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 of related applications> This application claims priority to a Chinese patent application filed with the China National Intellectual Property Administration on December 31, 2021, with patent application number 202111668463.1 and the title of invention "GPRC5D antibody and its applications." The aforementioned prior application is incorporated into this application in its entirety by reference.

[0002] <Technical field> This application relates to the field of biopharmaceuticals, and more specifically to GPRC5D antibodies and their applications. [Background technology]

[0003] Multiple myeloma (MM) is a plasma cell malignancy characterized by the uncontrolled proliferation of plasma cells in the bone marrow, resembling tumor cells, accompanied by the secretion of monoclonal immunoglobulins, leading to a range of clinical symptoms including multiple osteolytic lesions, hypercalcemia, anemia, renal damage, and recurrent infections. Hematopoietic stem cell transplantation, as well as combination therapies with immunomodulators and protease inhibitors, significantly improve the prognosis. In recent years, CAR-T therapy has made remarkable progress in the treatment of multiple myeloma, and CAR-T cells targeting B-cell maturation antigen (BCMA) in particular have shown positive clinical efficacy in treating multiple myeloma. However, for BCMA-negative or BCMA-low-expressing multiple myeloma patients, relapses still occur even after BCMA-targeted CAR-T cell therapy, highlighting the need to find other more promising novel targets for the treatment of multiple myeloma.

[0004] GPRC5D is a G protein-coupled receptor, class C, group 5 member D, belonging to the orphan receptor category, and is a 7-transmembrane protein. GPRC5D is a novel multiple myeloma target following BCMA. Tissue expression profiling studies have revealed that GPRC5D is specifically highly expressed in plasma cells of multiple myeloma, but is lowly expressed in normal tissues and limited to immune-tolerant hair follicle regions. Furthermore, the expression of GPRC5D and BCMA do not overlap. In tumor recurrence models where BCMA is lost, GPRC5D CAR-T still exhibits therapeutic efficacy and overcomes tumor evasion. Theoretically, dual targeting of GPRC5D and BCMA could target a wider population and potentially lead to better clinical responses. Therefore, there is a huge potential clinical need for the development of GPRC5D-specific antibodies and related biologics. [Overview of the project] [Means for solving the problem]

[0005] This 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, manufacturing methods, pharmaceutical compositions, pharmaceutical uses, and methods for treating tumors or cancers (e.g., B-cell lymphoma or multiple myeloma).

[0006] In one embodiment, 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 comprises a heavy chain variable region and a light chain variable region, (1) The light chain variable region includes LCDR1, LCDR2 and LCDR3, wherein LCDR1 has any one of the LCDR1 sequences shown below or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the aforementioned sequence, LCDR2 has any one of the LCDR2 sequences shown below or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the aforementioned sequence, and LCDR3 has any one of the LCDR3 sequences shown below or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the aforementioned sequence,

[0007] [Table 1] and, (2) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3, wherein HCDR1 has any one of the following HCDR1 sequences or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the aforementioned sequence; HCDR2 has any one of the following HCDR2 sequences or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the aforementioned sequence; and HCDR3 has any one of the following HCDR3 sequences or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the aforementioned sequence.

[0008] [Table 2]

[0009] In another embodiment, the application provides a multispecific antigen-binding molecule comprising the aforementioned antibody or its antigen-binding fragment and another antigen-binding molecule that binds to an antigen other than GPRC5D or to a GPRC5D epitope different from the aforementioned antibody or its antigen-binding fragment.

[0010] In another embodiment, the present application provides a chimeric antigen receptor (CAR), wherein the 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, wherein the extracellular antigen-binding domain comprises the aforementioned GPRC5D antibody or its antigen-binding fragment, or the aforementioned multispecific antigen-binding molecule.

[0011] In another embodiment, the present application provides immune effector cells, wherein the immune effector cells express or encode the aforementioned chimeric antigen receptor.

[0012] In another embodiment, the present application provides an isolated nucleic acid fragment that encodes the aforementioned antibody or its antigen-binding fragment, the aforementioned multispecific antigen-binding molecule, or the aforementioned chimeric antigen receptor.

[0013] In another embodiment, the present application provides a vector comprising the nucleic acid fragment described above.

[0014] In another embodiment, the present application provides a host cell, wherein the host cell includes the aforementioned vector.

[0015] In another embodiment, the present application provides a method for producing the aforementioned antibody or its antigen-binding fragment or the aforementioned multispecific antigen-binding molecule, the method comprising culturing the aforementioned cells and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by the cells.

[0016] In another embodiment, the present application provides a method for producing the aforementioned immune effector cells, the method comprising introducing a nucleic acid fragment encoding the aforementioned CAR into the immune effector cells.

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

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

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

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

[0021] [[ID=1,6]]In another aspect, the present application provides a kit, wherein the kit comprises the aforementioned antibody or its antigen-binding fragment, the aforementioned multispecific antigen-binding molecule, the aforementioned immune effector cell, the aforementioned nucleic acid fragment, the aforementioned vector, or a product produced according to the aforementioned method or the aforementioned 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 aforementioned antibody or an antigen-binding fragment thereof under conditions that allow 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 aforementioned antibody or an antigen-binding fragment thereof in the manufacture of a GPRC5D detection reagent.

[0024] BCMA negativity or low expression is a common cause of recurrence or refractoriness in multiple myeloma. The present application focuses on another target, GPRC5D, in relapsed / refractory multiple myeloma, and provides an antibody or an antigen-binding fragment thereof that targets human GPRC5D, which binds to human or monkey GPRC5D with high affinity but does not bind or binds with low affinity to GPRC5A, GPRC5B or GPRC5C proteins. This has important significance for the further development of antibodies (monospecific antibodies or multispecific antibodies) against GPRC5D targets and cell therapy products. <000——102>

Brief Description of the Drawings

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

[0026] <Detailed description of the invention> Definitions and explanations of terms Unless otherwise defined herein, scientific and technical terms relating to this application have the meanings understood by those skilled in the art.

[0027] Furthermore, unless otherwise explicitly stated herein, singular terms herein should include plural forms, and plural terms herein should include singular forms. More specifically, unless otherwise clearly indicated, the singular forms “one” and “such” as used herein and in the appended claims include multiple references.

[0028] In this specification, the terms “includes,” “contains,” and “have” are used synonymously and are intended to indicate the comprehensiveness of a scheme, meaning that the scheme may contain elements other than those listed. At the same time, it should be understood that the statements “includes,” “contains,” and “have” as used herein also provide a scheme consisting of…

[0029] In this specification, the terms "and / or" include, when used herein, "and," "or," and "all or any other combination of the elements linked by the terms to which they belong."

[0030] In this specification, the term GPRC5D refers to G protein-coupled receptor class C group 5 member D, belonging to the orphan receptor category, and is a 7-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. Studies have shown that GPRC5D has an expression threshold of over 50% in 65% of multiple myeloma patients, and this characteristic has made GPRC5D a potential target for treating MM.

[0031] In this specification, the term "specific binding" refers to the fact that an antigen-binding molecule (e.g., an antibody) typically binds specifically to an antigen and substantially the same 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] In this specification, the term “antigen-binding molecule” is used in its broadest sense to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen-binding molecules include, but are not limited to, antibodies or antibody mimetic molecules. “Antibody mimetic” refers to an organic compound or binding domain that can specifically bind to an antigen but is unrelated to the antibody structure. Exemplarily, antibody mimetic molecules include, but are not limited to, affibody, affitin, affilin, designed ankyrin repeat protein (DARPin), nucleic acid aptamers, or Kunitz-type domain peptides.

[0033] In this specification, the term “antibody” is used in its broadest sense to refer to a polypeptide or combination of polypeptides comprising a sufficient sequence from the immunoglobulin heavy chain variable region and / or a sufficient sequence from the immunoglobulin light chain variable region, thereby capable of specifically binding to an antigen. In this specification, “antibody” encompasses a variety of forms and structures, as long as they exhibit the desired antigen-binding activity. In this specification, “antibody” includes alternative protein scaffolds or artificial scaffolds having implanted complementarity-determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (including, for example, mutations introduced to stabilize the three-dimensional structure of the antibody) and totally synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003), and Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds may further include non-antibody-derived scaffolds, such as scaffold proteins known in this field that can be used for CDR transplantation, including but not limited to tenascin, fibronectin, and peptide aptamers.

[0034] The term "antibody" includes the entire antibody and any antigen-binding fragment thereof (i.e., "antigen-binding portion") or single chain. "Antibody" refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked to each other via disulfide bonds, or its antigen-binding portion. Each heavy chain consists of a heavy chain variable region (abbreviated here 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 here 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 scattered within more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the 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 region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, which include various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The constant regions of immunoglobulin heavy chains differ in their antigenicity due to their different amino acid composition and sequence. Thus, the “immunoglobulins” as used herein can be divided into five classes, or may be called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. Ig of the same class can be divided into various subclasses based on differences in the amino acid composition of its 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. The light chain can be divided into a κ chain or a λ chain depending on the constant region. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0035] In this specification, "antibody" further includes light chain-free antibodies, such as heavy-chain antibodies (HCAbs) produced from camelid animals such as dromedarius, Bactrianus, Lama glama, Lama guanicoe, and Vicugna pacos, as well as immunoglobulin new antigen receptors (IgNARs) found in cartilaginous fish such as sharks.

[0036] In this specification, the term “antibody” may be derived from any animal, including but not limited to humans and non-human animals, and such non-human animals may be selected from primates, mammals, rodents and vertebrates, for example, camelids, llamas, guanacos, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (for example, sharks).

[0037] In this specification, the term "heavy-chain antibody" refers to an antibody that lacks the light chain of a conventional antibody. Specifically, this term includes, but is not limited to, homodimer antibodies that contain a VH antigen-binding domain and constant CH2 and CH3 domains in the absence of a CH1 domain.

[0038] In this specification, the term "nanoantibody" refers to the fact that in the bodies of animals such as camels, there are naturally occurring heavy chain antibodies lacking light chains, and by cloning their variable region, a single-domain antibody consisting only of the heavy chain variable region can be obtained. This is also called VHH (Variable domain of heavy chain antibody) and is the smallest functional antigen-binding fragment.

[0039] In this specification, the terms "VHH domain," "nanobody," and "single-domain antibody (sdAb)" have the same meaning and are used synonymously. They refer to the construction of a single-domain antibody consisting of only one heavy-chain variable region by cloning the variable region of a heavy-chain antibody, which is the smallest antigen-binding fragment with complete function. Typically, a single-domain antibody consisting of only one heavy-chain variable region is constructed by obtaining a heavy-chain antibody that naturally lacks the light chain and heavy-chain constant region 1 (CH1), and then cloning the variable region of the antibody heavy chain.

[0040] For further explanations regarding "heavy chain antibodies," "single-domain antibodies," "VHH domains," and "nano antibodies," please refer to: Hamers-Casterman et al., Nature. 1993;363;446-8, Muyldermans' overview article (Reviews in Molecular Biotechnology 74:277-302, 2001), and the following patent applications mentioned as general background technology: 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 06 / 122825 and other prior art referenced in these applications.

[0041] In this specification, the term “multispecificity” refers to the ability of an antibody or its antigen-binding fragment to bind to, for example, different antigens or at least two different epitopes on the same antigen. Therefore, terms such as “bispecificity,” “tripspecificity,” and “quadrispecificity” refer to the number of different epitopes to which an antibody can bind. For example, a typical monospecific IgG antibody has two identical antigen-binding sites (complementary sites) and can therefore bind only to the same epitope (rather than to different epitopes). In contrast, a multispecific antibody has at least two different types of complementary sites / binding sites and can therefore bind to at least two different epitopes. As described herein, “complementarity-determining region” refers to the antigen-binding site of an antibody. Also, monospecificity can refer to one, two, three, or more identical complementarity-determining regions in a single antibody (the actual number of complementarity-determining regions / binding sites in a single antibody molecule is called the “valence”). For example, a single natural IgG antibody has two identical complementary sites and is therefore monospecific and bivalent. Accordingly, a multispecific antibody contains at least two (different) complementarity-determining regions / binding sites. Therefore, the term “multispecific antibody” refers to an antibody that has more than one complementary site and the ability to bind to two or more different epitopes. The term “multispecific antibody” specifically includes bispecific antibodies as defined above, but usually further includes antibodies, scaffolds, that specifically bind to three or more different epitopes, i.e., antibodies that have three or more complementary sites / binding sites.

[0042] In this specification, the term "valency" refers to the presence of a predetermined number of binding sites in an antibody / antigen-binding molecule. Therefore, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites in an antibody / antigen-binding molecule, respectively.

[0043] In this specification, "full-length antibody," "intact antibody," and "whole antibody" are used synonymously and refer to an antibody having a structure substantially similar to that of a natural antibody.

[0044] In this specification, the terms “antigen-binding fragment” and “antibody fragment” are used synonymously and include only local or local variants of the entire antibody, which do not have all the structures of the entire antibody, and which have the ability to bind to the antigen. Exemplarily, in this specification, “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 antibodies.

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

[0046] In this specification, the term "humanized antibody" refers to a genetically modified non-human antibody whose amino acid sequence is modified to improve homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies typically retain or partially retain desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, or ability to enhance the immune response.

[0047] In this specification, "fully human antibody" refers to an antibody in which both the FR and CDR have variable regions derived from human germline immunoglobulin sequences. Furthermore, if the antibody includes a constant region, the constant region also originates from a human germline immunoglobulin sequence. In this specification, a fully human antibody may include amino acid residues not encoded by a human germline immunoglobulin sequence (e.g., mutations introduced by in vitro random or site-directed induction or in vivo somatic mutation). However, in this specification, a "fully human antibody" does not include an antibody in which a CDR sequence derived from the germline of another mammalian species (e.g., mouse) has been transplanted into a human framework sequence.

[0048] In this specification, "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. "Heavy chain variable region" is used synonymously with "VH" and "HCVR," and "light chain variable region" is used synonymously with "VL" and "LCVR." The variable domains of the heavy and light chains of natural antibodies generally have similar structures, and each domain contains four conservative framework regions (FRs) and three hypervariable regions (HVRs). For example, Kindt et al., Kuby Immunology, 6. th See ed., WH Freeman and Co., p.91 (2007). A single VH or VL domain is sufficient to confer antigen-binding specificity.

[0049] In this specification, “complementarity-determining region” is used synonymously with “CDR” and typically refers to the hypervariable region (HVR) found in both the light and heavy chain variable domains. The more conserved portion of the variable domain is called the framework region (FR). As understood in the art, the amino acid positions representing the hypervariable region of an antibody can vary depending on the context and various definitions known in the art. Some positions within the variable domain may be considered heterozygous hypervariable positions, as they 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). One or more of these positions may also be found in the extended hypervariable region. This application includes antibodies in which modifications are included at these heterozygous hypervariable positions. The heavy chain variable region CDR may be abbreviated as HCDR, and the light chain variable region may be abbreviated as LCDR. The variable domains of the natural heavy and light chains each contain four framework regions that primarily utilize sheet structures, linked via three CDRs (CDR1, CDR2, and CDR3), which form loops linking the sheet structures and, in some cases, form parts of them. The CDRs in each chain are tightly held via FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and contribute to the formation of antigen-binding sites on antibodies with 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), and Lefranc See MP et al., Dev.Comp.Immunol., 27:55-77 (2003), and Honegger and Pluckthun, J.Mol.Biol., 309:657-670 (2001). In this specification, “CDR” may be labeled and defined in a manner well known in the art, and includes, but is not limited to, the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. The tool sites 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). In this specification, CDR includes overlaps and subsets of amino acid residues under different definitions.

[0051] In this specification, the term “Kabat numbering system” usually refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0052] In this specification, the term “Chothia numbering system” usually refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying the boundaries of CDR regions based on the location of loop regions in a structure (see, e.g., Chothia & Lesk (1987) J.Mol.Biol.196:901-917, Chothia et al. (1989) Nature 342:878-883).

[0053] In this specification, the term "IMGT numbering system" usually refers to the 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] In this specification, “heavy chain constant region” refers to the carboxyl-terminal portion of the antibody heavy chain, which does not directly participate in antibody-antigen binding but represents effector functions such as interaction with the Fc receptor and has an amino acid sequence that is more conserved than that of the antibody's variable domain. The “heavy chain constant region” may be selected from the CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. The “heavy chain constant region” includes “full-length heavy chain constant region” and “heavy chain constant region fragments,” the former having a structure substantially similar to the constant region of a native antibody, while the latter includes only “a portion 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, further including a CH4 domain if the antibody is IgE, and not including a CH1 domain if the antibody is a heavy chain antibody. Exemplarily, a typical “heavy chain constant region fragment” may be selected from the Fc or CH3 domain.

[0055] In this specification, the term “light chain constant region” refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen, and the light chain constant region may be selected from a constant κ domain or a constant λ domain.

[0056] In this specification, the term “Fc region” is used to define the C-terminal region of an antibody heavy chain that includes at least a portion of the constant region. The term includes both native sequence Fc regions and mutant Fc regions. Exemplarily, the human IgG heavy chain Fc region may extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells undergo posttranslational cleavage, which can cleave one or more, in particular one or two, amino acids from the C-terminus of the heavy chain. Thus, by expression of certain nucleic acid molecules encoding a full-length heavy chain, antibodies produced by host cells may contain a full-length heavy chain or a cleaved variant of a 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), or C-terminal glycine (Gly446) and lysine (Lys447), may or may not be present in the Fc region. Typically, the IgG Fc region includes the IgG CH2 and IgG CH3 domains, and optionally further includes a complete or partial hinge region, but does not include the CH1 domain. The "CH2 domain" of the human IgG Fc region usually extends from an amino acid residue at approximately position 231 to an amino acid residue at approximately position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain as used herein may be a native sequence CH2 domain or a mutant CH2 domain. The "CH3 domain" includes the residue at the C-terminus of the CH2 domain in the Fc region (i.e., from an amino acid residue at approximately position 341 to an amino acid residue at approximately position 447 of IgG). The CH3 region as used herein may be a native sequence CH3 domain or a mutant CH3 domain (for example, a CH3 domain having a “knob” introduced on one strand and a “cavity” or “hole” introduced on the other strand, see U.S. Patent No. 5,821,333, which is 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, the 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] In this specification, the term “Fc variant” refers to a change in Fc structure or function caused by the presence of one or more amino acid substitutions, insertions, or deletions at appropriate sites in Fc. “Interactions between Fc variants” refers to the potential for space-filling effects, electrostatic induction, hydrogen bonding, and hydrophobic interactions to form between mutation-designed Fc variants. These interactions facilitate the formation of stable heterodimeric proteins. Preferred mutations are those in the “Knob-into-Hole” form.

[0059] The technology for designing mutations in Fc variants is widely applied in this field to the production of 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 Amgen engineers using electrostatic steering (US 20100286374 A1); the heterodimer form formed by IgG / Ig chain exchange proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp.1-8, 2010) (SEEDbodies); the bispecific molecule formed by Genmab's DuoBody platform technology (Science, 2007.317(5844)); and the heterodimer protein form formed by Xencor engineers integrating structural calculations and Fc amino acid mutations, combining different modes of action (mAbs 3:6,546-557;November / December Other genetic engineering methods that achieve the formation of heterodimeric functional proteins based on Fc amino acid changes or functional modification means include the heterodimeric protein morphology obtained by the charge network-based Fc modification method (CN201110459100.7) by Suzhou Kangning Jierui Co., Ltd. in 2011. The Knob / Hole structure in the Fc mutant fragments described in this application refers to the fact that two Fc fragments are each mutated and can be joined in a "Knob-into-Hole" configuration after the mutations. It is preferable to perform site mutation modification in the Fc region using Cater et al.'s "knob-into-hole" model so that the obtained first Fc mutant and second Fc mutant can be joined together in a "knob-into-hole" configuration to form a heterodimer. Selecting a specific immunoglobulin Fc region from a specific immunoglobulin class and subclass is within the scope of those skilled in the art. Preferably, it is the Fc region of human antibodies IgG1, IgG2, IgG3, and IgG4, and more preferably, it is the Fc region of human antibody IgG1.A randomly selected mutant is chosen from either the first or second Fc mutant, with one undergoing a knob mutation and the other undergoing a hole mutation.

[0060] In this specification, the term “conservative amino acid” usually refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, back chain conformation, and rigidity). Exemplarily, the amino acids in each of the following groups belong to the conservative amino acid residues of each other, and substitutions of amino acid residues within a group belong to the substitutions of conservative amino acids: 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] In this specification, "identity" is calculated by the following method: aligning two amino acid sequences or two nucleic acid sequences for the purpose of optimal comparison to determine the proportion of "identity" between them (for example, gaps may be introduced into one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be rejected for comparison); then comparing amino acid residues or nucleotides at corresponding amino acid or nucleotide positions; if a position in the first sequence is occupied by the same amino acid residue or nucleotide at a corresponding position in the second sequence, the molecules are the same at that 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 proportion of identity between the two sequences changes according to the same positional changes common to the sequences.

[0062] Sequence comparison and the calculation of identity ratio between two sequences can be achieved using mathematical algorithms. For example, the Needlema and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm (available from www.gcg.com), incorporated into the GAP program of the GCG software package, can be used to determine the identity ratio between two amino acid sequences using the Blossum 62 matrix or PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6. Furthermore, for example, the GAP program in the GCG software package (available from www.gcg.com), the NWSgapdna.CMP matrix, and gap weights 40, 50, 60, 70, or 80 and length weights 1, 2, 3, 4, 5, or 6 can be used to determine the identity ratio between two nucleotide sequences. A particularly preferred parameter set (and the parameter set to be used unless otherwise specified) is the Blossum62 score matrix using a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Furthermore, the percentage of identity between two amino acid or nucleotide sequences can be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) incorporated into the ALIGN program (version 2.0), using the PAM120 weighted remainder 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 this application may be further used as “search sequences” to perform searches against a common database to identify, for example, other family member sequences or related sequences. For example, such searches can be performed using 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 this application, a BLAST nucleotide search may be performed using the NBLAST program with a score of 100 and a word length of 12. To obtain amino acid sequences homologous to the protein molecules of this application, a BLAST protein search may be performed using the XBLAST program with a score of 50 and a word length of 3. To obtain gapped alignments for comparison purposes, gap BLAST may be used as described in Altschul et al., (1997) Nucleic Acids Res.25:3389-3402. When using BLAST and GapBLAST programs, you can use the default parameters of the corresponding programs (e.g., XBLAST and NBLAST). See www.ncbi.nlm.nih.gov.

[0064] In this specification, the term “chimeric antigen receptor (CAR)” refers to an artificial cell surface receptor expressed on immune effector cells and modified to specifically bind to an antigen, comprising at least (1) an extracellular antigen-binding domain, e.g., the heavy chain variable region and / or light chain variable region of an antibody; (2) a transmembrane domain that anchors the CAR to enter immune effector cells; and (3) an intracellular signaling domain. The CAR can utilize its 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] In this specification, the term “nucleic acid” includes any compound and / or substance of a polymer containing nucleotides. Each nucleotide consists of a base, in particular a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or livos), and a phosphate group. Typically, nucleic acid molecules are described by the sequence of bases, thereby representing the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually represented as 5'-3'. In this specification, the term nucleic acid molecule includes, for example, deoxyribose nucleic acids (DNA) containing complementary DNA (cDNA) and genomic DNA, livos nucleic acids (RNA), in particular 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 cyclic. The term nucleic acid molecule also includes both sense strands and antisense strands, and single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may include naturally occurring or unnaturally occurring nucleotides. Examples of unnaturally occurring nucleotides include modified nucleotide bases having derivatized sugars or phosphate backbone links or chemically modified residues. The nucleic acid molecules further encompass DNA and RNA molecules and are suitable as vectors for the direct expression of the antibodies of this application in vitro and / or in vivo, for example, 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, thereby allowing the mRNA to be injected into a subject to produce antibodies in the body (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP2101823B1).

[0066] In this specification, “isolated” nucleic acid refers to nucleic acid molecules isolated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules contained in the following cells, which typically contain such nucleic acid molecules, but which are located outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0067] In this specification, the term “vector” refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. The term includes vectors as self-replicating nucleic acid structures, and vectors that are integrated into the genome of a host cell into which they are introduced. Some vectors can guide the expression of nucleic acids to which they are manipulably ligated. Such vectors are referred to herein as “expression vectors.”

[0068] In this specification, “host cell” refers to a cell into which an exogenous nucleic acid has been introduced, and includes the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” which, without regard to passage number, include the primary transformed cell and its offspring. Offspring may not be exactly the same as the parent cell in terms of nucleic acid material and may include mutations. In this specification, this includes mutant offspring having the same function or biological activity as those screened or selected from the initially transformed cells.

[0069] In this specification, “drug composition” means a formulation in which the active ingredient contained herein exists in a form that allows for the effective biological activity of the active ingredient contained herein, and which does not contain any other ingredients that are unacceptably toxic to the subject to which the drug composition is administered.

[0070] In this specification, “pharmaceutically acceptable carriers” include any and all solvents, dispersions, coating materials, surfactants, antioxidants, preservatives (e.g., antimicrobial and antifungal agents), isotonic agents, absorption retarders, salts, antiseptics, drug stabilizers, adhesives, excipients, disintegrants, lubricants, sweeteners, fragrances, dyes, etc., and combinations thereof, which are known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, MackPrinting Company, 1990, pp. 1289–1329). The application of any conventional carrier in a therapeutic or drug composition is considered unless it is incompatible with the active ingredient.

[0071] In this specification, “treatment” means surgical or therapeutic treatment aimed at preventing or slowing (reducing) undesirable physiological changes or lesions in the subject of treatment, such as cancer and tumors. Beneficial or desirable clinical outcomes include, but are not limited to, symptom relief, disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete remission), whether detectable or undetectable. Subjects requiring treatment include those suffering from a disability or disease, those susceptible to a disability or disease, or those seeking prevention of a disability or disease. Where terms such as slowing, mitigation, reduction, mitigation, and remission are used, their meaning also includes removal, disappearance, and non-occurrence.

[0072] In this specification, the term “subject” refers to a living organism receiving treatment for a specific disease or disorder described herein. Exemplary examples include mammals receiving treatment for a disease or disorder, such as humans, primates (e.g., monkeys), or primate mammals.

[0073] In this specification, the term “effective dose” means a therapeutic dose that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or relieving a disease disorder or the progression of such disease. “Effective dose” further means a dose of a compound sufficient to relieve symptoms, for example, treat, cure, prevent or relieve an associated medical disorder, or increase the rate at which such disorders are treated, cured, prevented or relieved. When an active ingredient is administered alone to an individual, the therapeutic effective dose refers to that ingredient alone. When a combination is applied, the therapeutic effective dose refers to the combined dose of the active ingredients that leads to the therapeutic effect, whether in combination, sequentially, or concurrently.

[0074] In this specification, the term “cancer” refers to or describes a physiological condition typically characterized by uncontrolled cell growth in mammals. This definition includes benign and malignant cancers. In this specification, “tumor” or “surgery” refers to all neoplastic cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms “cancer” and “tumor” are not mutually exclusive when used herein.

[0075] In this specification, the term "EC50" refers to the half-maximal effective concentration, which includes the antibody concentration that induces an intermediate response between 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. [Modes for carrying out the invention]

[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 comprises a heavy chain variable region and a light chain variable region, (1) The light chain variable region includes LCDR1, LCDR2 and LCDR3, wherein LCDR1 has any one of the LCDR1 sequences shown below or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the aforementioned sequence, LCDR2 has any one of the LCDR2 sequences shown below or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the aforementioned sequence, and LCDR3 has any one of the LCDR3 sequences shown below or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the aforementioned sequence,

[0077] [Table 3] and, (2) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3, wherein HCDR1 has any one of the following HCDR1 sequences or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the aforementioned sequence; HCDR2 has any one of the following HCDR2 sequences or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the aforementioned sequence; and HCDR3 has any one of the following HCDR3 sequences or a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the aforementioned sequence.

[0078] [Table 4]

[0079] In a preferred embodiment, the antibody or its antigen-binding fragment comprises a sequence of six CDRs in combinations of light chain variable regions and heavy chain variable regions, namely 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 a sequence of six CDRs having one, two, three or more amino acid insertions, deletions and / or substitutions compared to the sequence of the six CDRs.

[0080] In one specific embodiment, this application provides an antibody or an antigen-binding fragment thereof, and here, (1) The light chain variable region sequence includes any one of the sequences shown in SEQ ID NO: 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 with the sequence. and, (2) The heavy chain variable region sequence includes any one of the sequences shown in SEQ ID NO: 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 with the sequence.

[0081] In a preferred embodiment, the antibody or its antigen-binding fragment has the following light chain variable region and heavy chain variable region, (1) The light chain variable region and the heavy chain variable region each include 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 each include the sequences shown in SEQ ID NO:16 and SEQ ID NO:15, (3) The light chain variable region and the heavy chain variable region each include the sequences shown in SEQ ID NO:18 and SEQ ID NO:17, (4) The light chain variable region and the heavy chain variable region each include the sequences shown in SEQ ID NO:20 and SEQ ID NO:19, (5) The light chain variable region and the heavy chain variable region each include 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 each include 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 each include the sequences shown in SEQ ID NO:26 and SEQ ID NO:25, (8) The light chain variable region and the heavy chain variable region each include the sequences shown in SEQ ID NO:28 and SEQ ID NO:27, respectively. (9) The light chain variable region includes the sequence shown in any one of SEQ ID NO: 118 to 120, and the heavy chain variable region includes the sequence shown in any one of SEQ ID NO: 121 to 125. (10) The light chain variable region includes the sequence shown in any one of SEQ ID NO: 130 to 131, and the heavy chain variable region includes the sequence shown in any one of SEQ ID NO: 132 to 137. (11) The light chain variable region includes the sequence shown in any one of SEQ ID NO: 144 to 145, and the heavy chain variable region includes the sequence shown in any one of SEQ ID NO: 146 to 152. (12) The light chain variable region includes the sequence shown in any one of SEQ ID NO: 160 to 163, and the heavy chain variable region includes the sequence shown in any one of SEQ ID NO: 164 to 166. (13) The light chain variable region includes the sequence shown in any one of SEQ ID NO: 172 to 175, and the heavy chain variable region includes the sequence shown in any one of SEQ ID NO: 176 to 178. (14) The light chain variable region includes the sequence shown in any one of SEQ ID NO: 184 to 187, and the heavy chain variable region includes the sequence shown in any one of SEQ ID NO: 188 to 190. (15) The light chain variable region includes the sequence shown in any one of SEQ ID NO: 196 to 197, and the heavy chain variable region includes the sequence shown in any one of SEQ ID NO: 198 to 201. (16) The light chain variable region includes the sequence shown in any one of SEQ ID NO:206~208, and the heavy chain variable region includes the sequence shown in any one of SEQ ID NO:209~212. (17) The light chain variable region includes the sequence shown in any one of SEQ ID NO:218~221, and the heavy chain variable region includes the sequence shown in SEQ ID NO:222. (18) The light chain variable region includes the sequence shown in any one of SEQ ID NO:231~233, and the heavy chain variable region includes the sequence shown in any one of SEQ ID NO:234~238. Or (19) the light chain variable region includes 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 includes 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.

[0082] In one specific embodiment, the antibody or its antigen-binding fragment is a chimeric, humanized, or entirely human-derived antibody.

[0083] In one specific embodiment, the antibody or its antigen-binding fragment can be bound to human or monkey GPRC5D.

[0084] In one specific embodiment, the antibody or its antigen-binding fragment comprises a constant region sequence of any one of the human or mouse antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD, preferably comprising a constant region sequence of human or mouse antibody IgG1, IgG2, IgG3, or IgG4, or comprising 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, and further comprising a therapeutic agent or tracer The therapeutic agent is conjugated, and preferably the therapeutic agent is selected from radioisotopes, chemotherapeutic agents or immunomodulators, and the tracer is selected from radiocontrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers, and more preferably the cytotoxic agent is selected from alkaloids, methotrexate, anthracycline antibiotics (doxorubicin), taxanes, pyrrolobenzodiazepines (PBD) or toxin compounds.

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

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

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

[0088] Preferably, the multispecific antigen-binding molecule may be bispecific, triplicate, or quadruplicate.

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

[0090] In a third aspect, the application further provides 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 a GPRC5D antibody or its antigen-binding fragment as described in any one of the preceding paragraphs, or the aforementioned multispecific antigen-binding molecule.

[0091] In a fourth aspect, the present application further provides immune effector cells, the immune effector cells expressing the aforementioned chimeric antigen receptor or comprising a nucleic acid fragment encoding the aforementioned chimeric antigen receptor, preferably the immune effector cells being selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells, or mast cells, the T cells being selected from cytotoxic T cells (CTLs), regulatory T cells, or helper T cells, and preferably the immune effector cells being autologous immune effector cells or allogeneic immune effector cells.

[0092] In a fifth aspect, the application further provides isolated nucleic acid fragments, the nucleic acid fragments encoding the aforementioned antibody or its antigen-binding fragment, a multispecific antigen-binding molecule, or a chimeric antigen receptor.

[0093] In a sixth aspect, the present application further provides a vector comprising the aforementioned nucleic acid fragment.

[0094] In a seventh aspect, the present application further provides a host cell comprising the aforementioned vector, preferably the cell being a prokaryotic or eukaryotic cell, such as a bacterium (Escherichia coli), a fungus (yeast), an insect cell, or a mammalian cell (CHO cell line or 293T cell line).

[0095] In an eighth aspect, the application further provides a method for producing the aforementioned antibody or its antigen-binding fragment or multispecific antigen-binding molecule, the method comprising culturing the aforementioned cells and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by the cells.

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

[0097] In a tenth aspect, the application further provides a pharmaceutical composition comprising the aforementioned antibody or its antigen-binding fragment, a multispecific antigen-binding molecule, an immunoeffector cell, a nucleic acid fragment, a vector, or a product manufactured according to the method described above, optionally further comprising a pharmaceutically acceptable carrier, diluent, or adjuvant, optionally further comprising an additional antitumor agent.

[0098] In some embodiments, the pharmaceutically acceptable carrier is a carrier that does not impair the viability and function of immune cells and does not affect the specific binding of antibodies or their antigen-binding fragments to antigens, and includes, but is not limited to, cell culture media, buffers, physiological saline, and equilibrium salt solutions. Examples of buffers include isotonic phosphates, acetates, citrates, borates, carbonates, etc. In a specific embodiment, the pharmaceutically acceptable carrier is a phosphate buffer containing 1% serum.

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

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

[0101] In a twelfth aspect, the present application further provides applications for the aforementioned antibodies or their antigen-binding fragments, multispecific antigen-binding molecules, immune effector cells, nucleic acid fragments, vectors, products manufactured according to the aforementioned methods, or pharmaceutical compositions in the manufacture of pharmaceuticals for the treatment of tumors or cancer. In a preferred embodiment, the tumor or cancer is a tumor or cancer expressing GPRC5D, preferably a B-cell lymphoma, and more preferably multiple myeloma (MM).

[0102] In a thirteenth aspect, the application further provides a kit comprising the aforementioned antibody or its antigen-binding fragment, a multispecific antigen-binding molecule, an immunoeffector cell, a nucleic acid fragment, a vector, a product manufactured according to the method described above, or a pharmaceutical composition.

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

[0104] In a fifteenth aspect, the present application further provides applications for the aforementioned antibody or its antigen-binding fragment in the production of GPRC5D detection reagents.

[0105] Examples The present application will be further described below with reference to specific examples, and the advantages and features of this application will become clear through this description. Unless specific conditions are specified in the examples, the procedures will be carried out according to conventional conditions or conditions suggested by the manufacturer. Unless the manufacturer is specified, the reagents or equipment used are all common products that can be purchased commercially.

[0106] The embodiments of this application are illustrative and do not limit the scope of this application. Those skilled in the art will understand that modifications or substitutions can be made to the details and forms of the technical invention of this application without departing from the spirit and scope of this application, and that any such modifications or substitutions will fall within the scope of protection of this application.

[0107] Example 1: Production of control antibody, identification of endogenous cells, and production of overexpression cell line 1.1 Production of control antibody The sequence of JNJ7564 was derived from international patent application WO 2018017786A2, and the sequence of 5F11 was derived from international patent application WO 2019154890A1. Cloned VH and VL sequences that recognize 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 shown in Table 1.

[0108] Plasmid and transfection reagent PEI (Polysciences, catalog number: 24765-1) were added to OPTI-MEM (Gibco, catalog number: 11058021) and homogeneously mixed. After standing for 15 minutes, the mixture was added to Expi293 cells (manufacturer: Thermofisher, catalog number: A14527) and cultured in a shaker at 5% CO2, 120 rpm, and 37°C. The day after transfection, OPM-293 ProFeed (Shanghai Aopura Mai, catalog number: F081918-001) and 6 g / L glucose (manufacturer: Sigma, catalog number: G7528) were added. On day 6 after transfection, the cell supernatant was collected.

[0109] The sample was filtered through a 0.22 μm disposable filter, then the system was disinfected with 0.5 M NaOH for 30 minutes, and the chromatography column was disinfected with 0.1 M NaOH for 5 minutes. Each channel of the pure150 is filled with the corresponding buffer solution, in order: A2 is filled with Mab affinity rinse solution (anhydrous disodium hydrogen phosphate 2.69 g / L (No. 20040618, Sinopharm Chemical Reagents Co., Ltd.), sodium dihydrogen phosphate dihydrate 0.17 g / L (No. 20040718, Sinopharm Chemical Reagents Co., Ltd.), sodium chloride 58.44 g / L (No. 10019318, Sinopharm Chemical Reagents Co., Ltd.)), B1 is filled with Mab affinity eluate (citric acid monohydrate 3.92 g / L (No. 10007118, Sinopharm Chemical Reagents Co., Ltd.), trisodium citrate dihydrate 1.88 g / L (No. 10019418, Sinopharm Chemical Reagents Co., Ltd.)), and A1 is filled with Mab affinity equilibrium solution (20 × PBS). Add ddH2O to Buffer and dilute to 1×PBS (item number B548117-050, Bioengineering Shanghai Co., Ltd.), fill the Buffer pump with Mab affinity equilibrium solution (20×PBS), and fill the Buffer pump with Mab affinity equilibrium solution (20×PBS). Dilute Buffer with ddH2O to 1×PBS (No. B548117-050, Bioengineering Shanghai Co., Ltd.) and fill the container with it. Inject the sample into S1, add the sample at a flow rate of 5 mL / min, and after the sample addition is complete, use the buffer pump to perform wash1 (Mab affinity equilibrium solution) until the UV level drops below 10 mAU. Switch the inlet to A2 and perform wash2 (Mab affinity rinse solution) with a high-salt solution to remove impurities. Switch the inlet to A1 and replace the high-salt solution in the flow path until the electrical conductivity drops to approximately 20 ms / cm. Switch the inlet to B1 to elute the target protein, collect the target peak, add 1 M Tris (121.2 g / L, pH 8.0) to adjust the pH to neutral, measure the concentration using Nano Drop 8000 and wait.

[0110] [Table 5]

[0111] 1.2 Identification of cell lines that endogenously express human GPRC5D protein Cells endogenously expressing human GPRC5D protein were cultured in T-75 cell culture flasks until the logarithmic growth phase, then centrifuged, the supernatant was discarded, and the cell pellet was washed twice with PBS. 20 nM JNJ7564-hIgG1 and 5F11-hIgG1 antibodies were used as primary antibodies, and Alexa Fluorine was used as a secondary antibody. (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 Canto™, purchased from BD). The results are shown in Table 2 and Figure 1, indicating that NCI-H929, MolP-8, and RPMI-8226 cells all endogenously express human GPRC5D protein, and that the JNJ7564-hIgG1 and 5F11-hIgG1 control antibodies have binding activity to all cells.

[0112] [Table 6]

[0113] 1.3 Production of CHO-K1 recombinant cell lines expressing human GPRC5A, GPRC5B, GPRC5C, and GPRC5D proteins The nucleotide sequences encoding the amino acid sequences of humanGPRC5A (Uniprot:Q8NFJ5-1, SEQ ID NO:8), humanGPRC5B (Uniprot:Q9NZH0-1, SEQ ID NO:9), humanGPRC5C (Uniprot:Q9NQ84, SEQ ID NO:10), and humanGPRC5D (Uniprot:Q9NZD1, SEQ ID NO:11) were cloned into pLVX lentiviral vectors, and viral particles were produced in HEK293T cells.CHOK1 cell lines (purchased from the Chinese Academy of Sciences) were infected with lentivirus, and then 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) with 10 μg / ml puromycin (Gibco, catalog number A1113803). The cells were then subjected to the following treatments: humanGPRC5A Alexa Fluor 488-conjugated antibody (R&D systems, catalog number IC5239G-100UG), humanGPRC5B Alexa Fluor 488-conjugated antibody (R&D systems, catalog number FAB10253G-100UG), and humanGPRC5C Alexa Fluor 488-conjugated antibody (R&D CHOK1 cells, transfected with humanGPRC5D, were stained with human anti-human GPRC5D antibody (JNJ7564, proprietary) and goat anti-human IgG(H+L) antibody (Jackson, catalog number: 109605088). Subsequently, the cells were stained using a FACS Aria III flow cytometer (BD). High-expression positive cell populations (purchased from Biosciences) were sorted into 96-well plates and cultured at 37°C in 5% (v / v) CO2. After approximately two weeks, a portion of the cells were selected and amplified. A positive cell population with good growth, high fluorescence intensity, and high uniformity was selected and continued to be cultured, then cryopreserved in liquid nitrogen. The results of the expression level identification are shown in Table 3 and Figures 2A-2D. The results indicate that CHOK1 stable transfection cell lines expressing human GPRC5A, GPRC5B, GPRC5C, and GPRC5D, which underwent pressurized screening using puromycin, have a relatively single positive peak and can be used for FACS detection of the binding activity between antibodies and human GPRC5A, GPRC5B, GPRC5C, and GPRC5D proteins.

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

[0115] 1.4 Production of HEK293T recombinant cell lines expressing human GPRC5D protein For specific procedures, please refer to Example 1.3. HEK293T cell lines were infected with a lentivirus packaging a human GPRC5D plasmid vector, and selectively cultured for 2 weeks in DMEM Medium (Gibco, catalog number 10569044) containing 10% (v / v) fetal bovine serum (ExCell Bio, catalog number FND500) with 2 μg / ml puromycin (Gibco, catalog number A1113803). Human anti-human GPRC5D antibody (JNJ7564, proprietary) and goat anti-human IgG(H+L) antibody (Jackson, catalog number: 109605088) were used in a flow cytometer FACS CantoII (BD). The results of identifying the expression levels of cell lines that were detected and showed good expression (purchased from Biosciences) are shown in Table 4 and Figure 3. HEK293T-hGPRC5D, which underwent pressurized screening using puromycin, had a relatively single positive peak, indicating that it can be used for FACS detection of the binding activity between the antibody and the human GPRC5D protein.

[0116] [Table 8]

[0117] 1.5 Production of CHO-K1 and HEK293T recombinant cell lines expressing monkey GPRC5D protein For specific details on the production of a CHO-K1 recombinant cell line that stably expresses monkey GPRC5D (cynoGPRC5D), please refer to Example 1.3. The amino acid sequence of cynoGPRC5D is NCBI: XP_005570249.1 (SEQ ID NO: 12). Using a human anti-cynoGPRC5D antibody (JNJ7564, proprietary) and a goat anti-human IgG(H+L) antibody (Jackson, catalog number: 109605088), the expression levels of cell lines showing good expression were detected using a FACS Canto II flow cytometer (purchased from BD Biosciences). The results are shown in Table 5 and Figure 4A. The results indicate that CHOK1-cynoGPRC5D, which underwent pressurized screening using puromycin, had a relatively single positive peak, suggesting that it can be used for FACS detection of cross-activity between the antibody and the monkey GPRC5D protein.

[0118] [Table 9]

[0119] For specific details on the production of a HEK293T recombinant cell line that stably expresses monkey GPRC5D (cynoGPRC5D), please refer to Example 1.4. The results of identifying the expression levels of cell lines showing good expression are shown in Table 6 and Figure 4B. The results indicate that HEK293T-cynoGPRC5D, which underwent pressurized screening using puromycin, had a relatively single positive peak and can be used for FACS detection of cross-activity between the antibody and the monkey GPRC5D protein.

[0120] [Table 10] Full-length amino acid sequence of monkey GPRC5D (NCBI:XP_005570249.1, SEQ ID NO:12): [ka]

[0121] 1.6 Binding experiment between recombinant cell lines and control antibodies FACS detection of the binding activity of the control antibody to cells expressing human GPRC5D and monkey GPRC5D is shown in Figures 5A-5C, with human IgG1 being the IgG subtype control. JNJ7564-hIgG1 and 5F11-hIgG1 showed good binding activity to NCI-H929 tumor cells expressing human GPRC5D protein, as well as to HEK293T-hGPRC5D and HEK293T-cynoGPRC5D recombinant cells. 5F11-hIgG1 showed relatively high binding activity to NCI-H929 cells and HEK293T-hGPRC5D recombinant cells, while JNJ7564-hIgG1 showed relatively high binding activity to HEK293T-cynoGPRC5D recombinant cells.

[0122] Example 2: Production of anti-human GPRC5D hybridoma monoclonal antibody 2.1 Animal immunity Anti-human GPRC5D monoclonal antibodies were produced by immunization of mice. Six-to-eight-week-old female SJL mice (purchased from Shanghai SJL Laboratory Animals Co., Ltd.) were used in the experiment, and the mice were cultured under SPF conditions. After purchase, the mice were housed in a laboratory environment for one week, adjusted to a 12 / 12-hour light / dark cycle, with a temperature of 20-25°C and humidity of 40-60%. The mice adapted to the environment were immunized according to the following regimen. For the initial immunization, a human GPRC5D overexpressing cell line (HEK293T-hGPRC5D, in-house) was washed twice with PBS and then mixed with oligonucleotide CpG (ODN 1826, synthesized from Shanghai Biotechnology). First, 50 μl of emulsified Titer max (purchased from Sigma, Cat.T2684) was intraperitoneally injected into each mouse, and after a 15-minute wait, 5 × 10¹⁶ IVs were administered to each mouse. 6 Cells were injected intraperitoneally at a dose of 100 μl / 100. For the initial booster immunization, human GPRC5D overexpressing cell lines were washed twice with PBS, then mixed with CpG, and 5 × 10⁶ cells were administered to each mouse. 6Cells were injected intraperitoneally at a dose of 100 μl / cell. Subsequent booster immunizations were performed alternately, similar to the initial immunization and the first booster immunization, using alternately monkey GPRC5D overexpressing cell lines (HEK293T-cynoGPRC5D, in-house) and human GPRC5D overexpressing cell lines (HEK293T-hGPRC5D) as immunogens. With a 7-day interval between each immunization, blood was collected from mice on day 5 after the second and fourth booster immunizations, serum was separated, and the titer of specific antibodies in the serum was measured using a cell-based enzyme-linked immunosorbent assay (ELISA).

[0123] To detect the titer of specific antibodies in serum, the HEK293T-hGPRC5D, HEK293T-cynoGPRC5D, CHOK1-hGPRC5D, CHOK1-cynoGPRC5D recombinant cells and blank control cells obtained in Example 1 were used in 4 × 10⁶ wells. 4Cells were inoculated into a 96-well cell plate (manufacturer: Corning, catalog number: 3599), incubated overnight in a cell incubator, the supernatant was discarded, 50 μl of fixative (manufacturer: Beyotime, catalog number: P0098-500ML) was added per well, the plate was left in a fume hood at room temperature for 30 minutes, the supernatant fixative was discarded, the plate was washed twice with PBST, then 0.5% skimmed milk (manufacturer: Seikou, catalog number: A600669-0250) was added, the plate was blocked at room temperature for 2 hours, the mounting medium was discarded, the plate was washed twice with PBST, and 50 μl / well of mouse serum, which was initially diluted to a dilution of 1:100 and diluted 3-fold, was added. After incubation at room temperature for 1 hour, the plate was washed three times with PBST. A secondary antibody labeled with HRP (horseradish peroxidase) (purchased from Jackson, catalog number: 109-035-088) was added, incubated at room temperature for 1 hour, and then the plate was washed 5 times with PBST. 50 μl / well of TMB substrate was added, incubated at room temperature for 5-10 minutes, and then 50 μl / well of stop solution (1.0N HCl) was added. The OD450nm value was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer), and according to the cell-based ELISA detection results, the post-immunization serum of mice immunized with the above cells all showed varying degrees of binding to the immunogen, indicating antigen-antibody reactions.

[0124] 2.2 Splenocyte Fusion and Hybridoma Screening 5x10 for each selected mouse 6A number of HEK293T-hGPRC5D cells were intraperitoneally injected into the mice, and after 3 days the mice were sacrificed and splenocytes and lymphocytes were collected. After centrifugation at 1500 rpm, the supernatant was discarded, and ACK lysate (Gibco, Cat. A1049201) was added to the cells to lyse the doped erythrocytes and obtain a cell suspension. The cells were washed three times at 1500 rpm with DMEM basal medium (purchased from Gibco, catalog number 10569044), the cells were counted, and the viable cells were mixed with mouse myelocyte aneurysm cells SP2 / 0 (purchased from ATCC, Cat. CRL-1581) in a 2:1 ratio, and cell fusion was performed using electrofusion. 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 Yearson, Cat. 40107ES25), and NEAA (purchased from Gibco, Cat. 11140050), and then condensed into 5×10⁶ cells. 4 Cells were added to a 96-well cell culture plate with cells per 200 μL well and cultured in an incubator at 5% (v / v) CO2 and 37°C. After 7 days, the fusion plate supernatant was screened using cell-based ELISA to confirm binding activity to human GPRC5D overexpressing cells. Binding activity to monkey GPRC5D overexpressing cells was confirmed using cell-based ELISA on the positive clonal supernatant, and binding activity to endogenous cells NCI-H929 was confirmed using FACS.

[0125] Based on the screening results, positive clones that met the criteria were selected, and subcloning was performed 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, amplified in 24-well plates, and cultured further. After 3 days, the culture supernatant was further detected, and its binding activity to 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 this optimal clone was expanded in DMEM medium containing 10% (v / v) FBS under conditions of 37°C and 5% (v / v) CO2. By cryopreserving in liquid nitrogen, the hybridoma cells of this application were obtained.

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

[0127] [Table 11]

[0128] The CDRs of the sequences in Table 7 were split 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 Tongyong Biosystems (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 GGGGSGGGGSGGGGS (SEQ ID NO: 117), and cloned them into an 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 antibody 4.1 Detection of binding of chimeric antibodies to human GPRC5D protein by cell-based enzyme-linked immunosorbent assay (ELISA) Please refer to Example 2.1 for the specific detection method. The CHOK1-hGPRC5D, CHOK1-cynoGPRC5D recombinant cells and CHOK1 cells obtained in Example 1 were fixed, and chimeric antibodies and control antibodies with an initial concentration of 100 nM and diluted in a 3-fold gradient were added at 50 μl / well for detection. The cell-based ELISA detection results of the binding activity between the chimeric antibody and the human GPRC5D protein were as shown in Figures 6A-6C, 7A-7C, and 8A-8C. The results showed that the purified and produced chimeric antibody binds to the human GPRC5D protein at a different level at the ELISA level. Here, the negative control antibody hIgG1 is an antibody anti-hel-hIgG1 against hen egg lysozyme (purchased from Beyotime, catalog number: B117901), and the data in the figure were OD450nm values.

[0132] 4.2 Detection of the binding between the chimeric antibody and endogenous cells by flow cytometry (FACS) The required cells were expanded in a T-75 cell culture flask until the logarithmic growth phase. NCI-H929, MolP-8, and RPMI-8226 cells were taken, washed twice with PBS buffer, and after cell counting, the cell pellet was resuspended in [PBS + 2% (w / v) BSA] blocking solution to 6 2 × 10 cells / ml, added to a 96-well FACS reaction plate at 50 μl / well, the detection target sample of the chimeric antibody was added at 50 μl / well, and incubated on ice for 1 hour. Centrifuged and washed three times with PBS buffer, and 50 μl / well of Alexa Fluor (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 washed by centrifugation three times with PBS buffer and detected by FACS (FACS CantoII, purchased from BD), and the results were analyzed. The data was analyzed using software (Flowjo) to obtain the mean fluorescence intensity (MFI) of the cells. Next, the data was analyzed using software (GraphPad Prism8), and the analysis results are shown in Table 9 and Figures 9A-9C, 10A-10C, and 11A-11C. The chimeric antibodies all 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 the binding activity of chimeric antibodies to CHOK1-hGPRC5A, CHOK1-hGPRC5B, and CHOK1-hGPRC5C by flow cytometry (FACS) For specific methods, please refer to Example 4.2. CHOK1-hGPRC5A, CHOK1-hGPRC5B, and CHOK1-hGPRC5C cells were taken, and the results are shown in Table 10. The results indicate that none of the chimeric antibodies bound to CHOK1-hGPRC5A, CHOK1-hGPRC5B, or CHOK1-hGPRC5C cells, and that they did not cross-bind with these subtypes.

[0135] [Table 14]

[0136] 4.4 Identification of competitive strength between chimeric antibodies and the positive control antibody 5F11 To identify the binding ability of chimeric antibodies to antigens, competitive binding of GPRC5D chimeric antibodies to the antigen with the positive control molecule 5F11 was analyzed using a competitive ELISA method. The positive molecule 5F11 was biotin-labeled, and ELISA plates were coated with 2 μg / mL of hGPRC5D-LVP protein (manufacturer: Kactus Biosystems, catalog number: GPR-HM05P). Biotin-5F11 antibody was added starting at 100 nM, followed by 3-fold gradient dilution. 450 The values ​​were measured, a curve was fitted using GraphPad Prism software, and the EC80 concentration was calculated to serve as the reference concentration in competitive ELISA.

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

[0138] [Table 15]

[0139] Example 5 Humanization of antibodies derived from GPRC5D mice 5.1 Humanization of GPRC5D-mab01 By aligning the germline gene databank of human antibody heavy and light chain variable regions from IMGT (http: / / imgt.cines.fr), germline genes of the heavy and light chain variable regions, which have high homology to mouse-derived antibodies, were selected as templates. The CDRs of mouse-derived antibodies were transplanted into the corresponding human templates, forming the variable region sequences FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in order. Where necessary, reverse mutations were performed on key amino acids in the skeletal sequence (FR region sequence) to form amino acids corresponding to the mouse-derived antibody, ensuring the original affinity. In addition, since antibodies have sites that are prone to chemical modification, point mutations were already performed on these sites to eliminate the risk of modification, thereby obtaining humanized monoclonal antibodies. Here, the CDR amino acid residues of the antibodies were determined and annotated using the Kabat numbering system.

[0140] The humanized light chain templates for 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 are shown in Table 12.

[0141] [Table 16]

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

[0143] This application describes how to select and combine different light and heavy chain sequences from mutation designs in the light and heavy chain variable regions of the humanized antibody GPRC5D-mab01 described above, ultimately obtaining various GPRC5D-mab01 humanized antibodies. The variable region amino acid sequences of each antibody are shown in Table 13 below:

[0144] [Table 17]

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

[0146] [Table 18]

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

[0148] [Table 19]

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

[0150] This application describes how to obtain various GPRC5D-mab02 humanized antibodies by selecting and combining different light and heavy chain sequences from mutation designs of the light and heavy chain variable regions of the GPRC5D-mab02 humanized antibody, and the variable region amino acid sequences of each antibody are shown in Table 16 below:

[0151] [Table 20]

[0152] According to the Kabat numbering system, the analysis results of the 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 that in 5.1. The humanized light chain templates of the 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 humanized GPRC5D-mab03 antibody are as follows: The amino acid sequence of GPRC5D-mab03.VL1 is as shown in SEQ ID NO:144:

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

[0157] This application describes how to select and combine different light and heavy chain sequences from mutation designs of the light and heavy chain variable regions of the humanized antibody GPRC5D-mab03 described above, ultimately obtaining various GPRC5D-mab03 humanized antibodies. The variable region amino acid sequences of each antibody are shown in Table 19 below:

[0158] [Table 23]

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

[0160] [Table 24]

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

[0162] [Table 25]

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

[0164] This application describes how to select and combine different light and heavy chain sequences from mutation designs of the light and heavy chain variable regions of the humanized antibody GPRC5D-mab04 described above, ultimately obtaining various GPRC5D-mab04 humanized antibodies. The variable region amino acid sequences of each antibody are shown in Table 22 below:

[0165] [Table 26]

[0166] According to the Kabat numbering system, the analysis results of the 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, with the humanized light chain templates for the mouse-derived antibody GPRC5D-mab05 being IGKV6-21*01 / IGKV3-11*01 and IGKJ4*01, and the humanized heavy chain templates being IGHV1-69*02 and IGHJ6*01. The specific mutation designs are shown in Table 24.

[0169] [Table 28]

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

[0171] This application describes how to select and combine different light and heavy chain sequences from mutation designs of the light and heavy chain variable regions of the humanized antibody GPRC5D-mab05 described above, ultimately obtaining various GPRC5D-mab05 humanized antibodies. The variable region amino acid sequences of each antibody are shown in Table 25 below:

[0172] [Table 29]

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

[0174] [Table 30]

[0175] 5.6 GPRC5D-mab06 Humanization 5.6.1 Humanized Design for the First Round of GPRC5D-mab06 The method was the same as in 5.1, with the humanized light chain templates for the mouse-derived antibody GPRC5D-mab06 being IGKV4-1*01 / IGKV1-27*01 and IGKJ2*01, and the humanized heavy chain templates being IGHV2-26*01 and IGHJ6*01. The specific mutation designs are shown in Table 27.

[0176] [Table 31]

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

[0178] This application involves selecting and combining different light and heavy chain sequences from the first round of mutation designs for the humanized antibody light and heavy chain variable regions of GPRC5D-mab06, ultimately obtaining various GPRC5D-mab06 humanized antibodies. The variable region amino acid sequences of each antibody are shown in Table 28.

[0179] [Table 32]

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

[0181] [Table 33]

[0182] 5.6.2 Humanized Design for the Second Round of GPRC5D-mab06 The method was the same as in 5.1, with the second round of humanized light chain templates for the mouse-derived antibody GPRC5D-mab06 being IGKV1-27*01 and IGKJ2*01, and the second round of humanized heavy chain templates being IGHV2-26*01 and IGHJ6*01. The specific mutation designs are shown in Table 30.

[0183] [Table 34]

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

[0185] This application involves selecting and combining different light and heavy chain sequences from the second round of mutation designs for the humanized antibody light and heavy chain variable regions of GPRC5D-mab06, ultimately obtaining various GPRC5D-mab06 humanized antibodies. The variable region amino acid sequences of each antibody are shown in Table 31.

[0186] [Table 35]

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

[0188] [Table 36]

[0189] 5.7 GPRC5D-mab07 Humanization 5.7.1 Humanized Design for the First Round of GPRC5D-mab07 The method was the same as in 5.1, with the humanized light chain templates for the mouse-derived antibody GPRC5D-mab07 being IGKV2-40*01 and IGKJ4*01, and the humanized heavy chain templates being IGHV1-69*02 and IGHJ6*01. The specific mutation designs are shown in Table 33.

[0190] [Table 37]

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

[0192] This application involves selecting and combining different light and heavy chain sequences from the first round of humanized antibody light and heavy chain variable region designs of GPRC5D-mab07 described above, ultimately obtaining various GPRC5D-mab07 humanized antibodies. The variable region amino acid sequences of each antibody are shown in Table 34.

[0193] [Table 38]

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

[0195] [Table 39]

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

[0197] [Table 40]

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

[0199] This application involves selecting and combining different light and heavy chain sequences from the second round of mutation designs for the humanized antibody light and heavy chain variable regions of GPRC5D-mab07, ultimately obtaining various GPRC5D-mab07 humanized antibodies. The variable region amino acid sequences of each antibody are shown in Table 37.

[0200] [Table 41]

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

[0202] [Table 42]

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

[0204] [Table 43]

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

[0206] This application describes how to select and combine different light and heavy chain sequences from mutation designs of the light and heavy chain variable regions of the humanized antibody GPRC5D-mab08 described above, ultimately obtaining various GPRC5D-mab08 humanized antibodies. The variable region amino acid sequences of each antibody are shown in Table 40.

[0207] [Table 44]

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

[0209] [Table 45]

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

[0211] 6.2 Detection of the binding activity of humanized antibodies to NCI-H929 cells endogenously expressing human GPRC5D by flow cytometry (FACS) To detect the binding activity of the GPRC5D humanized antibody to the human GPRC5D full-length protein, the binding of the humanized antibody to the human GPRC5D protein was detected using a detection method similar to that used in FACS in Example 4. The detection results are shown in Figures 15A to 15J. The results indicate that the humanized antibodies were able to bind to NCI-H929 cells to varying degrees, demonstrating good specificity.

Claims

1. 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 comprises a heavy chain variable region and a light chain variable region, where, (1) The light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein LCDR1 consists of the sequence shown in SEQ ID NO: 79, LCDR2 consists of the sequence shown in SEQ ID NO: 80, and LCDR3 consists of the sequence shown in SEQ ID NO:

81. and, (2) An antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 consists of the sequence shown in SEQ ID NO: 73, HCDR2 consists of the sequence shown in SEQ ID NO: 74, and HCDR3 consists of the sequence shown in SEQ ID NO:

75.

2. The antibody or its antigen-binding fragment has the following light chain variable region and heavy chain variable region, (1) The light chain variable region and the heavy chain variable region each include the sequences shown in SEQ ID NO: 175 and SEQ ID NO: 178, Or (2) the antibody or antigen-binding fragment according to claim 1, wherein the light chain variable region includes a sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the light chain variable region shown in (1) above, and the heavy chain variable region includes a sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the heavy chain variable region shown in (1) above.

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

4. An antibody or antigen-binding fragment thereof according to claim 1 or 2, which can bind to human or monkey GPRC5D.

5. The antigen-binding fragment is F(ab) ２ An antibody or antigen-binding fragment thereof according to claim 1 or 2, selected from one or more of Fab', Fab, Fv, or scFv.

6. The antibody or antigen-binding fragment according to claim 1 or 2, comprising a constant region sequence of a human or mouse antibody IgG1, IgG2, IgG3, or IgG4, or comprising a sequence having 90%, 95%, 96%, 97%, 98%, 99% or higher identity with a constant region sequence of a human or mouse antibody IgG1, IgG2, IgG3, or IgG4, and further comprising a therapeutic agent or tracer conjugated to the antibody or antigen-binding fragment thereof.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the therapeutic agent is selected from a radioisotope, a chemotherapeutic agent, or an immunomodulator, and the tracer is selected from a radiocontrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasonic contrast agent, and a photosensitizer.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the chemotherapeutic agent is selected from alkaloids, methotrexate, anthracycline antibiotics, taxanes, pyrrolobenzodiazepines, or toxin compounds.

9. A multispecific antigen-binding molecule comprising an antibody or antigen-binding fragment thereof as described in claim 1 or 2, and another antigen-binding molecule that binds to an antigen other than GPRC5D, or to a GPRC5D epitope different from the antibody or antigen-binding fragment described in claim 1 or 2.

10. The other antigen-binding molecule is an antibody or an antigen-binding fragment thereof, The multispecific antigen-binding molecule may be bispecific, triplicate, or quadruplicate. Furthermore, the multispecific antigen-binding molecule according to claim 9 may be divalent, trivalent, tetravalent, pentavalent, or hexavalent.

11. A chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises at least a signal peptide, an extracellular antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signal transduction domain, the extracellular antigen-binding domain comprising the GPRC5D antibody described in claim 1 or an antigen-binding fragment thereof.

12. An immune effector cell, wherein the immune effector cell expresses the chimeric antigen receptor described in claim 11, or comprises a nucleic acid fragment encoding the chimeric antigen receptor described in claim 11.

13. The immune effector cell according to claim 12, wherein the immune effector cell is selected from T cells, NK cells, NKT cells, DNT cells, monocytes, macrophages, dendritic cells, or mast cells.

14. The immune effector cell according to claim 13, wherein the T cell is selected from cytotoxic T cells (CTLs), regulatory T cells, or helper T cells.

15. The immune effector cell according to claim 12, wherein the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell.

16. An isolated nucleic acid fragment, wherein the nucleic acid fragment encodes the antibody or its antigen-binding fragment as described in claim 1.

17. A vector comprising the nucleic acid fragment described in claim 16.

18. A host cell, wherein the host cell contains the vector described in claim 17, and the cell is a prokaryotic cell or a eukaryotic cell.

19. The host cell according to claim 18, wherein the cell is a bacterium, fungus, insect cell, or mammalian cell.

20. A method for producing an antibody or an antigen-binding fragment thereof according to claim 1 or 2, the method comprising culturing the cells according to claim 18 and isolating an antibody, an antigen-binding fragment, or a multispecific antigen-binding molecule expressed by the cells.

21. A method for producing the aforementioned immune effector cells, the method comprising introducing a nucleic acid fragment encoding the CAR described in claim 11 into the immune effector cells.

22. The method according to claim 21, further comprising priming the immune effector cells to express the CAR described in claim 11.

23. A pharmaceutical composition, The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof as described in claim 1 or 2; The invention comprises a multispecific antigen-binding molecule that includes the antibody or antigen-binding fragment thereof described in claim 1 or 2, and another antigen-binding molecule that binds to an antigen other than GPRC5D, or to a GPRC5D epitope different from the antibody or antigen-binding fragment described in claim 1 or 2; Alternatively, a pharmaceutical composition comprising 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 signal transduction domain, wherein the extracellular antigen-binding domain comprises a chimeric antigen receptor (CAR) comprising the GPRC5D antibody or its antigen-binding fragment as described in claim 1 or 2.

24. The pharmaceutical composition according to claim 23, further comprising a pharmaceutically acceptable carrier, diluent or auxiliary agent.

25. The pharmaceutical composition according to claim 23, further comprising an additional antitumor agent.

26. An antibody or antigen-binding fragment thereof according to claim 1 or 2, for treating a tumor or cancer, The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the tumor or cancer is a tumor or cancer expressing GPRC5D.

27. ​​The antibody or antigen-binding fragment thereof according to claim 26, wherein the tumor or cancer is a B-cell lymphoma.

28. The antibody or antigen-binding fragment thereof according to claim 26, wherein the tumor or cancer is multiple myeloma (MM).

29. A kit comprising the antibody or antigen-binding fragment thereof according to claim 1 or 2.

30. A method for detecting GPRC5D expression in a biological sample, the method comprising contacting the biological sample with the antibody or antigen-binding fragment described in claim 1 or 2 under conditions that allow for the formation of a complex between the antibody or antigen-binding fragment described therein and GPRC5D.

31. The method according to claim 30, further comprising detecting the formation of the complex and indicating the presence or expression level of GPRC5D in the sample.

Images