1 by cell-based ELISA. [Figure 8B] FIG. 1 shows detection of binding reaction between chimeric antibody and CHOK1 by cell-based ELISA. [Figure 8C] FIG. 1 shows detection of binding reaction between chimeric antibody and CHOK1 by cell-based ELISA. [Figure 9A] FIG. 1 shows the binding reaction between a chimeric antibody and NCI-H929 (high expression) cells detected by FACS. [Figure 9B] FIG. 1 shows the binding reaction between a chimeric antibody and NCI-H929 (high expression) cells detected by FACS. [Figure 9C] FIG. 1 shows the binding reaction between a chimeric antibody and NCI-H929 (high expression) cells detected by FACS. [Figure 10A] FIG. 1 shows the binding reaction between a chimeric antibody and MolP-8 (moderately expressing) cells detected by FACS. [Figure 10B] FIG. 1 shows the binding reaction between a chimeric antibody and MolP-8 (moderately expressing) cells detected by FACS. [Figure 10C] FIG. 1 shows the binding reaction between a chimeric antibody and MolP-8 (moderately expressing) cells detected by FACS. [Figure 11A] FIG. 1 shows the binding reaction between a chimeric antibody and RPMI-8226 (low expression) cells detected by FACS. [Figure 11B] FIG. 1 shows the binding reaction between a chimeric antibody and RPMI-8226 (low expression) cells detected by FACS. [Figure 11C] FIG. 1 shows the binding reaction between a chimeric antibody and RPMI-8226 (low expression) cells detected by FACS. [Figure 12A] FIG. 1 shows the detection by ELISA of the competitive binding reaction between the chimeric antibody and the positive control antibody 5F11. [Figure 12B] FIG. 1 shows the detection by ELISA of the competitive binding reaction between the chimeric antibody and the positive control antibody 5F11. [Figure 12C] FIG. 1 shows the detection by ELISA of the competitive binding reaction between the chimeric antibody and the positive control antibody 5F11. [Figure 13A] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-hGPRC5D by cell-based ELISA. [Figure 13B] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-hGPRC5D by cell-based ELISA. [Figure 13C] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-hGPRC5D by cell-based ELISA. [Figure 13D] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-hGPRC5D by cell-based ELISA. [Figure 13E] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-hGPRC5D by cell-based ELISA. [Figure 13F] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-hGPRC5D by cell-based ELISA. [Figure 13G] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-hGPRC5D by cell-based ELISA. [Figure 13H] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-hGPRC5D by cell-based ELISA. [Figure 13I] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-hGPRC5D by cell-based ELISA. [Figure 13J] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-hGPRC5D by cell-based ELISA. [Figure 14A] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-cynoGPRC5D by cell-based ELISA. [Figure 14B]FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-cynoGPRC5D by cell-based ELISA. [Figure 14C] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-cynoGPRC5D by cell-based ELISA. [Figure 14D] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-cynoGPRC5D by cell-based ELISA. [Figure 14E] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-cynoGPRC5D by cell-based ELISA. [Figure 14F] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-cynoGPRC5D by cell-based ELISA. [Figure 14G] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-cynoGPRC5D by cell-based ELISA. [Figure 14H] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-cynoGPRC5D by cell-based ELISA. [Figure 14I] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-cynoGPRC5D by cell-based ELISA. [Figure 14J] FIG. 1 shows detection of the binding reaction between humanized antibodies and CHOK1-cynoGPRC5D by cell-based ELISA. [Figure 15A] FIG. 1 shows the binding reaction between GPRC5D humanized antibody and NCI-H929 detected by FACS. [Figure 15B] FIG. 1 shows the binding reaction between GPRC5D humanized antibody and NCI-H929 detected by FACS. [Figure 15C] FIG. 1 shows the binding reaction between GPRC5D humanized antibody and NCI-H929 detected by FACS. [Figure 15D]FIG. 1 shows the binding reaction between GPRC5D humanized antibody and NCI-H929 detected by FACS. [Figure 15E] FIG. 1 shows the binding reaction between GPRC5D humanized antibody and NCI-H929 detected by FACS. [Figure 15F] FIG. 1 shows the binding reaction between GPRC5D humanized antibody and NCI-H929 detected by FACS. [Figure 15G] FIG. 1 shows the binding reaction between GPRC5D humanized antibody and NCI-H929 detected by FACS. [Figure 15H] FIG. 1 shows the binding reaction between GPRC5D humanized antibody and NCI-H929 detected by FACS. [Figure 15I] FIG. 1 shows the binding reaction between GPRC5D humanized antibody and NCI-H929 detected by FACS. [Figure 15J] FIG. 1 shows the binding reaction between GPRC5D humanized antibody and NCI-H929 detected by FACS.

[0026] <Detailed Description of the Invention> Definitions and Explanations of Terms Unless otherwise defined in this application, scientific and technical terms associated with this application have the meanings that are understood by those skilled in the art.

[0027] Also, unless specifically stated otherwise herein, singular terms herein shall include the plural and plural terms shall include the singular. More specifically, the singular forms "one," "an," and "such," as used in this specification and the appended claims, include plural referents unless clearly stated otherwise.

[0028] The terms "comprise", "include", and "have" are used interchangeably herein and are intended to indicate the inclusiveness of a scheme, meaning that there may be other elements in the scheme than the recited elements. At the same time, it should be understood that the statements "comprise", "include", and "have" as used herein also provide a scheme "consisting of".

[0029] The term "and / or" as used herein includes the meaning of "and", "or" and "all or any other combination of the elements linked by the term to which it belongs".

[0030] The term GPRC5D herein refers to G protein-coupled receptor class C group 5 member D, which belongs to orphan receptors and is a seven-transmembrane protein. GPRC5D is highly expressed on the surface of primary multiple myeloma cells, but its expression in normal tissues is limited to the hair follicle region. Research has shown that 65% of multiple myeloma patients have GPRC5D with an expression threshold of more than 50%, and this characteristic makes GPRC5D a potential target for treating MM.

[0031] As used herein, the term "specific binding" refers to an antigen-binding molecule (e.g., an antibody) that typically specifically binds to an antigen and a substantially identical antigen with high affinity, but does not bind to unrelated antigens with high affinity. The equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis assays.

[0032] As used herein, the term "antigen-binding molecule" is used in the broadest sense and refers to a molecule that specifically binds to an antigen. Exemplary antigen-binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refers to organic compounds or binding domains that can specifically bind to an antigen but are unrelated to antibody structure, and exemplary antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

[0033] As used herein, the term "antibody" is used in the broadest sense to refer to a polypeptide or combination of polypeptides that contains sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region and is thereby capable of specifically binding to an antigen. As used herein, "antibody" encompasses a variety of forms and a variety of structures, so long as it exhibits the desired antigen-binding activity. As used herein, "antibody" includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which, for example, contain mutations introduced to stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds, including, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds may further include non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used for CDR grafting, including, but not limited to, tenascin, fibronectin, peptide aptamers, and the like.

[0034] The term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together via disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions may be further divided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each VH and VL is composed 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 variable regions of the heavy and light chains contain binding domains that can interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The immunoglobulin heavy chain constant regions differ in amino acid composition and sequence, and therefore in their antigenicity. This allows the "immunoglobulins" herein to be divided into five classes, or may be referred to as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. The same class Ig can be divided into various subclasses depending on the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds, for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are divided into κ chains and λ chains depending on the constant region. Each of the five classes of Ig can have either κ chains or λ chains.

[0035] As used herein, "antibody" further includes antibodies that do not contain light chains, for example heavy-chain antibodies (HCAbs) produced by camelids such as Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and alpaca (Vicugna pacos), and Ig new antigen receptors (IgNARs) found in chondrichthyes such as sharks.

[0036] As used herein, the term "antibody" may be derived from any animal, including, but not limited to, humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, guanacos, alpacas, sheep, rabbits, mice, rats or chondrichthyes (e.g., sharks).

[0037] As used herein, the term "heavy chain antibody" refers to an antibody lacking the light chain of a conventional antibody. The term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen-binding domain in the absence of a CH1 domain, and CH2 and CH3 constant domains.

[0038] As used herein, the term "nanobody" refers to the existence of naturally occurring heavy chain antibodies lacking light chains in the bodies of animals such as camels, and cloning of the variable regions of these antibodies can result in single domain antibodies consisting only of the heavy chain variable region, which are also called VHH (variable domain of heavy chain of heavy chain antibody) and are the smallest functional antigen-binding fragments.

[0039] As used herein, the terms "VHH domain", "nanobody" and "single domain antibody (sdAb)" have the same meaning and are used interchangeably, and refer to cloning the variable region of a heavy chain antibody to construct a single domain antibody consisting of only one heavy chain variable region, which is the minimum antigen-binding fragment with complete functionality. Typically, a heavy chain antibody that is naturally missing the light chain and heavy chain constant region 1 (CH1) is obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0040] For further explanation of "heavy chain antibodies", "single domain antibodies", "VHH domains" and "nanobiodies" see: Hamers-Casterman et al., Nature. 1993;363;446-8, the review article by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001), and the following patent applications which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103, WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193, WO97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527, WO 03 / 050531, WO 01 / 90190, WO03 / 025020, and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787, WO No. 06 / 122825 and other prior art cited in these applications.

[0041] As used herein, the term "multispecific" refers to the ability of an antibody or antigen-binding fragment thereof to bind at least two different epitopes, for example, on different antigens or on the same antigen. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes to which an antibody can bind. For example, a typical monospecific IgG-type antibody has two identical antigen-binding sites (complementary sites), and therefore can only bind to the same epitope (rather than binding to different epitopes). In contrast, a multispecific antibody has at least two different types of complementary sites / binding sites, and therefore can bind to at least two different epitopes. As explained herein, "complementarity determining region" refers to the antigen-binding site of an antibody. Also, a single "specificity" can refer to one, two, three, or more than three identical complementary determining regions in a single antibody (the actual number of complementary determining regions / binding sites in one single antibody molecule is referred to as "valency"). For example, a single natural IgG antibody is monospecific and bivalent, since it has two identical complementary sites. Accordingly, a multispecific antibody comprises at least two (different) complementarity determining regions / binding sites. The term "multispecific antibody" therefore refers to an antibody having more than one complementary site and thus the ability to bind to two or more different epitopes. The term "multispecific antibody" particularly includes bispecific antibodies as defined above, but also generally includes proteins, e.g. antibodies, scaffolds, i.e. antibodies having three or more complementary sites / binding sites.

[0042] As used herein, the term "valency" refers to the presence of a given number of binding sites on an antibody / antigen-binding molecule. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of one binding site, two binding sites, four binding sites, and six binding sites, respectively, on an antibody / antigen-binding molecule.

[0043] As used herein, "full length antibody," "intact antibody," and "whole antibody" are used interchangeably and refer to having a structure substantially similar to that of a native antibody.

[0044] As used herein, the terms "antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of a whole antibody, but only include local or regional variants of a whole antibody, which have the ability to bind to an antigen. Exemplarily, as used herein, "antigen-binding fragment" or "antibody fragment" includes, but is not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabody, and single domain antibody.

[0045] As used herein, the term "chimeric antibody" refers to an antibody that has immunoglobulin variable sequences derived from one source organism (e.g., rat, mouse, rabbit, or alpaca) and immunoglobulin constant regions derived from a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, Bio Techniques 4:214-221; Gillies et al., 1985 J Immunol Methods 125:191-202, all of which are incorporated herein by reference.

[0046] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to improve homology with the sequence of a human antibody. Typically, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FR and / or constant region) are derived from a human immunoglobulin (acceptor antibody). A humanized antibody typically retains or partially retains the desired properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, the ability to improve immune cell activity or enhance immune responses, etc.

[0047] As used herein, a "fully human antibody" refers to an antibody having variable regions in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. As used herein, a fully human antibody may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific induction or in vivo somatic mutation). However, as used herein, a "fully human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

[0048] As used herein, the term "variable region" refers to a region in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen, and "heavy chain variable region" is used synonymously with "VH" and "HCVR", and "light chain variable region" is used synonymously with "VL" and "LCVR". The heavy and light chain variable domains of a natural antibody generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6 th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain is sufficient to confer antigen-binding specificity.

[0049] As used herein, "complementarity determining region" is used synonymously with "CDR" and generally refers to the hypervariable regions (HVRs) found in both light and heavy chain variable domains. The more conserved portions of the variable domains are called framework regions (FRs). As understood in the art, the amino acid positions that represent the hypervariable regions of an antibody can vary depending on the context and the various definitions known in the art. Some positions within a variable domain may be considered to be within the hypervariable region under one set of standards (e.g., IMGT or KABAT), while being outside the hypervariable region under a different set of standards (e.g., KABAT or IMGT), and thus may be considered heterozygous hypervariable positions. One or more of these positions may also be found in an extended hypervariable region. The present application includes antibodies that include modifications at these heterozygous hypervariable positions. Heavy chain variable region CDRs may be abbreviated as HCDRs and light chain variable regions may be abbreviated as LCDRs. Each of the variable domains of naturally occurring heavy and light chains contains four framework regions that mainly adopt a sheet structure, which are connected through three CDRs (CDR1, CDR2 and CDR3) that form loops connecting, and in some cases forming part of, the sheet structure. The CDRs in each chain are closely held together through the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and contribute to the formation of the antigen-binding site of antibodies with the CDRs from other antibody chains (see Kabat et al., Sequences of Protein sofImmunological Interest, National Institute of Health, Bethesda, Md. 1987, which is incorporated herein by reference).

[0050] Further descriptions of CDRs can be found in Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol., 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc See MP et al., Dev. Comp. Immunol., 27:55-77 (2003), and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001). As used herein, "CDRs" may be labeled and defined in any manner known in the art, including, but not limited to, the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, and tool sites that may be used include, but are not limited to, the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). As used herein, CDRs include overlapping and subsets of amino acid residues in different defined ways.

[0051] As used herein, the term "Kabat numbering system" generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0052] As used herein, the term "Chothia numbering system" generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical convention for identifying boundaries of CDR regions based on the location of loop regions in the structure (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0053] As used herein, the term "IMGT numbering system" generally refers to a numbering system based on the international ImMunoGeneTics information system (IMGT) proposed by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0054] As used herein, "heavy chain constant region" refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in binding an antibody to an antigen but exhibits effector functions such as interaction with an Fc receptor, and has an amino acid sequence more conservative than that of the variable domain of an antibody. The "heavy chain constant region" may be selected from a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof. The "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment", the former having a structure substantially similar to that of the constant region of a natural antibody, while the latter includes only a "part of the full-length heavy chain constant region". Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain, and if the antibody is an IgE, it further includes a CH4 domain, and if the antibody is a heavy chain antibody, it does not include a CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" may be selected from an Fc or CH3 domain.

[0055] As used herein, the term "light chain constant region" refers to the carboxyl-terminal portion of an antibody light chain, which is not directly involved in binding the antibody to an antigen, said light chain constant region may be selected from a constant κ domain or a constant λ domain.

[0056] As used herein, the term "Fc region" is intended to define a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Exemplarily, a human IgG heavy chain Fc region can extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by a host cell can undergo post-translational cleavage to remove one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, upon expression of a particular nucleic acid molecule encoding a full-length heavy chain, an antibody produced by a host cell may comprise a full-length heavy chain or a truncated variant of the full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (Lys447), may or may not be present. Typically, an IgG Fc region comprises an IgG CH2 and an IgG CH3 domain, and may optionally further comprise a complete or partial hinge region, but does not comprise a CH1 domain. The "CH2 domain" of a human IgG Fc region usually extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises residues at the C-terminus of the CH2 domain in the Fc region (i.e., from an amino acid residue at about position 341 to an amino acid residue at about position 447 of IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "knob" introduced in one chain and a corresponding "cavity" or "hole" introduced in another chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference).As described herein, such mutant CH3 domains may be used to promote heterodimerization of two different antibody heavy chains.

[0057] Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0058] As used herein, the term "Fc variant" refers to the presence of one or more amino acid substitutions, insertions or deletions at appropriate sites in Fc, resulting in changes in Fc structure or function. "Interaction between Fc variants" refers to the space-filling effect, electrostatic induction, hydrogen bonding effect, and hydrophobic interactions that may be formed between Fc variants designed by mutation. The interaction between Fc variants helps to form a stable heterodimeric protein. The preferred mutation is a "knob-into-hole" type of mutation design.

[0059] Mutational design techniques for Fc variants have been widely applied in the art to generate bispecific antibodies or heterodimeric Fc fusion protein forms. Representative examples include the "Knob-into-Hole" form proposed by Cater et al. (Protein Engineering vol.9 no.7 pp .617-621, 1996); the Fc-containing heterodimer form formed by electrostatic steering by Amgen engineers (US 20100286374 A1); the heterodimer form formed by IgG / Ig chain exchange (SEEDbodies) proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp.1-8, 2010); the bispecific molecule formed by Genmab's DuoBody (Science, 2007.317(5844)) platform technology; and the heterodimer protein form formed by Xencor engineers by integrating structural calculations and Fc amino acid mutations to integrate different action modes (mAbs 3:6,546-557; November / December 2011), the heterodimeric protein form obtained by the charge network-based Fc remodeling method (CN201110459100.7) by Suzhou Kangning Jierui Co., Ltd., and other genetic engineering methods to achieve the formation of heterodimeric functional proteins based on Fc amino acid changes or function remodeling means. The knob / hole structure in the Fc variant fragment described in this application refers to the fact that the two Fc fragments are mutated respectively and can be bound in the form of "knob-into-hole" after mutation. It is preferable to carry out site mutation remodeling in the Fc region using the "knob-into-hole" model of Cater et al., so that the obtained first Fc variant and second Fc variant can be bound together in the form of "knob-into-hole" to form a heterodimer. It is within the knowledge of a person skilled in the art to select a specific immunoglobulin Fc region from a specific immunoglobulin class and subclass. Preferably, it is the Fc region of human antibody IgG1, IgG2, IgG3, or IgG4, and more preferably, it is the Fc region of human antibody IgG1.A first Fc variant or a second Fc variant is randomly selected, one of which is mutated with a knob and the other with a hole.

[0060] As used herein, the term "conservative amino acids" generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation and rigidity). Illustratively, the following groups of amino acids belong to the conserved amino acid residues of each other, and substitutions of amino acid residues within the groups belong to the conservative amino acid substitutions: 1) Alanine (A), Serine (S), Threonine (T), 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), Glutamine (Q), 4) arginine (R), lysine (K), histidine (H); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0061] As used herein, "identity" is calculated according to the following method: To determine the percentage of "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position. Taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap, the percentage of identity between the two sequences varies according to the change in the same position common to the sequences.

[0062] The sequence comparison and identity percentage calculation between two sequences can be realized by using a mathematical algorithm. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm incorporated in the GAP program of the GCG software package (available at www.gcg.com) is used to determine the identity percentage between two amino acid sequences, using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6. Furthermore, for example, the GAP program of the GCG software package (available at www.gcg.com) is used to determine the identity percentage between two nucleotide sequences, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and that should be used unless otherwise specified) is a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Additionally, the percent identity between two amino acid or nucleotide sequences can be determined utilizing the E. Meyers and W. Miller algorithm, ((1989) CABIOS, 4:11-17), implemented in the ALIGN program (version 2.0), using a PAM120 weighting covariance table, a gap length penalty of 12, and a gap penalty of 4.

[0063] Additionally or alternatively, the nucleic acid and protein sequences described in the present application may be further used as "query sequences" to perform searches against common databases, for example, to identify other family member sequences or related sequences. For example, such searches can be performed with the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. To obtain nucleotide sequences homologous to the nucleic acid molecules of the present application, BLAST nucleotide searches may be performed using the NBLAST program, score=100, wordlength=12. To obtain amino acid sequences homologous to the protein molecules of the present application, BLAST protein searches may be performed using the XBLAST program, score=50, wordlength=3. Gapped BLAST may be used to obtain gapped alignments for comparison purposes, as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0064] As used herein, the term "chimeric antigen receptor (CAR)" refers to an artificial cell surface receptor engineered to be expressed in immune effector cells and specifically bind to an antigen, which includes at least (1) an extracellular antigen-binding domain, e.g., an antibody heavy and / or light chain variable region, (2) a transmembrane domain that anchors the CAR to enter the immune effector cell, and (3) an intracellular signaling domain. CARs can utilize the extracellular antigen-binding domain to redirect T cells and other immune effector cells to selected targets, e.g., cancer cells, in a non-MHC-restricted manner.

[0065] As used herein, the term "nucleic acid" includes any compound and / or substance that is a polymer containing nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or riboso) and a phosphate group. A nucleic acid molecule is usually described by the sequence of bases, whereby said bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is usually represented 5' to 3'. As used herein, the term nucleic acid molecule includes, for example, deoxyribose nucleic acid (DNA), including complementary DNA (cDNA) and genomic DNA, ribosomal nucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers containing mixtures of two or more of these molecules. Nucleic acid molecules may be linear or circular. The term nucleic acid molecule also includes both sense and antisense strands, and single-stranded and double-stranded forms. And the nucleic acid molecules described herein may comprise naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules further encompass DNA and RNA molecules, suitable as vectors to directly express the antibodies of the present application in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, whereby the mRNA can be injected into a subject to produce the antibody in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP2101823B1).

[0066] As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell, which normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0067] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Some vectors are capable of directing the expression of nucleic acids that are operably linked to them. Such vectors are referred to herein as "expression vectors."

[0068] As used herein, "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and the progeny derived therefrom, without regard to the number of passages. The progeny may not be completely identical to the parent cell in terms of nucleic acid material and may contain mutations. As used herein, mutant progeny that have the same function or biological activity as screened or selected from the initially transformed cell are included.

[0069] As used herein, a "drug composition" refers to a formulation that is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain other ingredients that are unacceptably toxic to a subject to which the drug composition is administered.

[0070] As used herein, "pharmaceutical acceptable carrier" includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial and antifungal agents), isotonicity agents, absorption retardants, salts, preservatives, drug stabilizers, adhesives, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and the like, and combinations thereof, which are known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Edition. Mack Printing Company, 1990, pages 1289-1329). Except insofar as it is incompatible with the active ingredient, the use of any conventional carrier in the therapeutic or pharmaceutical compositions is contemplated.

[0071] As used herein, "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions, such as cancer and tumors, in the subject being treated. Beneficial or desirable clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete remission), whether detectable or undetectable. Subjects in need of treatment include those suffering from a disorder or disease, and those susceptible to a disorder or disease, or those in whom a disorder or disease is to be prevented. When terms such as slow down, alleviation, reduction, mitigation, remission are mentioned, the meaning also includes removal, disappearance, non-occurrence, and the like.

[0072] As used herein, the term "subject" refers to a living organism receiving treatment for a particular disease or disorder as described herein. Exemplary, a "subject" includes a mammal, such as a human, a primate (e.g., a monkey), or a primate mammal, receiving treatment for a disease or disorder.

[0073] As used herein, the term "effective amount" refers to a therapeutic dose that can effectively prevent or ameliorate disease disorders or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject. "Effective amount" also refers to a dose of a compound that is sufficient to ameliorate symptoms, for example, treat, cure, prevent, or ameliorate related medical disorders, or increase the rate at which these disorders are treated, cured, prevented, or ameliorated. When an active ingredient is administered alone to an individual, the therapeutically effective dose refers to the ingredient alone. When a combination is applied, the therapeutically effective dose refers to the combined dose of active ingredients that leads to a therapeutic effect, whether combined, administered sequentially, or administered simultaneously.

[0074] As used herein, the term "cancer" refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. This definition includes benign and malignant cancers. As used herein, "tumor" or "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.

[0075] As used herein, the term "EC50" refers to the half-maximal effective concentration, which includes the antibody concentration that induces a response halfway between the baseline and maximum after a particular exposure time. EC50 essentially represents 50% of the antibody concentration at which its maximum effect is observed, and can be measured by methods known in the art. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] In a first aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to G protein-coupled receptor class C group 5 member D (GPRC5D), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: (1) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 has any one of the LCDR1 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to said sequence, the LCDR2 has any one of the LCDR2 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to said sequence, and the LCDR3 has any one of the LCDR3 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to said sequence,

[0077] [Table 3] and, (2) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 has any one of the HCDR1 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to the sequence; the HCDR2 has any one of the HCDR2 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to the sequence; and the HCDR3 has any one of the HCDR3 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to the sequence.

[0078] [Table 4]

[0079] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises the sequence of the six CDRs in the combination of light chain variable region and heavy chain variable region of: L1+H1, L2+H2, L3+H3, L4+H4, L5+H5, L6+H6, L7+H7, L8+H8, L9+H9, L10+H10, L11+H11, L12+H12, L13+H13, L14+H14, L15+H15, L16+H16, L3+H17, L3+H18, L5+H19, L5+H20, L17+H13, L18+H13, L19+H13, L20+H13 or L21+H13, or the sequence of the six CDRs having one, two, three or more amino acid insertions, deletions and / or substitutions compared to the sequences of the six CDRs.

[0080] In one specific embodiment, the present application provides an antibody or antigen-binding fragment thereof, wherein: (1) the light chain variable region sequence comprises any one of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 118-120, 130-131, 144-145, 160-163, 172-175, 184-187, 196-197, 206-208, 218-221, and 231-233, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto; and, (2) The heavy chain variable region sequence includes any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 121 to 125, 132 to 137, 146 to 152, 164 to 166, 176 to 178, 188 to 190, 198 to 201, 209 to 212, 222, and 234 to 238, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to the above sequence.

[0081] In a preferred embodiment, the antibody or antigen-binding fragment thereof has the light chain variable region and heavy chain variable region shown below: (1) the light chain variable region and the heavy chain variable region comprise the sequences set forth in SEQ ID NO:14 and SEQ ID NO:13, respectively; (2) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO:16 and SEQ ID NO:15, respectively; (3) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO:18 and SEQ ID NO:17, respectively; (4) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO:20 and SEQ ID NO:19, respectively; (5) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO:22 and SEQ ID NO:21, respectively; (6) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO:24 and SEQ ID NO:23, respectively; (7) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 25, respectively; (8) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 28 and SEQ ID NO: 27, respectively; (9) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 118 to 120, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 121 to 125; (10) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 130 to 131, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 132 to 137; (11) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 144 to 145, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 146 to 152; (12) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 160 to 163, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 164 to 166; (13) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 172 to 175, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 176 to 178; (14) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 184 to 187, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 188 to 190; (15) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 196 to 197, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 198 to 201; (16) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 206 to 208, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 209 to 212; (17) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 218 to 221, and the heavy chain variable region comprises a sequence shown in SEQ ID NO: 222; (18) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 231 to 233, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 234 to 238; or (19) the light chain variable region comprises a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with any one of the light chain variable regions shown in (1) to (18) above, and the heavy chain variable region comprises a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with any one of the heavy chain variable regions shown in (1) to (18) above.

[0082] In a specific embodiment, the antibody or antigen-binding fragment thereof is chimeric, humanized or fully human.

[0083] In a specific embodiment, the antibody or antigen-binding fragment thereof is capable of binding to human or monkey GPRC5D.

[0084] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises any one of the constant region sequences of human or mouse antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, and preferably comprises the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4, or comprises a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4, and further, the antibody or antigen-binding fragment thereof further comprises a therapeutic agent or a tracer. Preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic agent or an immunomodulatory agent, the tracer is selected from a radiocontrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer, and more preferably, the cytotoxic agent is selected from alkaloids, methotrexate, anthracycline antibiotics (doxorubicin), taxanes, pyrrolobenzodiazepine (PBD) or a toxin compound.

[0085] In one specific embodiment, said antigen-binding fragment is selected from one or more of a F(ab')2, a Fab', a Fab, an Fv, a scFv, a nanobody or an affibody.

[0086] In a second aspect, the present application further provides a multispecific antigen-binding molecule, comprising the above-mentioned antibody or antigen-binding fragment thereof and another antigen-binding molecule that binds to an antigen other than GPRC5D or binds to a GPRC5D epitope different from the above-mentioned antibody or antigen-binding fragment thereof.

[0087] Preferably, the other antigen-binding molecule is an antibody or an antigen-binding fragment thereof.

[0088] Preferably, said multispecific antigen-binding molecule may be bispecific, trispecific or tetraspecific.

[0089] Preferably, said multispecific antigen-binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent.

[0090] In a third aspect, the present application further provides a chimeric antigen receptor (CAR), said chimeric antigen receptor comprising at least a signal peptide, an extracellular antigen-binding domain, a hinge region, a transmembrane domain and an intracellular signaling domain, said extracellular antigen-binding domain comprising the GPRC5D antibody or antigen-binding fragment thereof described in any one of the preceding claims, or said multispecific antigen-binding molecule.

[0091] In a fourth aspect, the present application further provides an immune effector cell, said immune effector cell expressing said chimeric antigen receptor or comprising a nucleic acid fragment encoding said chimeric antigen receptor, preferably said immune effector cell is selected from a T cell, a natural killer cell (NK cell), a natural killer T cell (NKT cell), a double negative T cell (DNT cell), a monocyte, a macrophage, a dendritic cell or a mast cell, said T cell is preferably selected from a cytotoxic T cell (CTL), a regulatory T cell or a helper T cell, preferably said immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell.

[0092] In a fifth aspect, the present application further provides an isolated nucleic acid fragment, wherein said nucleic acid fragment encodes the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, or chimeric antigen receptor.

[0093] In a sixth aspect, the present application further provides a vector, said vector comprising the nucleic acid fragment as described above.

[0094] In a seventh aspect, the present application further provides a host cell, said host cell comprising the above-mentioned vector, preferably said cell is a prokaryotic or eukaryotic cell, such as a bacterial (E. coli), fungal (yeast), insect cell or mammalian cell (CHO cell line or 293T cell line).

[0095] In an eighth aspect, the present application further provides a method of producing the above-mentioned antibody or antigen-binding fragment thereof, or multispecific antigen-binding molecule, the method comprising culturing the above-mentioned cell and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by the cell.

[0096] In a ninth aspect, the present application further provides a method of producing said immune effector cell, said method comprising introducing a nucleic acid fragment encoding said CAR into said immune effector cell, and optionally, said method further comprises priming said immune effector cell to express said CAR.

[0097] In a tenth aspect, the present application further provides a pharmaceutical composition, the pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, nucleic acid fragment, vector, or product produced according to the above-mentioned method, optionally, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier, diluent or adjuvant, and optionally, the pharmaceutical composition further comprises an additional anti-tumor agent.

[0098] In some embodiments, the pharma- ceutically acceptable carrier is a carrier that does not reduce the viability and function of immune cells and does not affect the specific binding between an antibody or an antigen-binding fragment thereof and an antigen, and includes, but is not limited to, cell culture medium, buffer, saline, balanced salt solution, etc. Examples of buffers include isotonic phosphate, acetate, citrate, borate, carbonate, etc. In a specific embodiment, the pharma-ceutically acceptable carrier is a phosphate buffer containing 1% serum.

[0099] In an eleventh aspect, the present application further provides a method of treating a tumor or cancer, the method comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, nucleic acid fragment, vector, product produced according to the aforementioned method, or pharmaceutical composition.

[0100] In a preferred embodiment, the tumor or cancer is a tumor or cancer that expresses GPRC5D, preferably a B-cell lymphoma, more preferably multiple myeloma (MM).

[0101] In a twelfth aspect, the present application further provides the use of said antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, nucleic acid fragment, vector, product produced according to said method or pharmaceutical composition in the manufacture of a medicament for treating tumor or cancer, In a preferred embodiment, the tumor or cancer is a tumor or cancer that expresses GPRC5D, preferably a B-cell lymphoma, more preferably multiple myeloma (MM).

[0102] In a thirteenth aspect, the present application further provides a kit, said kit comprising the above-mentioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, nucleic acid fragment, vector, product produced according to the above-mentioned method, or pharmaceutical composition.

[0103] In a fourteenth aspect, the present application further provides a method for detecting GPRC5D expression in a biological sample, the method comprising contacting the biological sample with the antibody or its antigen-binding fragment under conditions allowing the formation of a complex between the antibody or its antigen-binding fragment and GPRC5D, and preferably the method further comprises detecting the formation of the complex and indicating the presence or expression level of GPRC5D in the sample.

[0104] In a fifteenth aspect, the present application further provides a use of the aforementioned antibody or antigen-binding fragment thereof in the manufacture of a reagent for detecting GPRC5D.

[0105] Working Example In the following, the present application will be further described with reference to specific examples, and the advantages and features of the present application will be apparent from the description. If no specific conditions are specified in the examples, they will be carried out according to conventional conditions or conditions suggested by the manufacturer. If no manufacturer is specified, the reagents or equipment used are all general products that can be purchased commercially.

[0106] The examples of the present application are merely illustrative and do not limit the scope of the present application. Those skilled in the art will understand that modifications or replacements can be made to the details and forms of the technical solutions of the present application without departing from the spirit and scope of the present application, and all of these modifications and replacements are within the protection scope of the present application.

[0107] Example 1: Production of control antibodies, identification of endogenous cells, and production of overexpressing cell lines 1.1 Preparation of control antibodies The sequence of JNJ7564 was derived from International Patent Application No. WO 2018017786A2, and the sequence of 5F11 was derived from International Patent Application No. WO 2019154890A1. The cloned VH and VL sequences recognizing the human GPRC5D epitope were recombined into human IgG1 CH and CL expression vectors to obtain recombinant plasmids. The control antibody, CH, CL and Fc sequences are as shown in Table 1.

[0108] The plasmid and transfection reagent PEI (Polysciences, Catalog No.: 24765-1) were added to OPTI-MEM (Gibco, Catalog No.: 11058021) and mixed uniformly, then left to stand for 15 min, added to Expi293 cells (manufacturer: Thermofisher, Catalog No.: A14527), and cultured in a shaker at 5% CO2, 120 rpm, and 37°C. The day after transfection, OPM-293 ProFeed (Shanghai Aopumai, Catalog No.: F081918-001) and 6 g / L glucose (manufacturer: Sigma, Catalog No.: G7528) were added. On the 6th day after transfection, the cell supernatant was collected.

[0109] The samples were filtered through a 0.22 μm disposable filter, after which the system was sanitized with 0.5 M NaOH for 30 min, the chromatography column was sanitized with 0.1 M NaOH for 5 min, and the column was sterilized with AKTA Each flow path of the pure150 was filled with the corresponding buffer solution. A2 was filled with Mab affinity rinse solution (anhydrous disodium hydrogen phosphate 2.69 g / L (No. 20040618, China National Pharmaceutical Group Chemical Reagents Co., Ltd.), sodium dihydrogen phosphate dihydrate 0.17 g / L (No. 20040718, China National Pharmaceutical Group Chemical Reagents Co., Ltd.), sodium chloride 58.44 g / L (No. 10019318, China National Pharmaceutical Group Chemical Reagents Co., Ltd.)), B1 was filled with Mab affinity elution solution (citric acid monohydrate 3.92 g / L (No. 10007118, China National Pharmaceutical Group Chemical Reagents Co., Ltd.), trisodium citrate dihydrate 1.88 g / L (No. 10019418, China National Pharmaceutical Group Chemical Reagents Co., Ltd.)), and A1 was filled with Mab affinity equilibration solution (20× PBS Add ddH2O to the buffer to dilute it to 1x PBS (No. B548117-050, Shanghai Biotechnology Co., Ltd.) and fill the buffer pump with Mab affinity equilibration solution (20x PBS The buffer is diluted with ddH2O to 1x PBS (No. B548117-050, Shanghai Bioengineering Co., Ltd.), the sample is injected into S1, the sample is added at a flow rate of 5mL / min, and after the sample addition is completed, the buffer pump is used to perform wash1 (Mab affinity equilibration solution) until the UV falls below 10mAU, the inlet is switched to A2, and wash2 (Mab affinity rinse solution) is performed with high salt solution to remove impurities, the inlet is switched to A1, and the high salt solution in the flow path is replaced until the electrical conductivity falls to about 20ms / cm, the inlet is switched to B1, the target protein is eluted, the target peak is collected, 1M Tris (121.2g / L, pH8.0) is added to adjust the pH to neutral, and the concentration is measured using Nano drop 8000 and waited.

[0110] [Table 5]

[0111] 1.2 Identification of cell lines that endogenously express human GPRC5D protein Cells endogenously expressing human GPRC5D protein were expanded to logarithmic growth phase in a T-75 cell culture flask, centrifuged to discard the culture supernatant, and the cell pellet was washed twice with PBS. 20 nM of JNJ7564-hIgG1 and 5F11-hIgG1 antibodies were used as primary antibodies, and Alexa Fluor 5000 (F5000) was used as secondary antibodies. (R) 647 AffiniPure Goat Anti-Human IgG(H+L) (purchased from Jackson Immuno, catalog number: 109-605-088) was used for detection and analysis by FACS (FACS CantoTM, purchased from BD). The results are shown in Table 2 and Figure 1, and indicate that NCI-H929, MolP-8 and RPMI-8226 cells all endogenously express human GPRC5D protein, and that JNJ7564-hIgG1 and 5F11-hIgG1 control antibodies have binding activity with all cells.

[0112] [Table 6]

[0113] 1.3 Production of CHO-K1 recombinant cell lines expressing human GPRC5A, GPRC5B, GPRC5C, and GPRC5D proteins Nucleotide sequences encoding the amino acid sequences of human GPRC5A (Uniprot: Q8NFJ5-1, SEQ ID NO: 8), human GPRC5B (Uniprot: Q9NZH0-1, SEQ ID NO: 9), human GPRC5C (Uniprot: Q9NQ84, SEQ ID NO: 10) and human GPRC5D (Uniprot: Q9NZD1, SEQ ID NO: 11) were each cloned into a pLVX lentiviral vector, and viral particles were produced in HEK293T cells.CHOK1 cell line (purchased from the Chinese Academy of Sciences) was infected with lentivirus and selectively cultured for one week in Advanced DMEM / F12 Medium (Gibco, catalog number 12634028) containing 10% (v / v) fetal bovine serum (ExCell Bio, catalog number FND500) containing 10 μg / ml puromycin (Gibco, catalog number A1113803). The cells were then incubated with human GPRC5A Alexa Fluor 488-conjugated antibody (R&D systems, catalog number IC5239G-100UG), human GPRC5B Alexa Fluor 488-conjugated antibody (R&D systems, catalog number FAB10253G-100UG), and human GPRC5C Alexa Fluor 488-conjugated antibody (R&D systems, catalog number FAB10253G-100UG). CHOK1 transfected with human GPRC5D was stained with human anti-human GPRC5D antibody (JNJ7564, in-house) and goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109605088), and then analyzed using a flow cytometer FACS Aria III (BD A positive cell population with high expression levels was sorted into a 96-well plate and cultured at 37°C and 5% (v / v) CO2. After about two weeks, some cells were selected and amplified. A positive cell population with good growth, high fluorescence intensity, and high uniformity was selected and expanded, and then frozen and stored in liquid nitrogen. The expression level identification results are shown in Table 3 and Figures 2A to 2D. The results show that the CHOK1 stable transfected cell line expressing human GPRC5A, GPRC5B, GPRC5C, and GPRC5D, which was subjected to pressure screening using puromycin, has a relatively single positive peak and can be used to detect the binding activity of antibodies with human GPRC5A, GPRC5B, GPRC5C, and GPRC5D proteins by FACS.

[0114] [Table 7] Human GPRC5A full length amino acid sequence (Uniprot:Q8NFJ5-1, SEQ ID NO:8): [ka] Human GPRC5B full length amino acid sequence (Uniprot: Q9NZH0-1, SEQ ID NO: 9): [ka] Human GPRC5C full length amino acid sequence (Uniprot: Q9NQ84, SEQ ID NO: 10): [ka] Human GPRC5D full length amino acid sequence (Uniprot: Q9NZD1, SEQ ID NO: 11): [ka]

[0115] 1.4 Preparation of HEK293T recombinant cell line expressing human GPRC5D protein For specific methods, see Example 1.3. HEK293T cell line was infected with lentivirus packaging human GPRC5D plasmid vector, selectively cultured in DMEM Medium (Gibco, Catalog No. 10569044) containing 10% (v / v) fetal bovine serum (ExCell Bio, Catalog No. FND500) containing 2 μg / ml puromycin (Gibco, Catalog No. A1113803) for 2 weeks, and analyzed by flow cytometer FACS CantoII (BD) using human anti-human GPRC5D antibody (JNJ7564, in-house production) and goat anti-human IgG (H+L) antibody (Jackson, Catalog No. 109605088). The expression levels of the cell lines that showed good expression were identified using a FACS-based screening method (purchased from Biosciences) as shown in Table 4 and Figure 3. HEK293T-hGPRC5D, which underwent pressure screening using puromycin, had a relatively single positive peak, indicating that it can be used to detect the binding activity of the antibody to the human GPRC5D protein by FACS.

[0116] [Table 8]

[0117] 1.5 Preparation of CHO-K1 and HEK293T recombinant cell lines expressing monkey GPRC5D protein For the specific method of producing CHO-K1 recombinant cell line stably expressing monkey GPRC5D (cynoGPRC5D), please refer to Example 1.3. cynoGPRC5D amino acid sequence, NCBI: XP_005570249.1 (SEQ ID NO: 12). Using human anti-cynoGPRC5D antibody (JNJ7564, self-produced) and goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109605088), detection was performed using flow cytometer FACS CantoII (purchased from BD Biosciences). The expression level of the cell line with good expression was identified as shown in Table 5 and Figure 4A. The results showed that CHOK1-cynoGPRC5D, which was subjected to pressure screening using puromycin, had a relatively single positive peak, and could be used to detect the cross-reactivity of the antibody with monkey GPRC5D protein by FACS.

[0118] [Table 9]

[0119] For the specific method of constructing the HEK293T recombinant cell line stably expressing monkey GPRC5D (cynoGPRC5D), please refer to Example 1.4. The expression level of the cell line that shows good expression is identified as shown in Table 6 and Figure 4B. The results show that HEK293T-cynoGPRC5D that has been subjected to pressure screening using puromycin has a relatively single positive peak, and can be used to detect the cross-reactivity of antibody with monkey GPRC5D protein by FACS.

[0120] [Table 10] Monkey GPRC5D full length amino acid sequence (NCBI: XP_005570249.1, SEQ ID NO: 12): [ka]

[0121] 1.6 Binding experiments with recombinant cell lines and control antibodies The binding activity of the control antibody to cells expressing human GPRC5D and monkey GPRC5D was detected by FACS as shown in Figures 5A to 5C, and the IgG subtype control was human IgG1. JNJ7564-hIgG1 and 5F11-hIgG1 had good binding activity to NCI-H929 tumor cells expressing human GPRC5D protein, HEK293T-hGPRC5D and HEK293T-cynoGPRC5D recombinant cells, 5F11-hIgG1 had relatively high binding activity to NCI-H929 cells and HEK293T-hGPRC5D recombinant cells, and JNJ7564-hIgG1 had relatively high binding activity to HEK293T-cynoGPRC5D recombinant cells.

[0122] Example 2: Preparation of anti-human GPRC5D hybridoma monoclonal antibody 2.1 Animal immunity Anti-human GPRC5D monoclonal antibodies were produced by immunization of mice. 6-8 week-old female SJL mice (purchased from Shanghai Slice Laboratory Animal Co., Ltd.) were used in the experiment, and the mice were cultured under SPF conditions. After purchase, the mice were kept in a laboratory environment for 1 week, regulated with a 12 / 12-h light / dark cycle, with a temperature of 20-25°C and a humidity of 40-60%. The mice adapted to the environment were immunized according to the following regimen. In the first immunization, human GPRC5D-overexpressing cell line (HEK293T-hGPRC5D, in-house) was washed twice with PBS, then mixed with oligonucleotide CpG (ODN 1826, synthesized from Shanghai Biotechnology Co., Ltd.), and first each mouse was intraperitoneally injected with 50 μl of emulsified Titer max (purchased from sigma, Cat. T2684), waited 15 minutes, and then each mouse was injected with 5 × 10 6 For the first booster immunization, human GPRC5D overexpressing cell lines were washed twice with PBS, mixed with CpG, and injected into each mouse at 5 × 10 6The mice were intraperitoneally injected with 100 μl / cell. Subsequent boosts were alternated between the primary and first boosts, with monkey GPRC5D overexpressing cell lines (HEK293T-cynoGPRC5D, in-house) and human GPRC5D overexpressing cell lines (HEK293T-hGPRC5D) used as immunogens. After a 7-day interval between each boost, the mice were bled on the 5th day after the second and fourth boosts, respectively, and the sera were separated and the titers of specific antibodies in the sera were measured using a cell-based enzyme-linked immunosorbent assay (ELISA) method.

[0123] To detect the titer of specific antibodies in the serum, the HEK293T-hGPRC5D, HEK293T-cynoGPRC5D, CHOK1-hGPRC5D, CHOK1-cynoGPRC5D recombinant cells obtained in Example 1 and blank control cells were cultured at 4 × 10 per well. 4Cells were inoculated into a 96-well cell plate (manufacturer: corning, catalog number: 3599), incubated overnight in a cell incubator, the supernatant medium was discarded, 50 μl of fixative (manufacturer: Beyotime, catalog number: P0098-500ML) was added per well, and the plate was left at room temperature for 30 min in a fume hood, the supernatant fixative was discarded, the plate was washed twice with PBST, and then 0.5% skim milk (manufacturer: Seikoh, catalog number: A600669-0250) was added, and the plate was blocked for 2 hours at room temperature, the mounting solution was discarded, the plate was washed twice with PBST, and 50 μl / well of mouse serum diluted 3-fold with a starting dilution of 1:100 was added. After incubation at room temperature for 1 hour, the plate was washed three times with PBST. Secondary antibodies labeled with HRP (horseradish peroxidase) (purchased from Jackson, catalog number: 109-035-088) were added and incubated at room temperature for 1 hour, after which the plates were washed five times with PBST. TMB substrate was added at 50μl / well and incubated at room temperature for 5-10 minutes, after which stop solution (1.0N HCl) was added at 50μl / well. OD450nm was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer), and the cell-based ELISA detection results showed that the post-immunization sera from mice immunized with the above cells all showed different degrees of binding to the immunogen, indicating antigen-antibody reaction.

[0124] 2.2 Spleen cell fusion and hybridoma screening 5 × 10 for each selected mouse 6HEK293T-hGPRC5D cells were intraperitoneally injected in a number of cells, and the mice were sacrificed 3 days later to collect splenocytes and lymphocytes. After centrifugation at 1500 rpm, the supernatant was discarded, and ACK lysis solution (Gibco, Cat. A1049201) was added to the cells to lyse the red blood cells doped in the cells to obtain a cell suspension, and the cells were washed three times at 1500 rpm with DMEM basal medium (purchased from Gibco, Cat. No. 10569044), counted, and mixed with mouse myeloma cells SP2 / 0 (purchased from ATCC, Cat. CRL-1581) in a live cell number of 2:1, and cell fusion was performed using the electric fusion method. The fused cells were diluted in DMEM medium containing 20% ​​(v / v) fetal bovine serum (purchased from ExCell Bio, Cat. FND500), 1× HAT (purchased from sigma, Cat. H0262-10VL), bovine insulin (purchased from Yeason, Cat. 40107ES25), and NEAA (purchased from Gibco, Cat. 11140050), and then diluted to 5×10 4 The cells were added to a 96-well cell culture plate at 200 μL per well and cultured in an incubator with 5% (v / v) CO2 at 37°C. After 7 days, the fusion plate supernatants were screened by cell-based ELISA to confirm their binding activity to human GPRC5D-overexpressing cells, and the positive clone supernatants were screened for their binding activity to monkey GPRC5D-overexpressing cells by cell-based ELISA and to endogenous cells NCI-H929 by FACS.

[0125] Based on the screening results, positive clones that meet the conditions were selected and subcloned using semi-solid medium (purchased from stemcell, Cat. 03810). After 7 days, the grown clones were transferred one by one to 96-well culture plates and expanded in DMEM medium containing 10% (w / w) fetal bovine serum and 1×HT (purchased from sigma, Cat. H0137-10VL). After 1 day, initial screening was performed using cell-based ELISA, and positive monoclones were selected and expanded in 24-well plates for continued culture. After 3 days, the culture supernatant was further detected and its binding activity with monkey GPRC5D overexpressing cells and endogenous cells NCI-H929 was evaluated. Based on the detection results of the 24-well plate samples, the optimal clone was selected and expanded in DMEM medium containing 10% (v / v) FBS under conditions of 37°C and 5% (v / v) CO2, and frozen and stored in liquid nitrogen to obtain the hybridoma cells of the present application.

[0126] Example 3 Amino acid sequence determination of the light and heavy chain variable regions of hybridoma positive clones Hybridoma cells in the logarithmic growth phase were collected, thoroughly lysed with Trizol (Invitrogen, Cat No. 15596-018), and then stored at -80°C. The samples were entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. to complete amino acid sequencing of the light and heavy chain variable regions of hybridoma-positive clones. The sequencing results were analyzed using MOE software, and a phylogenetic tree was constructed based on the amino acid sequences of the variable region-encoding proteins. Close sequences in the phylogenetic tree were removed based on sequence similarity, and then eight clones, named GPRC5D-mab01-08 (sequences are shown in Table 7, SEQ ID NO: 13-28), were obtained by screening.

[0127] [Table 11]

[0128] The CDRs of the sequences in Table 7 are divided using the Kabat and IMGT numbering systems, respectively, and the sequences of the CDRs are shown in Table 8:

[0129] [Table 12-1] [Table 12-2]

[0130] We commissioned General Biological Systems (Anhui) Co., Ltd. to synthesize the nucleotide sequences encoding the heavy and light chain variable regions of the above eight clones, linked them via a linker with the sequence GGGGSGGGSGGGGS (SEQ ID NO:117), and cloned them into the expression vector pTT5-huFc (C220S) (heavy chain constant region sequence SEQ ID NO:5) containing a signal peptide and human-derived antibody Fc to obtain an expression vector for a human-mouse chimeric antibody. For the expression and purification steps, please refer to Example 1.1.

[0131] Example 4 Identification of GPRC5D human-mouse chimeric antibodies 4.1 Detection of the binding of chimeric antibodies to human GPRC5D protein by cell-based enzyme-linked immunosorbent assay For specific detection methods, please refer to Example 2.1. CHOK1-hGPRC5D, CHOK1-cynoGPRC5D recombinant cells and CHOK1 cells obtained in Example 1 were fixed, and the chimeric antibody and control antibody, which were diluted in a 3-fold gradient with an initial concentration of 100 nM, were added at 50 μl / well for detection. The cell-based ELISA detection results of the binding activity of the chimeric antibody to human GPRC5D protein are shown in Figures 6A-6C, 7A-7C and 8A-8C. The results showed that the purified and produced chimeric antibody bound to human GPRC5D protein to different degrees at the ELISA level. Here, the negative control antibody hIgG1 was an antibody against hen egg lysozyme, anti-hel-hIgG1 (purchased from Baiying, catalog number: B117901), and the data in the figures were OD450nm values.

[0132] 4.2 Detection of chimeric antibody binding to endogenous cells by flow cytometry (FACS) The required cells were expanded to the logarithmic growth phase in a T-75 cell culture flask, and NCI-H929, MolP-8, and RPMI-8226 cells were taken and washed twice with PBS buffer. After performing a cell count on the cells, the cell pellet was diluted to 2 × 10 with a mounting solution of [PBS + 2% (w / v) BSA]. 6 The cells were resuspended to 100 cells / ml and added to a 96-well FACS reaction plate at 50 μl / well, and a sample to be detected with the chimeric antibody was added at 50 μl / well. The plate was then incubated on ice for 1 hour. The plate was washed three times by centrifugation with PBS buffer, and then incubated with 50 μl / well of Alexa Fluor 100 μl / well. (R)647 AffiniPure Goat Anti-Human IgG, Fcγ fragment specific secondary antibody (purchased from Jackson, catalog number: 109-605-098) was added and incubated on ice for 1 hour. The cells were centrifuged and washed three times with PBS buffer, and detected by FACS (FACS CantoII, purchased from BD), and the results were analyzed. The data was analyzed by software (Flowjo) to obtain the mean fluorescence intensity (MFI) of the cells. Then, the analysis was performed by software (GraphPad Prism8), and the analysis results are shown in Table 9 and Figures 9A-9C, 10A-10C, and 11A-11C. All chimeric antibodies can bind well to NCI-H929 (high expression), MolP-8 (medium expression), and RPMI-8226 (low expression) cells.

[0133] [Table 13]

[0134] 4.3 Detection of binding activity of chimeric antibodies to CHOK1-hGPRC5A, CHOK1-hGPRC5B, and CHOK1-hGPRC5C by flow cytometry (FACS) For the specific method, please refer to Example 4.2. Take CHOK1-hGPRC5A, CHOK1-hGPRC5B and CHOK1-hGPRC5C cells, the results are shown in Table 10, which shows that the chimeric antibody does not bind to CHOK1-hGPRC5A, CHOK1-hGPRC5B, CHOK1-hGPRC5C cells, and does not cross-link with these subtypes.

[0135] [Table 14]

[0136] 4.4 Identification of competitive ability between chimeric antibodies and positive control antibody 5F11 To identify the binding ability of the chimeric antibody to the antigen, the competitive binding of the GPRC5D chimeric antibody to the antigen and the positive control molecule 5F11 was analyzed using a competitive ELISA method. The positive molecule 5F11 was biotin-labeled, and the ELISA plate was coated with hGPRC5D-LVP protein (manufacturer: Kactus Biosystems, catalog number: GPR-HM05P) at 2 μg / mL. The Biotin-5F11 antibody was diluted three-fold starting from 100 nM, and the OD of the Biotin-5F11 antibody was measured. 450 Values ​​were measured, curves were fitted using GraphPad Prism software, and the EC80 concentration was calculated to serve as the reference concentration in competitive ELISA.

[0137] hGPRC5D-LVP protein was diluted to 2 μg / mL and coated at 50 μL / well on a 96-well high-adsorption enzyme plate. After coating overnight at 4°C, 250 μL of mounting solution (PBS containing 2% (w / v) BSA) was used for blocking at room temperature for 2 hours. Chimeric antibodies diluted in a three-fold gradient starting from 100 nM were added, followed by Biotin-5F11 EC80 antibody (approximately 1 nM) and incubation for 1.5 hours. After washing five times with PBS, HRP-labeled Streptavidin Peroxidase Conjugate secondary antibody (purchased from sigma-millipore, catalog number: 189733) was added, incubated for 1 hour, and the plate was washed five times. TMB substrate was added at 50 μL / well and incubated at room temperature for 10 minutes, followed by addition of stop solution (1.0 M HCl) at 50 μL / well. The OD450nm value was read using an ELISA plate reader (Insight, purchased from PerkinElmer). According to the OD450nm value, the results are shown in Table 11 and Figures 12A to 12C, and the lower the value, the stronger the competitive power of the chimeric antibody. The results showed that all eight chimeric antibodies were in an epitope competitive relationship with the positive molecule 5F11-hIgG1.

[0138] [Table 15]

[0139] Example 5 Humanization of GPRC5D mouse-derived antibodies 5.1 Humanization of GPRC5D-mab01 By aligning the IMGT (http: / / imgt.cines.fr) germline gene data bank of human antibody heavy and light chain variable regions, germline genes of heavy and light chain variable regions with high homology to mouse-derived antibodies were selected as templates, and the CDRs of the mouse-derived antibodies were transplanted into the corresponding human-derived templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. If necessary, back mutations were performed on key amino acids in the framework sequence (FR region sequence) to form amino acids corresponding to the mouse-derived antibody, ensuring the original affinity, and at the same time, there are sites in the antibody that are prone to chemical modification, and point mutations were already performed on these sites to eliminate the risk of modification, to obtain a humanized monoclonal antibody. Here, the CDR amino acid residues of the antibody were determined and annotated according to the Kabat numbering system.

[0140] The humanized light chain templates of the mouse-derived antibody GPRC5D-mab01 were IGKV1-9*01 and IGKJ4*01, and the humanized heavy chain templates were IGHV2-26*01 and IGHJ6*01. The specific mutation designs were as shown in Table 12.

[0141] [Table 16]

[0142] The specific sequences of the variable regions of the GPRC5D-mab01 humanized antibody are as follows: The GPRC5D-mab01.VL1 amino acid sequence is as shown in SEQ ID NO:118: [ka] The GPRC5D-mab01.VL2 amino acid sequence is as shown in SEQ ID NO:119: [ka] The GPRC5D-mab01.VL3 amino acid sequence is as shown in SEQ ID NO:120: [ka] The GPRC5D-mab01.VH1 amino acid sequence is as shown in SEQ ID NO:121: [ka] The GPRC5D-mab01.VH2 amino acid sequence is as shown in SEQ ID NO:122: [ka] The GPRC5D-mab01.VH3 amino acid sequence is as shown in SEQ ID NO:123: [ka] The GPRC5D-mab01.VH4 amino acid sequence is as shown in SEQ ID NO:124: [ka] The GPRC5D-mab01.VH5 amino acid sequence is as shown in SEQ ID NO:125: [ka] The humanized light chain template IGKV1-9*01 amino acid sequence is as shown in SEQ ID NO:126: [ka] The humanized light chain template IGKJ4*01 amino acid sequence is as shown in SEQ ID NO:127: [ka] The humanized heavy chain template IGHV2-26*01 amino acid sequence is as shown in SEQ ID NO:128: [ka] The humanized heavy chain template IGHJ6*01 amino acid sequence is as shown in SEQ ID NO:129: [ka]

[0143] The present application selects and combines different light and heavy chain sequences from the above-mentioned mutation designs of the humanized antibody light and heavy chain variable regions of GPRC5D-mab01, respectively, to finally obtain various GPRC5D-mab01 humanized antibodies, the variable region amino acid sequences of each antibody being as shown in Table 13 below:

[0144] [Table 17]

[0145] According to the Kabat numbering system, the analysis of the above humanized antibody VH and VL sequences is shown in Table 14.

[0146] [Table 18]

[0147] 5.2 Humanization of GPRC5D-mab02 The method was the same as in 5.1, the humanized light chain templates of mouse-derived antibody GPRC5D-mab02 were IGKV3-20*02 and IGKJ2*01, and the humanized heavy chain templates were IGHV1-46*01 and IGHJ6*01. The specific mutation design was as shown in Table 15.

[0148] [Table 19]

[0149] The specific sequences of the variable regions of the GPRC5D-mab02 humanized antibody are as follows: The GPRC5D-mab02.VL1 amino acid sequence is as shown in SEQ ID NO:130: [ka] The GPRC5D-mab02.VL2 amino acid sequence is as shown in SEQ ID NO:131: [ka] The GPRC5D-mab02.VH1 amino acid sequence is as shown in SEQ ID NO:132: [ka] The GPRC5D-mab02.VH2 amino acid sequence is as shown in SEQ ID NO:133: [ka] The GPRC5D-mab02.VH2a amino acid sequence is as shown in SEQ ID NO:134: [ka] The GPRC5D-mab02.VH3 amino acid sequence is as shown in SEQ ID NO:135: [ka] The GPRC5D-mab02.VH3a amino acid sequence is as shown in SEQ ID NO:136: [ka] The GPRC5D-mab02.VH4 amino acid sequence is as shown in SEQ ID NO:137: [ka] The humanized light chain template IGKV3-20*02 amino acid sequence is as shown in SEQ ID NO:138: [ka] The humanized light chain template IGKJ2*01 amino acid sequence is as shown in SEQ ID NO:139: [ka] The humanized heavy chain template IGHV1-46*01 amino acid sequence is as shown in SEQ ID NO:140: [ka] The humanized heavy chain template IGHJ6*01 amino acid sequence is as shown in SEQ ID NO:141: [ka]

[0150] The present application selects and combines different light and heavy chain sequences from the above-mentioned GPRC5D-mab02 humanized antibody light and heavy chain variable region mutation designs, respectively, to finally obtain various GPRC5D-mab02 humanized antibodies, the variable region amino acid sequences of each antibody are as shown in Table 16 below:

[0151] [Table 20]

[0152] According to the Kabat numbering system, the analysis results of the above humanized antibody VH and VL sequences are shown in Table 17.

[0153] [Table 21]

[0154] 5.3 Humanization of GPRC5D-mab03 The method was the same as in 5.1, and the humanized light chain templates of mouse-derived antibody GPRC5D-mab03 were IGKV4-1*01 / IGKV1-39*01 and IGKJ4*01, and the humanized heavy chain templates were IGHV1-3*01 and IGHJ6*01. The specific mutation design was as shown in Table 18.

[0155] [Table 22]

[0156] The specific sequences of the variable regions of the GPRC5D-mab03 humanized antibody are as follows: The GPRC5D-mab03.VL1 amino acid sequence is as shown in SEQ ID NO:144: [ka] The GPRC5D-mab03.VL2 amino acid sequence is as shown in SEQ ID NO:145: [ka] The GPRC5D-mab03.VH1 amino acid sequence is as shown in SEQ ID NO:146: [ka] The GPRC5D-mab03.VH2 amino acid sequence is as shown in SEQ ID NO:147: [ka] The GPRC5D-mab03.VH2a amino acid sequence is as shown in SEQ ID NO:148: [ka] The GPRC5D-mab03.VH3 amino acid sequence is as shown in SEQ ID NO:149: [ka] The GPRC5D-mab03.VH4 amino acid sequence is as shown in SEQ ID NO:150: [ka] The GPRC5D-mab03.VH4a amino acid sequence is as shown in SEQ ID NO:151: [ka] The GPRC5D-mab03.VH5 amino acid sequence is as shown in SEQ ID NO:152: [ka] The humanized light chain template IGKV4-1*01 amino acid sequence is as shown in SEQ ID NO:153: [ka] The humanized light chain template IGKV1-39*01 amino acid sequence is as shown in SEQ ID NO:154: [ka] The humanized light chain template IGKJ4*01 amino acid sequence is as shown in SEQ ID NO:155: [ka] The humanized heavy chain template IGHV1-3*01 amino acid sequence is as shown in SEQ ID NO:156: [ka] The humanized heavy chain template IGHJ6*01 amino acid sequence is as shown in SEQ ID NO:157: [ka]

[0157] The present application selects and combines different light and heavy chain sequences from the above-mentioned mutation designs of the humanized antibody light and heavy chain variable regions of GPRC5D-mab03, respectively, to finally obtain various GPRC5D-mab03 humanized antibodies, the variable region amino acid sequences of each antibody being as shown in Table 19 below:

[0158] [Table 23]

[0159] According to the Kabat numbering system, the analysis of the above humanized antibody VH and VL sequences is shown in Table 20.

[0160] [Table 24]

[0161] 5.4 Humanization of GPRC5D-mab04 The method was the same as in 5.1, and the humanized light chain templates of mouse-derived antibody GPRC5D-mab04 were IGKV1-39*01 / IGKV2-28*01 and IGKJ4*01, and the humanized heavy chain templates were IGHV3-7*01 and IGHJ6*01. The specific mutation design was as shown in Table 21.

[0162] [Table 25]

[0163] The specific sequences of the variable regions of the GPRC5D-mab04 humanized antibody are as follows: The GPRC5D-mab04.VL1 amino acid sequence is as shown in SEQ ID NO:160: [ka] The GPRC5D-mab04.VL2 amino acid sequence is as shown in SEQ ID NO:161: [ka] The GPRC5D-mab04.VL3 amino acid sequence is as shown in SEQ ID NO:162: [ka] The GPRC5D-mab04.VL4 amino acid sequence is as shown in SEQ ID NO:163: [ka] The GPRC5D-mab04.VH1 amino acid sequence is as shown in SEQ ID NO:164: [ka] The GPRC5D-mab04.VH2 amino acid sequence is as shown in SEQ ID NO:165: [ka] The GPRC5D-mab04.VH3 amino acid sequence is as shown in SEQ ID NO:166: [ka] The humanized light chain template IGKV1-39*01 amino acid sequence is as shown in SEQ ID NO:167: [ka] The humanized light chain template IGKV2-28*01 amino acid sequence is as shown in SEQ ID NO:168: [ka] The humanized light chain template IGKJ4*01 amino acid sequence is as shown in SEQ ID NO:169: [ka] The humanized heavy chain template IGHV3-7*01 amino acid sequence is as shown in SEQ ID NO:170: [ka] The humanized heavy chain template IGHJ6*01 amino acid sequence is as shown in SEQ ID NO:171: [ka]

[0164] The present application selects and combines different light and heavy chain sequences from the above-mentioned GPRC5D-mab04 humanized antibody light and heavy chain variable region mutation designs, respectively, to finally obtain various GPRC5D-mab04 humanized antibodies, the variable region amino acid sequences of each antibody are as shown in Table 22 below:

[0165] [Table 26]

[0166] According to the Kabat numbering system, the analysis results of the above humanized antibody VH and VL sequences are shown in Table 23.

[0167] [Table 27]

[0168] 5.5 Humanization of GPRC5D-mab05 The method was the same as in 5.1, and the humanized light chain templates of mouse-derived antibody GPRC5D-mab05 were IGKV6-21*01 / IGKV3-11*01 and IGKJ4*01, and the humanized heavy chain templates were IGHV1-69*02 and IGHJ6*01. The specific mutation design was as shown in Table 24.

[0169] [Table 28]

[0170] The specific sequences of the variable regions of the GPRC5D-mab05 humanized antibody are as follows: The GPRC5D-mab05.VL1 amino acid sequence is as shown in SEQ ID NO:172: [ka] The GPRC5D-mab05.VL2 amino acid sequence is as shown in SEQ ID NO:173: [ka] The GPRC5D-mab05.VL3 amino acid sequence is as shown in SEQ ID NO:174: [ka] The GPRC5D-mab05.VL4 amino acid sequence is as shown in SEQ ID NO:175: [ka] The GPRC5D-mab05.VH1 amino acid sequence is as shown in SEQ ID NO:176: [ka] The GPRC5D-mab05.VH2 amino acid sequence is as shown in SEQ ID NO:177: [ka] The GPRC5D-mab05.VH3 amino acid sequence is as shown in SEQ ID NO:178: [ka] The humanized light chain template IGKV6-21*01 amino acid sequence is as shown in SEQ ID NO:179: [ka] The humanized light chain template IGKV3-11*01 amino acid sequence is as shown in SEQ ID NO:180: [ka] The humanized light chain template IGKJ4*01 amino acid sequence is as shown in SEQ ID NO:181: [ka] The humanized heavy chain template IGHV1-69*02 amino acid sequence is as shown in SEQ ID NO:182: [ka] The humanized heavy chain template IGHJ6*01 amino acid sequence is as shown in SEQ ID NO:183: [ka]

[0171] The present application selects and combines different light and heavy chain sequences from the above-mentioned mutation designs of the humanized antibody light and heavy chain variable regions of GPRC5D-mab05, respectively, to finally obtain various GPRC5D-mab05 humanized antibodies, the variable region amino acid sequences of each antibody being as shown in Table 25 below:

[0172] [Table 29]

[0173] According to the Kabat numbering system, the analysis of the above humanized antibody VH and VL sequences is shown in Table 26.

[0174] [Table 30]

[0175] 5.6 GPRC5D-mab06 humanization 5.6.1 First round humanization design of GPRC5D-mab06 The method was the same as in 5.1, and the humanized light chain templates of mouse-derived antibody GPRC5D-mab06 were IGKV4-1*01 / IGKV1-27*01 and IGKJ2*01, and the humanized heavy chain templates were IGHV2-26*01 and IGHJ6*01. The specific mutation design was as shown in Table 27.

[0176] [Table 31]

[0177] The specific sequences of the variable regions of the GPRC5D-mab06 humanized antibody are as follows: The GPRC5D-mab06.VL1 amino acid sequence is as shown in SEQ ID NO:184: [ka] The GPRC5D-mab06.VL2 amino acid sequence is as shown in SEQ ID NO:185: [ka] The GPRC5D-mab06.VL3 amino acid sequence is as shown in SEQ ID NO:186: [ka] The GPRC5D-mab06.VL4 amino acid sequence is as shown in SEQ ID NO:187: [ka] The GPRC5D-mab06.VH1 amino acid sequence is as shown in SEQ ID NO:188: [ka] The GPRC5D-mab06.VH2 amino acid sequence is as shown in SEQ ID NO:189: [ka] The GPRC5D-mab06.VH3 amino acid sequence is as shown in SEQ ID NO:190: [ka] The humanized light chain template IGKV4-1*01 amino acid sequence is as shown in SEQ ID NO:191: [ka] The humanized light chain template IGKV1-27*01 amino acid sequence is as shown in SEQ ID NO:192: [ka] The humanized light chain template IGKJ2*01 amino acid sequence is as shown in SEQ ID NO:193: [ka] The humanized heavy chain template IGHV2-26*01 amino acid sequence is as shown in SEQ ID NO:194: [ka] The humanized heavy chain template IGHJ6*01 amino acid sequence is as shown in SEQ ID NO:195: [ka]

[0178] The present application selects and combines different light and heavy chain sequences from the first round of mutation design of the humanized antibody light and heavy chain variable regions of the above GPRC5D-mab06, respectively, to finally obtain various GPRC5D-mab06 humanized antibodies, the variable region amino acid sequences of each antibody are as shown in Table 28:

[0179] [Table 32]

[0180] According to the Kabat numbering system, the analysis of the above humanized antibody VH and VL sequences is shown in Table 29.

[0181] [Table 33]

[0182] 5.6.2 Second round humanization design of GPRC5D-mab06 The method was the same as in 5.1, and the second round humanized light chain templates of mouse-derived antibody GPRC5D-mab06 were IGKV1-27*01 and IGKJ2*01, and the second round humanized heavy chain templates were IGHV2-26*01 and IGHJ6*01. The specific mutation design was as shown in Table 30.

[0183] [Table 34]

[0184] The specific sequences of the variable regions of the GPRC5D-mab06 humanized antibody are as follows: The GPRC5D-mab06.VL4 amino acid sequence is as shown in SEQ ID NO:196: [ka] The GPRC5D-mab06.VL5 amino acid sequence is as shown in SEQ ID NO:197: [ka] The GPRC5D-mab06.VH4 amino acid sequence is as shown in SEQ ID NO:198: [ka] The GPRC5D-mab06.VH5 amino acid sequence is as shown in SEQ ID NO:199: [ka] The GPRC5D-mab06.VH6 amino acid sequence is as shown in SEQ ID NO:200: [ka] The GPRC5D-mab06.VH7 amino acid sequence is as shown in SEQ ID NO:201: [ka] The humanized light chain template IGKV1-27*01 amino acid sequence is as shown in SEQ ID NO:202: [ka] The humanized light chain template IGKJ2*01 amino acid sequence is as shown in SEQ ID NO:203: [ka] The humanized heavy chain template IGHV2-26*01 amino acid sequence is as shown in SEQ ID NO:204: [ka] The humanized heavy chain template IGHJ6*01 amino acid sequence is as shown in SEQ ID NO:205: [ka]

[0185] The present application selects and combines different light and heavy chain sequences from the second round of humanized antibody light and heavy chain variable region mutation design of the above GPRC5D-mab06, respectively, to finally obtain various GPRC5D-mab06 humanized antibodies, the variable region amino acid sequences of each antibody are as shown in Table 31:

[0186] [Table 35]

[0187] According to the Kabat numbering system, the analysis of the above humanized antibody VH and VL sequences is shown in Table 32.

[0188] [Table 36]

[0189] 5.7 GPRC5D-mab07 humanization 5.7.1 First round humanization design of GPRC5D-mab07 The method was the same as in 5.1, and the humanized light chain templates of mouse-derived antibody GPRC5D-mab07 were IGKV2-40*01 and IGKJ4*01, and the humanized heavy chain templates were IGHV1-69*02 and IGHJ6*01. The specific mutation design was as shown in Table 33.

[0190] [Table 37]

[0191] The specific sequences of the variable regions of the GPRC5D-mab07 humanized antibody are as follows: The GPRC5D-mab07.VL1 amino acid sequence is as shown in SEQ ID NO:206: [ka] The GPRC5D-mab07.VL2 amino acid sequence is as shown in SEQ ID NO:207: [ka] The GPRC5D-mab07.VL2a amino acid sequence is as shown in SEQ ID NO:208: [ka] The GPRC5D-mab07.VH1 amino acid sequence is as shown in SEQ ID NO:209: [ka] The GPRC5D-mab07.VH2 amino acid sequence is as shown in SEQ ID NO:210: [ka] The GPRC5D-mab07.VH3 amino acid sequence is as shown in SEQ ID NO:211: [ka] The GPRC5D-mab07.VH4 amino acid sequence is as shown in SEQ ID NO:212: [ka] The humanized light chain template IGKV2-40*01 amino acid sequence is as shown in SEQ ID NO:213: [ka] The humanized light chain template IGKJ4*01 amino acid sequence is as shown in SEQ ID NO:214: [ka] The humanized heavy chain template IGHV1-69*02 amino acid sequence is as shown in SEQ ID NO:215: [ka] The humanized heavy chain template IGHJ6*01 amino acid sequence is as shown in SEQ ID NO:216: [ka]

[0192] The present application selects and combines different light and heavy chain sequences from the first round of humanized antibody light and heavy chain variable region back mutation design of the above GPRC5D-mab07, respectively, to finally obtain various GPRC5D-mab07 humanized antibodies, and the amino acid sequences of the variable regions of each antibody are as shown in Table 34:

[0193] [Table 38]

[0194] According to the Kabat numbering system, the analysis of the above humanized antibody VH and VL sequences is shown in Table 35.

[0195] [Table 39]

[0196] 5.7.2 Second round humanization design of GPRC5D-mab07 The method was the same as in 5.1, and the second round humanized light chain templates of mouse-derived antibody GPRC5D-mab07 were IGKV2-40*01 and IGKJ4*01, and the second round humanized heavy chain templates were IGHV1-69*02 and IGHJ6*01. The specific mutation design was as shown in Table 36.

[0197] [Table 40]

[0198] The specific sequences of the variable regions of the GPRC5D-mab07 humanized antibody are as follows: The GPRC5D-mab07.VL1a amino acid sequence is as shown in SEQ ID NO:218: [ka] The GPRC5D-mab07.VL1b amino acid sequence is as shown in SEQ ID NO:219: [ka] The GPRC5D-mab07.VL1c amino acid sequence is as shown in SEQ ID NO:220: [ka] The GPRC5D-mab07.VL1d amino acid sequence is as shown in SEQ ID NO:221: [ka] The GPRC5D-mab07.VH4 amino acid sequence is as shown in SEQ ID NO:222: [ka] The humanized light chain template IGKV2-40*01 amino acid sequence is as shown in SEQ ID NO:223: [ka] The humanized light chain template IGKJ4*01 amino acid sequence is as shown in SEQ ID NO:224: [ka] The humanized heavy chain template IGHV1-69*02 amino acid sequence is as shown in SEQ ID NO:225: [ka] The humanized heavy chain template IGHJ6*01 amino acid sequence is as shown in SEQ ID NO:226: [ka]

[0199] The present application selects and combines different light and heavy chain sequences from the second round of mutation design of the humanized antibody light and heavy chain variable regions of the above GPRC5D-mab07, respectively, to finally obtain various GPRC5D-mab07 humanized antibodies, the variable region amino acid sequences of each antibody are as shown in Table 37:

[0200] [Table 41]

[0201] According to the Kabat numbering system, the analysis of the above humanized antibody VH and VL sequences is shown in Table 38.

[0202] [Table 42]

[0203] 5.8 GPRC5D-mab08 humanization The method was the same as in 5.1, and the humanized light chain templates of mouse-derived antibody GPRC5D-mab08 were IGKV1-27*01 / IGKV2-29*02 and IGKJ4*01, and the humanized heavy chain templates were IGHV1-3*01 and IGHJ6*01. The specific mutation design was as shown in Table 39.

[0204] [Table 43]

[0205] The specific sequences of the variable regions of the GPRC5D-mab08 humanized antibody are as follows: The GPRC5D-mab08.VL4 amino acid sequence is as shown in SEQ ID NO:231: [ka] The GPRC5D-mab08.VL5 amino acid sequence is as shown in SEQ ID NO:232: [ka] The GPRC5D-mab08.VL6 amino acid sequence is as shown in SEQ ID NO:233: [ka] The GPRC5D-mab08.VH6 amino acid sequence is as shown in SEQ ID NO:234: [ka] The GPRC5D-mab08.VH7 amino acid sequence is as shown in SEQ ID NO:235: [ka] The GPRC5D-mab08.VH8 amino acid sequence is as shown in SEQ ID NO:236: [ka] The GPRC5D-mab08.VH9 amino acid sequence is as shown in SEQ ID NO:237: [ka] The GPRC5D-mab08.VH10 amino acid sequence is as shown in SEQ ID NO:238: [ka] The humanized light chain template IGKV1-27*01 amino acid sequence is as shown in SEQ ID NO:239: [ka] The humanized light chain template IGKV2-29*02 amino acid sequence is as shown in SEQ ID NO:240: [ka] The humanized light chain template IGKJ4*01 amino acid sequence is as shown in SEQ ID NO:241: [ka] The humanized heavy chain template IGHV1-3*01 amino acid sequence is as shown in SEQ ID NO:242: [ka] The humanized heavy chain template IGHJ6*01 amino acid sequence is as shown in SEQ ID NO:243: [ka]

[0206] The present application selects and combines different light and heavy chain sequences from the above-mentioned GPRC5D-mab08 humanized antibody light and heavy chain variable region mutation designs, respectively, to finally obtain various GPRC5D-mab08 humanized antibodies, the variable region amino acid sequences of each antibody are as shown in Table 40:

[0207] [Table 44]

[0208] According to the Kabat numbering system, the analysis of the above humanized antibody VH and VL sequences is shown in Table 41.

[0209] [Table 45]

[0210] Example 6 Identification of GPRC5D humanized antibodies 6.1 Detection of binding of humanized antibodies to human GPRC5D protein by cell-based enzyme-linked immunosorbent assay To detect the binding activity of the GPRC5D humanized antibody to the full-length human GPRC5D protein, a detection method similar to the cell-based enzyme-linked immunosorbent assay (cell-based ELISA) of Example 4 was used to detect the binding between the humanized antibody and human GPRC5D protein, and the detection results are shown in Figures 13A to 13J and 14A to 14J. The results showed that the purified and produced humanized antibodies bind to different degrees to the full-length human GPRC5D protein at the ELISA level.

[0211] 6.2 Detection of binding activity between humanized antibodies and NCI-H929 cells that endogenously express human GPRC5D by flow cytometry (FACS) In order to detect the binding activity of the GPRC5D humanized antibody with the full-length human GPRC5D protein, a detection method similar to FACS in Example 4 was used to detect the binding between the humanized antibody and human GPRC5D protein, and the detection results are shown in Figures 15A to 15J. The results showed that all of the humanized antibodies can bind to NCI-H929 cells to different degrees and have good specificity.

Claims

1. 1. An antibody or antigen-binding fragment thereof that specifically binds to G protein-coupled receptor class C group 5 member D (GPRC5D), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein: (1) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 has any one of the LCDR1 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to said sequence, the LCDR2 has any one of the LCDR2 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to said sequence, and the LCDR3 has any one of the LCDR3 sequences shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to said sequence, 【Table 1】 and, (2) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 has any one of the sequences of HCDR1 shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to the sequence, the HCDR2 has any one of the sequences of HCDR2 shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to the sequence, and the HCDR3 has any one of the sequences of HCDR3 shown below or a sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to the sequence, 【Table 2】 Preferably, the antibody or antigen-binding fragment thereof is selected from the group consisting of L1+H1, L2+H2, L3+H3, L4+H4, L5+H5, L6+H6, L7+H7, L8+H8, L9+H9, L10+H10, L11+H11, L12+H12, L13+H13, L14+H14, L15+H15, L16+H16, L3+H17, L3+H18, L5+H19, L5+H20, L5+H21, L5+H22, L5+H23, L5+H24, L5+H25, L5+H26, L5+H27, L5+H28, L5+H29, L5+H30, L5+H31, L5+H32, L5+H33, L5+H34, L5+H35, L5+H36, L5+H37, L5+H38, L5+H39, L5+H40, L5+H41, L5+H42, L5+H43, L5+H44, L5+H45, L5+H46, L5+H47, L5+H48, L5+H49, L5+H50, L5+H51, L5+H52, L5+H53, L5+H54, L5+H55, L5+H56, L5+H57, L5+H58, L5+H59, L5+H60, L5+H61, L5+H62, L5+H63, L5+H64, L5+H65, L5+H66, L5+H67, L5+H68, L5+H69, L5+H70, L5+H71 An antibody or antigen-binding fragment thereof comprising a six CDR sequence in a combination of a light chain variable region and a heavy chain variable region of L17+H13, L18+H13, L19+H13, L20+H13 or L21+H13, or a six CDR sequence having one, two, three or more amino acid insertions, deletions and / or substitutions compared to the six CDR sequences.

2. (1) the light chain variable region sequence comprises any one of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 118-120, 130-131, 144-145, 160-163, 172-175, 184-187, 196-197, 206-208, 218-221, and 231-233, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto; and, (2) the heavy chain variable region sequence comprises any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 121-125, 132-137, 146-152, 164-166, 176-178, 188-190, 198-201, 209-212, 222, and 234-238, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto; Preferably, the antibody or antigen-binding fragment thereof has the light chain variable region and heavy chain variable region shown below: (1) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 14 and SEQ ID NO: 13, respectively; (2) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 16 and SEQ ID NO: 15, respectively; (3) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 18 and SEQ ID NO: 17, respectively; (4) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 20 and SEQ ID NO: 19, respectively; (5) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 21, respectively; (6) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 24 and SEQ ID NO: 23, respectively; (7) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 25, respectively; (8) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 28 and SEQ ID NO: 27, respectively; (9) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 118 to 120, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 121 to 125; (10) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 130 to 131, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 132 to 137; (11) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 144 to 145, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 146 to 152; (12) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 160 to 163, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 164 to 166; (13) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 172 to 175, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 176 to 178; (14) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 184 to 187, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 188 to 190; (15) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 196 to 197, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 198 to 201; (16) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 206 to 208, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 209 to 212; (17) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 218 to 221, and the heavy chain variable region comprises a sequence shown in SEQ ID NO: 222; (18) The light chain variable region comprises a sequence shown in any one of SEQ ID NOs: 231 to 233, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NOs: 234 to 238; or (19) the antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region comprises a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the light chain variable region shown in any one of (1) to (18) above, and the heavy chain variable region comprises a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the heavy chain variable region shown in any one of (1) to (18) above.

3. 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which is chimeric, humanized or fully human.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, capable of binding to human or monkey GPRC5D.

5. The antigen-binding fragment is an F(ab) ２ 5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is selected from one or more of the following: Fab', Fab, Fv, scFv, nanobody or affibody.

6. The antibody or antigen-binding fragment thereof further comprises any one of a constant region sequence of human or mouse antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD, and preferably comprises a constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4, or comprises a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein a therapeutic agent or tracer is conjugated to the antibody or antigen-binding fragment thereof, preferably the therapeutic agent is selected from a radioisotope, a chemotherapeutic drug or an immunomodulatory agent, the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer, and more preferably the cytotoxic agent is selected from alkaloids, methotrexate, an anthracycline antibiotic, a taxane, a pyrrolobenzodiazepine or a toxin compound.

7. A multispecific antigen-binding molecule, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and another antigen-binding molecule that binds to an antigen other than GPRC5D or binds to a GPRC5D epitope different from the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; Preferably, the other antigen-binding molecule is an antibody or an antigen-binding fragment thereof, Preferably, the multispecific antigen-binding molecule may be bispecific, trispecific or tetraspecific; Preferably, said multispecific antigen-binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent.

8. A chimeric antigen receptor (CAR), comprising at least a signal peptide, an extracellular antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises the GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the multispecific antigen-binding molecule according to claim 7.

9. 9. An immune effector cell, said immune effector cell expressing a chimeric antigen receptor according to claim 8 or comprising a nucleic acid fragment encoding the chimeric antigen receptor according to claim 8, preferably said immune effector cell being selected from a T cell, a NK cell, a NKT cell, a DNT cell, a monocyte, a macrophage, a dendritic cell or a mast cell, said T cell being preferably selected from a cytotoxic T cell (CTL), a regulatory T cell or a helper T cell, preferably said immune effector cell being an autologous or an allogeneic immune effector cell.

10. 9. An isolated nucleic acid fragment, wherein the nucleic acid fragment encodes the antibody or antigen-binding fragment thereof of any one of claims 1 to 6, the multispecific antigen-binding molecule of claim 7, or the chimeric antigen receptor of claim 8.

11. A vector comprising the nucleic acid fragment of claim 10.

12. 12. A host cell, said host cell comprising the vector according to claim 11, preferably said cell being a prokaryotic or eukaryotic cell, such as a bacterial (e.g. E. coli), a fungal (e.g. yeast), an insect cell or a mammalian cell (e.g. a CHO cell line or a 293T cell line).

13. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or a multispecific antigen-binding molecule according to claim 7, the method comprising culturing a cell according to claim 12, and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by the cell.

14. 10. A method of producing the immune effector cell, the method comprising introducing a nucleic acid fragment encoding a CAR of claim 8 into the immune effector cell, and optionally, the method further comprises priming the immune effector cell to express the CAR of claim 8.

15. 15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6, the multispecific antigen-binding molecule of claim 7, the immune effector cell of claim 9, the nucleic acid fragment of claim 10, the vector of claim 11, or a product produced according to the method of claim 13 or 14, optionally wherein the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier, diluent or adjuvant, and optionally wherein the drug composition further comprises an additional anti-tumor agent.

16. A method for treating a tumor or cancer, the method comprising administering to a subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the multispecific antigen-binding molecule according to claim 7, the immune effector cell according to claim 9, the nucleic acid fragment according to claim 10, the vector according to claim 11, the product produced according to the method of claim 13 or 14, or the pharmaceutical composition according to claim 15. Preferably, said tumor or cancer is a GPRC5D expressing tumor or cancer, preferably a B cell lymphoma, more preferably multiple myeloma (MM).

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the multispecific antigen-binding molecule according to claim 7, the immune effector cell according to claim 9, the nucleic acid fragment according to claim 10, the vector according to claim 11, the product produced according to the method of claim 13 or 14, or the pharmaceutical composition according to claim 15 in the manufacture of a medicament for treating tumors or cancer, comprising: Preferably, said tumor or cancer is a tumor or cancer that expresses GPRC5D, preferably a B cell lymphoma, more preferably multiple myeloma (MM).

18. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the multispecific antigen-binding molecule according to claim 7, the immune effector cell according to claim 9, the nucleic acid fragment according to claim 10, the vector according to claim 11, a product produced according to the method according to claim 13 or 14, or the pharmaceutical composition according to claim 15.

19. A method for detecting GPRC5D expression in a biological sample, the method comprising contacting the biological sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 under conditions allowing the formation of a complex between the antibody or antigen-binding fragment thereof and GPRC5D, preferably the method further comprising detecting the formation of the complex and indicating the presence or expression level of GPRC5D in the sample.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 in the manufacture of a GPRC5D detection reagent.

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