Antibodies that bind to GPRC5D and uses thereof

Abstract

Provided are antibodies that bind to GPRC5D and uses thereof. Specifically, provided are anti-GPRC5D antibodies and antigen-binding fragments thereof, nucleic acids encoding them, vectors containing the nucleic acids, cells containing the vectors, and pharmaceutical compositions containing them, as well as uses thereof in reducing tumors or inhibiting tumor cell proliferation, treating diseases, and the like.

Description

[Technical field]

[0001] The present disclosure relates to antibodies, particularly antibodies that bind to GPRC5D, methods of preparation and uses. [Background technology]

[0002] Multiple myeloma (MM) is a type of plasma cell malignancy characterized by the abnormal proliferation of bone marrow-derived plasma cells, which causes MM patients to experience many disease-related symptoms, such as bone necrosis, bone marrow infiltration, renal failure, and immunodeficiency. Currently, treatment options for multiple myeloma include proteasome inhibitors, immunomodulatory agents, monoclonal antibodies, and stem cell transplantation.

[0003] GPRC5D (G protein-coupled receptor class C group 5 member D) belongs to the retinoic acid-inducible orphan G protein-coupled receptor (RAIG) family and is a seven-transmembrane protein consisting of 345 amino acid residues. Its normal physiological function is related to the structure of sclerokeratin, but currently, the specific biological function and ligand of GPRC5D have not yet been clarified. Some studies have shown that the expression threshold of GPRC5D is more than 50% in malignant bone marrow-derived plasma cells of 65% of multiple myeloma patients and its expression is independent of BCMA (B cell maturation antigen), while other studies have shown that overexpression of GPRC5D is associated with tumor burden and poor prognosis in multiple myeloma patients. GPRC5D is not expressed at all or has very low expression in normal tissues, except for hair follicle tissue of the skin and bone marrow-derived plasma cells, which may provide a new treatment for multiple myeloma patients and its clinical value is significant.

[0004] Although several antibodies targeting GPRC5D as a potential therapeutic target are already in development, they are still limited and more available options are needed. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure provides antibodies that bind to GPRC5D, as well as related nucleic acids capable of encoding the provided antibodies, vectors, cells, compositions, methods of preparation and uses. [Means for solving the problem]

[0006] In one aspect, the disclosure provides an isolated anti-GPRC5D antibody or antigen-binding fragment thereof, the anti-GPRC5D antibody comprising: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, 9, 17, 25 or 33; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, 10, 18, 26 or 34, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, 11, 19, 27 or 35 and a heavy chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, 12, 20, 28 or 36; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, 13, 21, 29 or 37, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, 14, 22, 30 or 38 a light chain variable region comprising Includes.

[0007] In one embodiment of the disclosure, the anti-GPRC5D antibody is (i) HCDR1 comprising the amino acid sequence shown in SEQ ID NO:1; HCDR2 comprising the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:3 and a heavy chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO:4; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:6 and a light chain variable region comprising (ii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 9; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:11 and a heavy chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 12; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:14 and a light chain variable region comprising (iii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 17; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:19 and a heavy chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:22 and a light chain variable region comprising (iv) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:27 and a heavy chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 28; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:29, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:30 or a light chain variable region comprising (v) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:35 and a heavy chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 36; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:38 and a light chain variable region comprising:

[0008] In one aspect, the disclosure provides an isolated anti-GPRC5D antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO: 7, 15, 23, 31 or 39, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO: 8, 16, 24, 32 or 40.

[0009] In one aspect, the disclosure provides a fusion protein comprising an anti-GPRC5D antibody or antigen-binding fragment thereof described herein.

[0010] In one aspect, the disclosure provides a pharmaceutical composition comprising an anti-GPRC5D antibody or antigen-binding fragment thereof, or a fusion protein described herein, and further comprising a pharma- ceutically acceptable carrier.

[0011] In one aspect, the disclosure provides an isolated nucleic acid encoding an anti-GPRC5D antibody or antigen-binding fragment thereof described herein.

[0012] In one aspect, the disclosure provides a vector comprising a nucleic acid described herein.

[0013] In one aspect, the disclosure provides a host cell comprising a vector described herein or having a nucleic acid described herein integrated into its genome.

[0014] In one aspect, the disclosure provides a method for preparing an anti-GPRC5D antibody or antigen-binding fragment thereof described herein, the method comprising culturing a host cell and recovering the anti-GPRC5D antibody or antigen-binding fragment thereof from the host cell or host cell medium.

[0015] In one aspect, the disclosure provides the use of an anti-GPRC5D antibody or antigen-binding fragment thereof or a fusion protein as described above in the preparation of a medicament for treating a disease, preferably wherein said disease is cancer or an autoimmune disease.

[0016] In one aspect, the present disclosure provides a method for reducing a tumor or inhibiting tumor cell proliferation in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-GPRC5D antibody or antigen-binding fragment thereof, or a fusion protein or pharmaceutical composition described herein.

[0017] In one aspect, the present disclosure provides a method for treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-GPRC5D antibody or an antigen-binding fragment thereof or a fusion protein or pharmaceutical composition described herein, preferably wherein the disease is cancer or an autoimmune disease.

[0018] In one aspect, the disclosure provides a kit comprising an anti-GPRC5D antibody or antigen-binding fragment thereof, or an immunoconjugate or pharmaceutical composition described herein. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows OD450 measured by ELISA for some mouse anti-hGPRC5D antibodies and CHO-K1-hGPRC5Dhi, CHO-K1-cynoGPRC5D (CHO-K1-monkey GPRC5D), and CHO-K1-murineGPRC5D (CHO-K1-mouse GPRC5D) cells. [Diagram 2]FIG. 2 shows the MFI and EC50 values ​​of the binding of chimeric antibodies to cells with high hGPRC5D expression detected by FACS. [Diagram 3] FIG. 3 shows the MFI of the binding of the chimeric antibody to cells with low hGPRC5D expression, detected by FACS. [Figure 4A] FIG. 4A shows the MFI of binding of anti-GPRC5D antibody to H929 cells detected by FACS. [Figure 4B] FIG. 4B shows the MFI of binding of anti-GPRC5D antibody to MM1S cells detected by FACS. [Figure 5A] FIG. 5A shows the MFI of binding of anti-GPRC5D antibody to CHO-K1-cynoGPRC5D cells detected by FACS. [Figure 5B] FIG. 5B shows the MFI of binding of anti-GPRC5D antibody to CHO-K1-murineGPRC5D cells detected by FACS. [Figure 6A] FIG. 6A shows the MFI of binding of anti-GPRC5D antibody to CHO-K1-GPRC5A cells detected by FACS. [Figure 6B] FIG. 6B shows the MFI of binding of anti-GPRC5D antibody to CHO-K1-GPRC5B cells detected by FACS. [Figure 6C] FIG. 6C shows the MFI of binding of anti-GPRC5D antibody to CHO-K1-GPRC5C cells detected by FACS. [Figure 7] FIG. 7 shows the MFI of the binding of anti-GPRC5D antibody to CHO-K1 cells detected by FACS. [Figure 8] FIG. 8 shows an internalization activity assay of anti-GPRC5D antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Although exemplary embodiments of the present disclosure have been described in this specification, those skilled in the art will understand that the scope of protection of the present disclosure is not limited thereto, and various modifications, amendments, or changes can be made based on the spirit and concept of the present disclosure, and the contents after these modifications, amendments, or changes are still within the scope of the present disclosure.

[0021] term The term "antibody" is used in its broadest sense and covers a variety of antibody structures, including natural and artificial antibodies of various structures, including, but not limited to, monoclonal antibodies, single-specific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), single-chain antibodies, etc., so long as they exhibit the desired antigen-binding activity. An antigen-binding fragment refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen bound by the intact antibody. Antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)' fragments, Fv fragments, isolated CDR regions, single-chain Fv molecules (scFv), and other antibody fragments known in the art.

[0022] The term "isotype" refers to the class of antibody encoded by the heavy chain constant region genes. In one embodiment, the anti-GPRC5D antibody disclosed herein is of the IgG1 or IgG4 isotype. The anti-GPRC5D antibody and antigen-binding fragments thereof herein can be derived from any species, including, but not limited to, mouse, rat, rabbit, primate, llama, and human. The anti-GPRC5D antibody can be a chimeric antibody, a humanized antibody, or a fully human antibody. In certain embodiments, the anti-GPRC5D antibody provided herein is humanized. In certain embodiments, the anti-GPRC5D antibody provided herein is chimeric.

[0023] The term "chimeric antibody" is an antibody that has at least a portion of a heavy chain variable region and at least a portion of a light chain variable region derived from one species, and at least a portion of a constant region derived from another species. For example, in some embodiments, a chimeric antibody can include a murine variable region and a human constant region.

[0024] A "humanized antibody" is an antibody that contains complementarity determining regions (CDRs) from a non-human antibody and framework and constant regions from a human antibody. For example, a humanized antibody that binds to GPRC5D provided herein can contain one or more CDRs from a murine antibody, and human framework and constant regions.

[0025] The term "monoclonal antibody" ("mAb") refers to an antibody molecule of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope or, in the case of bispecific monoclonal antibodies, dual binding specificities for two different epitopes. A mAb is an example of an isolated antibody. mAbs can be produced by hybridoma, recombinant, transgenic, or other techniques known to those of skill in the art.

[0026] The term "variable domain" or "variable region" refers to the domain of an antibody that is involved in binding the antibody to an antigen. For example, naturally occurring four-chain antibodies (e.g., from humans, murines, etc.) have a heavy chain variable region (VH) and a light chain variable region (VL), while heavy chain antibodies (antibodies consisting only of heavy chains) from animals such as camelids and sharks have a single variable domain. In most cases, each variable domain of a naturally occurring antibody is essentially composed of four "framework regions" and three "complementarity determining regions". The four framework regions are respectively called framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3), and framework region 4 (FR4), and the framework regions are separated by what are referred to in the art and below as complementarity determining regions (CDRs). Thus, the general structure of a variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In one embodiment, the general structure of a heavy chain variable region can be represented as follows: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. In one embodiment, the general structure of a light chain variable region can be represented as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4.

[0027] "CDR" (complementarity determining region) is also called "hypervariable region (HVR)". For example, a natural four-chain antibody typically contains six CDRs, three of which are in the heavy chain variable region, namely heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and the remaining three are in the light chain variable region, namely light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), respectively. A heavy chain antibody or single variable domain typically has three CDRs (CDR1, CDR2, and CDR3).

[0028] Currently, there are many ways to classify and define CDRs. Among them, Kabat's definition, which classifies CDRs based on sequence variability, is the most commonly used (Elvin A. Kabat, et al, Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). And Chothia's definition is based on the location of structural loops (Cyrus Chothia, et al, Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol. Biol. 196:901-917 (1987)). The AbM definition is a compromise between the Kabat and Chothia definitions and is used by Oxford Molecular's AbM antibody modeling software. The "contact" CDR definition is based on the analysis of available complex crystal structures. However, it should be noted that the boundaries of CDRs of the same antibody variable region divided and defined based on different methods may be different, i.e., the CDR sequences of the same antibody variable region divided and defined by different methods may be different. Thus, when a specific CDR sequence defined by a specific division definition of the present disclosure is used to define an antibody, the scope of said antibody also includes antibodies defined by CDR sequences converted into any other definition (such as one or more combinations of the definitions of IMGT, Chothia, AbM, etc.). Although the CDRs sought to be protected in this disclosure are based on the sequences shown in Tables S1 and S2, corresponding amino acid sequences according to other CDR definition rules should also fall within the scope of protection of this disclosure. For example, according to another definition, the amino acid sequence of the heavy chain CDR1 of the chimeric antibody Chi-131 is NYVMH (SEQ ID NO: 71).

[0029] The term "framework region" (FR) refers to the amino acid residues of a variable domain other than the CDR residues as herein defined.

[0030] The term "isolated" refers to a compound of interest (e.g., an antibody or a nucleic acid) that has been separated from its natural environment.

[0031] As used herein, "EC 50 As used herein, the term "IC" refers to the effective concentration that is 50% of the maximal response of an antibody. 50 The term EC refers to the inhibitory concentration that is 50% of the maximal response of an antibody. 50 and IC 50 Both can be measured by ELISA or FACS analysis, or any other method known in the art.

[0032] As used herein, "K D The term " refers to the equilibrium dissociation constant, expressed as a molar concentration (M). D The K value can be measured using methods known in the art. D A preferred method of measuring is by using surface plasmon resonance, more preferably by using a biosensor system such as a Biacore system.

[0033] The term "test subject" as used herein includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including, for example, non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Preferably, a test subject according to the present disclosure is a human. Unless otherwise specified, the terms "patient" and "test subject" are used interchangeably.

[0034] As used herein, "cancer" refers to a physiological condition in mammals that is typically characterized by uncontrolled cell growth. Uncontrolled cell division and proliferation can lead to the formation of malignant tumors that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" may include tumors.

[0035] The term "treatment" refers to an attempt to alter the natural course of a disease in a treated individual and may be a clinical intervention performed for prophylaxis or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, alleviation of direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or palliation of the disease state, and regression or improvement of prognosis.

[0036] As used herein, the term "therapeutically effective amount" refers to the amount of a compound, composition, or pharmaceutical combination required to confer a therapeutic effect on a subject.

[0037] The term "pharmacologically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0038] A "pharmaceutical composition" refers to a composition comprising an active ingredient and a pharma- ceutically acceptable carrier.

[0039] "Percent identity" of an amino acid sequence refers to the percentage of amino acid residues that are identical to the amino acid residues of the sequence to be aligned and the specific amino acid sequence shown herein, after aligning the sequence to be aligned with the specific amino acid sequence shown herein and introducing gaps as necessary to achieve the maximum percent sequence identity, without any conservative substitutions being part of the sequence identity. Amino acid sequence identity alignment can be performed using a variety of methods within the skill of the art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to obtain maximum alignment over the entire length of the sequences being compared.

[0040] The terms "Xn" and "Xaa" are equivalent and refer to an Unspecified Amino Acid, the scope of which is specified by the subsequent definition in the relevant expression.

[0041] The terms "comprise," "contain," or "including," and variations thereof, should be understood to mean "including, but not limited to," and are meant to cover the listed elements, components, and steps, as well as other unspecified elements, components, and steps.

[0042] As used herein, singular terms include plural referents and vice versa unless the context clearly dictates otherwise.

[0043] All patents, patent applications, and other specific publications are expressly incorporated herein by reference for purposes of description and disclosure. These publications are provided solely for their disclosure prior to the filing date of the present disclosure. Any statement as to the date of these documents or the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Moreover, any reference to these publications in this specification is not an admission that the publications form part of the common general knowledge in any country in the art. Various aspects of the present disclosure are described in further detail below.

[0044] I. Anti-GPRC5D Antibodies and Antigen-Binding Fragments Thereof As used herein, the terms "human GPRC5D" and "hGPRC5D" are used interchangeably, and "antibody that binds to GPRC5D" and "anti-GPRC5D antibody" are used interchangeably.

[0045] The present disclosure provides novel antibodies that bind to GPRC5D. Anti-GPRC5D antibodies exhibit many other desirable properties for therapeutic and / or diagnostic applications. For example, in some embodiments, the anti-GPRC5D antibodies have good specificity for GPRC5D. In some embodiments, the anti-GPRC5D antibodies can bind to monkey GPRC5D or / and murine GPRC5D, which facilitates pharmacological or / and safety evaluation during antibody development. In some embodiments, the anti-GPRC5D antibodies provided herein exhibit better antigen binding properties compared to some known antibodies, such as better binding ability to cells with low GPRC5D expression. In some embodiments, the anti-GPRC5D antibodies have good killing properties against tumor cells, such as multiple myeloma cells.

[0046] In one aspect, the disclosure provides an isolated anti-GPRC5D antibody or antigen-binding fragment thereof, the anti-GPRC5D antibody comprising: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, 9, 17, 25 or 33; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, 10, 18, 26 or 34, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, 11, 19, 27 or 35 and a heavy chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, 12, 20, 28 or 36; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, 13, 21, 29 or 37, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, 14, 22, 30 or 38 and a light chain variable region comprising:

[0047] In some embodiments, the anti-GPRC5D antibody is A heavy chain variable region comprising HCDR1 comprising the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:3; and a light chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO:4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:6.

[0048] In some embodiments, the anti-GPRC5D antibody is A heavy chain variable region comprising HCDR1 comprising the amino acid sequence shown in SEQ ID NO:9, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:10, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:11; and a light chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 12, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 14.

[0049] In some embodiments, the anti-GPRC5D antibody is A heavy chain variable region comprising HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 18, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 19; and a light chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO:20, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:21, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:22.

[0050] In some embodiments, the anti-GPRC5D antibody is A heavy chain variable region comprising HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27; and a light chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO:28, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:29, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:30.

[0051] In some embodiments, the anti-GPRC5D antibody is A heavy chain variable region comprising HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 33, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 35; and a light chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO:36, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:37, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:38.

[0052] In some embodiments, an isolated anti-GPRC5D antibody or antigen-binding fragment thereof is provided, the anti-GPRC5D antibody comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO: 7, 15, 23, 31, or 39, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO: 8, 16, 24, 32, or 40. In some particular embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO: 7, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO: 8. In particular embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO: 15, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO: 16. In some particular embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO: 23, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO: 24. In some particular embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO: 31, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO: 32. In some particular embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO: 39, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO: 40. In some embodiments, in these particular embodiments, the CDR regions of the antibody may be those defined according to Kabat, Chothia, IMGT, or other definitions.

[0053] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 7, 15, 23, 31 or 39. In some specific embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, 15, 23, 31 or 39.

[0054] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 8, 16, 24, 32 or 40. In some specific embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8, 16, 24, 32 or 40.

[0055] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 7, 15, 23, 31 or 39, and the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 8, 16, 24, 32 or 40. In some specific embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7, 15, 23, 31, or 39, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, 16, 24, 32, or 40.

[0056] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO:8. Furthermore, in some such embodiments, the anti-GPRC5D antibody further comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO:1, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:2, an HCDR3 having the amino acid sequence set forth in SEQ ID NO:3, an LCDR1 having the amino acid sequence set forth in SEQ ID NO:4, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:5, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:6.

[0057] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO:15, and the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO:16. Furthermore, in some such embodiments, the anti-GPRC5D antibody further comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 9, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 10, an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 11, an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 12, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 13, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 14.

[0058] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO:23, and the light chain variable region comprises an amino acid sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO:24. Further, in some such embodiments, the anti-GPRC5D antibody further comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 17, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 18, an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 19, an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 20, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 21, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 22.

[0059] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO:31, and the light chain variable region comprises an amino acid sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO:32. Further, in some such embodiments, the anti-GPRC5D antibody further comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 25, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 26, an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 27, an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 28, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 29, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 30.

[0060] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO:39, and the light chain variable region comprises an amino acid sequence at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO:40. Further, in some such embodiments, the anti-GPRC5D antibody further comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 33, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 34, an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 35, an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 36, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 37, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 38.

[0061] In some specific embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8.

[0062] In some specific embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:15 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:16.

[0063] In some specific embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:23 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:24.

[0064] In some specific embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:31 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:32.

[0065] In some specific embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:39 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:40.

[0066] In some embodiments, the anti-GPRC5D antibodies provided herein comprise heavy and light chains that comprise the corresponding heavy and light chain variable regions as described herein plus a constant region, from N-terminus to C-terminus, the heavy chain consists of a heavy chain variable region and a heavy chain constant region, and the light chain consists of a light chain variable region and a light chain constant region.

[0067] In some embodiments, the light chain constant region of the anti-GPRC5D antibody is a human kappa chain constant region. In some embodiments, the light chain constant region of the anti-GPRC5D antibody is a human lambda chain constant region.

[0068] The heavy chain constant region of the anti-GPRC5D antibody can be derived from any type of constant region, such as IgG, IgM, IgD, IgA, and IgE, and any isotype, such as IgG1, IgG2, IgG3, and IgG4. In some embodiments, the anti-GPRC5D antibody is an IgG1 isotype. In some embodiments, the anti-GPRC5D antibody is an IgG4 isotype.

[0069] In some embodiments, the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO:69 and the amino acid sequence of the light chain constant region is set forth in SEQ ID NO:70.

[0070] In some embodiments, the heavy chain comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 49, and the light chain comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 51. In one particular embodiment, the anti-GPRC5D antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 49 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 51. In another embodiment, the heavy chain comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 53 and the light chain comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 55. In one particular embodiment, the anti-GPRC5D antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 53 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 55. In another embodiment, the heavy chain comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 57 and the light chain comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 59. In one particular embodiment, the anti-GPRC5D antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 57 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 59.In another embodiment, the heavy chain comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 61 and the light chain comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 63. In one particular embodiment, the anti-GPRC5D antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 61 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 63. In another embodiment, the heavy chain comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 65 and the light chain comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 67. In one particular embodiment, the anti-GPRC5D antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 65 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 67.

[0071] In some embodiments, the anti-GPRC5D antibodies cross-react with simian GPRC5D antigen and / or murine GPRC5D antigen.

[0072] In some embodiments, the anti-GPRC5D antibody or antigen fragment thereof binds to the human GPRC5D antigen, and optionally also has any one or more of the following properties: (1) Binds to monkey GPRC5D antigen; (2) binds to the murine GPRC5D antigen; or (3) does not bind to one or more of GPRC5A, GPRC5B, and GPRC5C.

[0073] In some embodiments, the anti-GPRC5D antibody or antigen fragment thereof binds to human GPRC5D antigen, and also binds to monkey GPRC5D antigen or / and murine GPRC5D antigen, and does not bind to GPRC5A, GPRC5B, and GPRC5C.

[0074] In some embodiments, the anti-GPRC5D antibody is a monoclonal antibody.

[0075] In some embodiments, the anti-GPRC5D antibody is a monospecific antibody.

[0076] In some embodiments, the anti-GPRC5D antibody is a multispecific antibody, such as a bispecific antibody or a trispecific antibody.

[0077] In another aspect, the present specification provides an anti-GPRC5D antibody or antigen-binding fragment thereof that binds to the same epitope on GPRC5D as any of the exemplary antibodies provided herein, e.g., the same epitope as Chi-131, e.g., the same epitope as Chi-169, e.g., the same epitope as Chi-89. In another aspect, the present specification provides an anti-GPRC5D antibody or antigen-binding fragment thereof that competes with any of the exemplary antibodies provided herein, e.g., competes with Chi-131 for binding to GPRC5D, e.g., competes with Chi-131 for binding to GPRC5D, e.g., competes with Chi-169 for binding to GPRC5D. Binding to GPRC5D can be measured by ELISA, flow cytometry, surface plasmon resonance (SPR) assay, or any other method known in the art.

[0078] Also provided herein are several exemplary monoclonal antibodies that bind to GPRC5D, including mouse anti-mu-89, mu-105, mu-128, mu-131, mu-164, mu-169, and mu-180, as well as constructed chimeric antibodies Chi-89, Chi-105, Chi-128, Chi-131, Chi-164, Chi-169, and Chi-180. The amino acid sequences of the HCDRs (HCDR1, HCDR2, and HCDR3) of several exemplary anti-GPRC5D antibodies provided herein are provided in Table S1 below, the amino acid sequences of the LCDRs (LCDR1, LCDR2, and LCDR3) are provided in Table S2 below, and the amino acid sequences of some antibody variable regions are provided in Table S3 below.

[0079] Table S1: Heavy chain CDR sequences [Table 1]

[0080] Table S2: Light chain CDR sequences [Table 2]

[0081] Table S3: Sequences of some antibody variable regions [Table 3]

[0082] II. Antibody Variants In some embodiments, a variant of the anti-GPRC5D antibody or an antigen-binding fragment thereof is provided. The amino acid sequence variant of the anti-GPRC5D antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the anti-GPRC5D antibody or by peptide synthesis. Such modifications include, for example, deletion of amino acid residues from, insertion of amino acid residues into, and / or substitution of amino acid residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to obtain the final construct, as long as the final variant has the desired properties, such as antigen-binding effect, for example, tumor-killing ability.

[0083] III. Fusion Proteins In some embodiments, the disclosure provides a fusion protein comprising an anti-GPRC5D antibody or antigen-binding fragment thereof described herein.

[0084] IV. Pharmaceutical Compositions The present disclosure provides a pharmaceutical composition comprising the anti-GPRC5D antibody or its antigen-binding fragment, or the fusion protein, and further comprising a pharma- ceutically acceptable carrier. In some embodiments, any one or more of Chi-89, Chi-105, Chi-128, Chi-131, Chi-164, Chi-169, and Chi-180 antibodies are prepared into a pharmaceutical composition together with a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other agents.

[0085] V. Isolated Nucleic Acids The present disclosure provides isolated nucleic acids encoding the anti-GPRC5D antibodies or antigen-binding fragments thereof described herein. In some embodiments, the nucleic acids encode antigen-binding fragments, such as Chi-89, Chi-105, Chi-128, Chi-131, Chi-164, Chi-169, or Chi-180. In some embodiments, the nucleic acids encode antibodies such as Chi-89, Chi-105, Chi-128, Chi-131, Chi-164, Chi-169, and Chi-180. The sequence listing provides exemplary nucleic acid sequences for some anti-GPRC5D antibodies or antigen-binding fragments thereof.

[0086] VI. Vector The present disclosure provides a vector comprising the isolated nucleic acid. In some embodiments, the vector is a cloning vector, and in other embodiments, the vector is an expression vector. The expression vector can optionally be any expression vector capable of expressing the anti-GPRC5D antibody or antigen-binding fragment thereof described herein, and in a specific example, the expression vector is pcDNA3.1.

[0087] VII.Host cells In some embodiments, the present disclosure provides a host cell comprising a vector described herein or having the nucleic acid integrated into its genome. The host cell is a host cell suitable for cloning or expressing an anti-GPRC5D antibody or antigen-binding fragment thereof. In some embodiments, the host cell is a prokaryotic cell. In other embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from a yeast cell, a mammalian cell, or other cells suitable for preparing an anti-GPRC5D antibody or antigen-binding fragment thereof. Mammalian cells are, for example, Chinese Hamster Ovary (CHO) cells and CHO-S cells.

[0088] VIII. Methods for Preparing Anti-GPRC5D Antibodies and Antigen-Binding Fragments Thereof In some embodiments, the present disclosure provides a method for preparing the anti-GPRC5D antibody or antigen-binding fragment thereof, the method comprising culturing a host cell described herein and recovering the anti-GPRC5D antibody or antigen-binding fragment thereof from the host cell or host cell culture medium.

[0089] To produce the anti-GPRC5D antibody or its antigen-binding fragment, the nucleic acid encoding the anti-GPRC5D antibody or its antigen-binding fragment is inserted into a vector for further cloning and / or expression in a host cell. The nucleic acid can be obtained by various methods well known in the art, such as gene splicing and chemical synthesis. The recovery can be performed using steps such as centrifugation and affinity chromatography.

[0090] IX.Use In some embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof, and fusion proteins provided herein can be used to treat cancer, reduce tumors, or inhibit tumor cell proliferation.

[0091] The present disclosure provides the use of the anti-GPRC5D antibody or its antigen-binding fragment, or fusion protein in the preparation of a medicament.In some embodiments, the use is for the preparation of a medicament for treating a disease.In some embodiments, the use is for the preparation of a medicament for reducing tumors or inhibiting the proliferation of tumor cells.

[0092] The present disclosure provides a method for reducing or inhibiting tumor cell proliferation in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-GPRC5D antibody or antigen-binding fragment thereof, a fusion protein, or a pharmaceutical composition.

[0093] The present disclosure provides a method of treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the anti-GPRC5D antibody or antigen-binding fragment thereof, the fusion protein, or the pharmaceutical composition. Subjects in need of treatment include subjects already suffering from a disease or condition, as well as subjects at risk of suffering from a disease or condition, in which the goal is to prevent, delay, or alleviate the disease or condition.

[0094] The disease is cancer or an autoimmune disease. In some embodiments, the autoimmune disease is, for example, systemic lupus erythematosus and / or rheumatoid arthritis. In some embodiments, the cancer is multiple myeloma.

[0095] In some embodiments, a method for detecting or measuring GPRC5D in a sample is provided, the method comprising contacting the sample with an anti-GPRC5D antibody or antigen-binding fragment thereof described herein and detecting or measuring the binding complex.

[0096] X.Kit The present disclosure provides kits comprising an anti-GPRC5D antibody or antigen-binding fragment thereof, a fusion protein, or a pharmaceutical composition described herein.

[0097] The present specification describes a kit comprising the anti-GPRC5D antibody or antigen-binding fragment thereof, the fusion protein, or the pharmaceutical composition. The kit can be used to carry out the use or other use of the anti-GPRC5D antibody or antigen-binding fragment thereof, the fusion protein, or the pharmaceutical composition provided herein. In some embodiments, the kit can include the anti-GPRC5D antibody or antigen-binding fragment thereof, the fusion protein, or the pharmaceutical composition described herein, and a reagent for detecting the presence of GPRC5D in a biological sample. The kit may also include instructions for use. The kit may also include other materials necessary from a commercial and user perspective, such as other buffers, diluents, needles, syringes, etc.

[0098] The present disclosure also provides the following specific embodiments, but the scope of protection of the present disclosure is not limited thereto.

[0099] Embodiment 1. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, 9, 17, 25 or 33; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, 10, 18, 26 or 34, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, 11, 19, 27 or 35 and a heavy chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, 12, 20, 28 or 36; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, 13, 21, 29 or 37, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, 14, 22, 30 or 38 a light chain variable region comprising An isolated anti-GPRC5D antibody, or an antigen-binding fragment thereof, comprising:

[0100] Embodiment 2. The anti-GPRC5D antibody is (i) HCDR1 comprising the amino acid sequence shown in SEQ ID NO:1; HCDR2 comprising the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:3 and a heavy chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO:4; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:6 and a light chain variable region comprising (ii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 9; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:11 and a heavy chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 12; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:14 and a light chain variable region comprising (iii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 17; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:19 and a heavy chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:22 and a light chain variable region comprising (iv) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:27 and a heavy chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 28; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:29, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:30 or a light chain variable region comprising (v) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:35 and a heavy chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 36; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:38 a light chain variable region comprising 2. The isolated anti-GPRC5D antibody or antigen-binding fragment thereof of embodiment 1, comprising:

[0101] Embodiment 3. An isolated anti-GPRC5D antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO: 7, 15, 23, 31 or 39, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO: 8, 16, 24, 32 or 40.

[0102] Embodiment 4. A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 comprising the variable region sequence shown in SEQ ID NO:7, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 comprising the variable region sequence shown in SEQ ID NO:8; a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 comprising the variable region sequence shown in SEQ ID NO:15, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 comprising the variable region sequence shown in SEQ ID NO:16; a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 comprising the variable region sequence shown in SEQ ID NO:23, and a light chain variable region comprising the variable region sequence shown in SEQ ID NO:24. An isolated anti-GPRC5D antibody or antigen-binding fragment thereof described in embodiment 3, comprising: a light chain variable region comprising CDR1, LCDR2, and LCDR3; a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO: 31, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO: 32; or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO: 39, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO: 40.

[0103] Embodiment 5. An isolated anti-GPRC5D antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 4, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 7, 15, 23, 31 or 39.

[0104] Embodiment 6. An isolated anti-GPRC5D antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 5, wherein the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 8, 16, 24, 32 or 40.

[0105] Embodiment 7. An isolated anti-GPRC5D antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 6, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% identity to the sequence shown in SEQ ID NO: 7, 15, 23, 31 or 39, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence shown in SEQ ID NO: 8, 16, 24, 32 or 40.

[0106] Embodiment 8. The heavy chain variable region and the light chain variable region are (i) the heavy chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:7 and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:8; (ii) the heavy chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 15, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 16; (iii) the heavy chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:23 and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:24; (iv) the heavy chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 31, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 32; or (v) the heavy chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 39, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 40. 8. The isolated anti-GPRC5D antibody or antigen-binding fragment thereof of embodiment 7, selected from any one of the following:

[0107] Embodiment 9. The heavy chain variable region and the light chain variable region are (i) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8; (ii) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16; (iii) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:23 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:24; (iv) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:31 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:32; or (v) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 39, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 40. 8. The isolated anti-GPRC5D antibody or antigen-binding fragment thereof of embodiment 7, selected from any one of the following:

[0108] Embodiment 10. An isolated anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9, wherein the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, an isolated CDR region or an scFv.

[0109] Embodiment 11. An isolated anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 10, wherein the anti-GPRC5D antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0110] Embodiment 12. An isolated anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 11, wherein the anti-GPRC5D antibody is of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0111] Embodiment 13. The anti-GPRC5D antibody or antigen fragment thereof binds to the human GPRC5D antigen and, optionally, also has any one or more of the following properties: (1) Binds to monkey GPRC5D antigen; (2) binds to the murine GPRC5D antigen; or (3) An isolated anti-GPRC5D antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 12, which does not bind to one or more of GPRC5A, GPRC5B, and GPRC5C.

[0112] Embodiment 14. An isolated anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 13, wherein the anti-GPRC5D antibody is a monospecific antibody or a multispecific antibody.

[0113] Embodiment 15. A fusion protein comprising the anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 14.

[0114] Embodiment 16. A pharmaceutical composition comprising an anti-GPRC5D antibody or an antigen-binding fragment thereof described in any one of embodiments 1 to 14, or an immune complex (fusion protein) described in embodiment 15, and further comprising a pharma- ceutical acceptable carrier.

[0115] Embodiment 17. An isolated nucleic acid encoding an anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 14.

[0116] Embodiment 18. A vector comprising the nucleic acid of embodiment 17.

[0117] Embodiment 19. A host cell comprising a vector according to embodiment 18 or having a nucleic acid according to embodiment 17 integrated into its genome.

[0118] Embodiment 20. A method for preparing an anti-GPRC5D antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 14, comprising culturing a host cell described in embodiment 19 and recovering the anti-GPRC5D antibody or antigen-binding fragment thereof from the host cell or host cell medium.

[0119] Embodiment 21. Use of an anti-GPRC5D antibody or an antigen-binding fragment thereof described in any one of embodiments 1 to 14, or a fusion protein described in embodiment 15, or a pharmaceutical composition described in embodiment 16 in the treatment of a disease, or in reducing tumors, or in inhibiting tumor cell proliferation, preferably wherein the disease is cancer or an autoimmune disease.

[0120] Embodiment 22. Use of an anti-GPRC5D antibody or an antigen-binding fragment thereof described in any one of embodiments 1 to 14 or a fusion protein described in embodiment 15 in the preparation of a medicament for treating a disease, reducing a tumor, or inhibiting tumor cell proliferation, preferably wherein the disease is cancer or an autoimmune disease.

[0121] Embodiment 23. A method for reducing tumors or inhibiting tumor cell proliferation in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-GPRC5D antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 14, or a fusion protein described in embodiment 15, or a pharmaceutical composition described in embodiment 16.

[0122] Embodiment 24. A method for treating a disease in a test subject, the method comprising administering to the test subject a therapeutically effective amount of an anti-GPRC5D antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 14, or a fusion protein described in embodiment 15, or a pharmaceutical composition described in embodiment 16, preferably wherein the disease is cancer or an autoimmune disease.

[0123] Embodiment 25 The method of embodiment 21, 22, or 24, wherein the cancer is multiple myeloma.

[0124] Embodiment 26. A kit comprising the anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 14, the fusion protein according to embodiment 15, or the pharmaceutical composition according to embodiment 16. Working Example

[0125] Example 1: Antigen Immunization 1. DNA antigen immunization The cDNA sequence encoding human GPRC5D protein (amino acid sequence shown in SEQ ID NO: 41) was obtained by gene synthesis and subcloned into expression vector pcDNA3.1(+) to construct plasmid pcDNA3.1-hGPRC5D. Large-scale plasmid preparation was carried out according to the instruction manual of the Endotoxin-Free Maxi Extraction Kit (QIAGEN, Catalog No.: 12391).

[0126] Plasmid pcDNA3.1-hGPRC5D was used as an antigen to immunize A / J mice (Nanjing University Institute of Model Zoology), BALB / c mice (Shanghai Lingcheng), and SJL mice (Beijing Charles River), respectively. First, hyaluronidase (Sigma, Catalog No.: H4272) was pre-injected into the left and right hind leg muscles of each mouse to pretreat them, and then CpG (InvivoGen, Catalog No.: tlrl-1826) and plasmid pcDNA3.1-hGPRC5D were mixed uniformly at a mass ratio of 1:1. The homogeneously mixed antigen complex was injected into the muscle of the pretreated mouse via a biogenetic transfection device (Type II, Shanghai Teresa Bio-Tech Co., Ltd.), and the same immunization as above was repeated once every 2 to 3 weeks, for a total of four immunizations. After completion of immunization, serum was collected from each mouse.

[0127] 2. Assessment of Mouse Serum Titers To evaluate the immune response of anti-human GPRC5D antibodies produced in mice immunized with DNA antigen, the antibody titers of mouse serum were detected by FACS. Mice with higher serum titers were selected for shock immunization before the subsequent splenocyte fusion.

[0128] 3. Cellular antigen immunity Transfect the plasmid pcDNA3.1-hGPRC5D into CHO-K1 cells according to the instruction manual of Lipofectamine 3000 transfection reagent (Thermo, catalog number: L3000015) to generate a stable and highly expressing hGPRC5D cell line, CHO-K1-hGPRC5D. hi The above CHO-K1-hGPRC5D hi Mice were immunized with the cells as antigens. Three to four days before spleen cell fusion, the final cell concentration was 5 × 10 7 Cells / mL CHO-K1-hGPRC5D hi The cell suspension and CpG (InvivoGen, catalog number: tlrl-1826) at a final concentration of 0.5 mg / mL were mixed uniformly and then injected intraperitoneally into the mice at 100 μL / mouse.

[0129] Example 2: Screening of mouse anti-human GPRC5D antibodies 1. Hybridoma Cell Preparation On the day of fusion, mouse spleens were aseptically removed and polished, red blood cell lysis solution (Sigma, catalog number: R7757) was added, and the cells were washed with PBS solution. The splenocytes were then resuspended in electrofusion buffer (BTX, catalog number: 47-0001), and the splenocytes and SP2 / 0 mouse myeloma cells were mixed uniformly at a cell number ratio of 2:1, and the splenocytes were fused using an electrofusion device (BTX). Hybridoma medium (Gibco, catalog number: 12045-076) containing 1×HAT (Gibco, catalog number: 21060-017) was added to dilute the fused cells, and the cells were cultured at 37°C and 5% CO2 for 7 to 10 days to prepare hybridoma cells. The culture supernatant of the hybridoma cells was collected and used for screening mouse anti-human GPRC5D antibodies.

[0130] 2. Construction of Stable Cell Lines To evaluate the interspecies cross-binding activity of mouse anti-human GPRC5D antibody at the cellular level, the following cell lines were constructed. The cDNAs encoding full-length monkey GPRC5D (amino acid sequence shown in SEQ ID NO: 42) and mouse GPRC5D (amino acid sequence shown in SEQ ID NO: 43) were obtained by gene synthesis, respectively, and subcloned into the vector pcDNA3.1(+) to obtain pcDNA3.1-cynoGPRC5D and pcDNA3.1-murineGPRC5D recombinant plasmids. The plasmids pcDNA3.1-cynoGPRC5D and pcDNA3.1-murineGPRC5D were transfected into CHO-K1 cells, respectively, according to the instruction manual of Lipofectamine 3000 transfection reagent (Thermo, catalog number: L3000015), to obtain CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D stable cell lines.

[0131] 3. Cell ELISA Preliminary Screening Logarithmic growth phase of CHO-K1-hGPRC5D hi The cells were harvested and resuspended in DMEM / F-12 (Gibco, Catalog No.: 11320033) medium containing 10% by volume FBS to a cell concentration of 1 × 10 6The cells were adjusted to cells / mL, plated at 100 μL / well in a 96-well flat-bottom plate, and incubated overnight at 37°C, 5% CO2. The next day, the cell plate was washed with PBS and fixed with 4% (volume percent) paraformaldehyde (Beyotime, Catalog No.: P0099) at room temperature for 30 minutes. After washing with PBS, 3% (volume percent) BSA in PBS was added at 200 μL / well and blocked at room temperature for 2 hours. After blocking, hybridoma cell culture supernatant was added to the cell plate at 100 μL / well and incubated at room temperature for 2 hours. After washing with PBS, HRP-conjugated goat anti-mouse IgG+IgM(H+L) antibody (Jackson Immuno Research, Catalog No.: 115-035-068) was added and incubated at room temperature for 1 hour. After washing with PBS, 100 μL / well of TMB reaction solution (Solarbio, Cat. No.: RP1200) was added and incubated at room temperature in the dark for 5 min, and the reaction was stopped with 0.5 M H2SO4. OD450 absorbance values ​​were read using a Bio-rad iMark microplate reader. Hybridoma cells corresponding to culture supernatants with absorbance greater than 1.0 were selected as positive clones for subsequent FACS screening.

[0132] 4. Flow Cytometric FACS Screening of Antibodies Cell concentration 5 x 10 in 96-well U-bottom plates 5CHO-K1-cynoGPRC5D cell suspension at 100 μL / well was plated, and the culture supernatant of the positive clone was added at 100 μL / well and mixed uniformly, and then incubated at 4° C. for 1 hour. After washing with PBS containing 2% (volume percent) FBS, a goat anti-mouse IgG+IgM (H+L) antibody conjugated with AlexaFluor488 (Jackson Immuno Research, Catalog No.: 115-545-044) was added and incubated at 4° C. for 1 hour. The cells were washed and resuspended in PBS containing 2% (volume percent) FBS, and the fluorescent signal was detected using a flow cytometer (Sartorius, IQue3), and the binding of the anti-hGPRC5D antibody to CHO-K1-cynoGPRC5D cells was analyzed by the mean fluorescence intensity (MFI) of staining.

[0133] 5. Preparation of Subcloning After screening by cell ELISA and FACS, CHO-K1-hGPRC5D hi Hybridoma cells positively binding to both CHO-K1-cynoGPRC5D and CHO-K1-cynoGPRC5D were picked up and selected for expansion. The expanded cells were mixed uniformly with semi-solid medium D (Stemcell, Catalog No.: 3810) at a volume ratio of 1:10 and cultured for 5 days under the conditions of 37°C and 5% CO2, and the monoclonal cell mass was aspirated under a 4x microscope and transferred to a 96-well plate covered with hybridoma medium (Gibco, Catalog No.: 12045-076), and continued to be cultured for 2 days under the conditions of 37°C and 5% CO2. The subclone cell culture supernatant was collected from each well and used for further screening of the subclone cells.

[0134] 6. Screening of Subclones (1) FACS screening of anti-hGPRC5D antibodies Cell concentration 3×10 5H929 (i.e., NCI-H929) cell suspensions of 100 μL / well of cells / mL were plated on a 96-well U-bottom plate, and 100 μL / well of subclone cell culture supernatant or an equivalent volume of medium (hereinafter abbreviated as Medium) was added, mixed uniformly, and then incubated at 4° C. for 1 hour. After washing the cells with PBS containing 2% (volume percent) FBS, a goat anti-mouse IgG+IgM (H+L) antibody conjugated with AlexaFluor488 (Jackson Immuno Research, Catalog No.: 115-545-044) was added and incubated at 4° C. for 1 hour. The cells were washed and resuspended in PBS containing 2% (volume percent) FBS, and the fluorescent signal was detected using a flow cytometer (Sartorius, IQue3), and the binding of mouse anti-hGPRC5D antibody to H929 cells was analyzed by the mean fluorescence intensity (MFI) of staining, and some results are shown in Table 1.

[0135] Table 1. FACS binding assay of some mouse anti-hGPRC5D antibodies with H929 cells [Table 4]

[0136] (2) Cell ELISA screening of anti-hGPRC5D antibodies Logarithmic growth phase of CHO-K1-hGPRC5D hi The cells, CHO-K1-cynoGPRC5D cells, CHO-K1-murineGPRC5D cells, and CHO-K1 cells were collected and resuspended in DMEM / F-12 (Gibco, catalog number: 11320033) medium containing 10% (volume percent) FBS to a cell concentration of 1 × 10 6The cells were adjusted to 100 μL / well of cells / mL and plated in a 96-well flat-bottom plate at 100 μL / well and incubated overnight at 37°C, 5% CO2. The next day, the cell plate was washed with PBS, and 4% (volume percent) paraformaldehyde (Beyotime, Catalog No.: P0099) was added and fixed at room temperature for 30 minutes. After washing with PBS, 200 μL / well of PBS solution containing 3% (volume percent) BSA was added and blocked at room temperature for 2 hours. After blocking, 50 μL / well of subclone cell culture supernatant was added to the cell plate and incubated at room temperature for 2 hours. After washing with PBS, HRP-conjugated goat anti-mouse IgG+IgM(H+L) antibody (Jackson Immuno Research, Catalog No.: 115-035-068) was added and incubated at room temperature for 1 hour. After washing with PBS, 100 μL / well of TMB reaction solution (Solarbio, Cat. No.: RP1200) was added and incubated at room temperature in the dark for 5 min, and the reaction was stopped with 0.5 M H2SO4. OD450 absorbance values ​​were read using a Bio-rad iMark microplate reader. Bar graphs of subclone absorbance values ​​were generated using Graphpad Prism. Figure 1 shows the typical mouse anti-hGPRC5D antibodies (e.g., mu-89, mu-105, mu-126, mu-128, mu-131, mu-153, mu-164, mu-169, mu-180, mu-183, etc.) and CHO-K1-hGPRC5D hi The absorbance values ​​of the cells, CHO-K1-cynoGPRC5D cells and / or CHO-K1-murineGPRC5D cells are more than 2-fold different from the absorbance values ​​of the control cells, CHO-K1.

[0137] Example 3: Preparation of anti-hGPRC5D chimeric antibody 1. Sequencing of the variable regions encoding mouse anti-hGPRC5D antibodies Total RNA in the screened hybridoma cells was isolated according to the instruction manual of the RNA extraction kit (Takara, Catalog No.: 9767), and the first strand cDNA was synthesized using a reverse transcription kit (Thermo, Catalog No.: K1652). The first strand cDNA was used as a template and mixed with mouse heavy and light chain constant region primers, respectively, and the obtained mouse anti-hGPRC5D antibody variable region sequence was cloned and sequenced using polymerase chain reaction (PCR) technology.

[0138] 2. Construction of chimeric antibody expression vector Using chemical synthesis, the nucleotide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of mouse anti-hGPRC5D antibody were linked to the nucleotide sequences of the human IgG1 heavy chain constant region and the kappa light chain constant region, respectively. Recombinant human-mouse chimeric antibody expression vectors were constructed using the pcDNA3.1(+) vector and used for transfection expression to prepare chimeric antibodies. The details of the full-length and VH / VL nucleotide and amino acid sequences of each chimeric antibody are shown in Table 2.

[0139] Table 2. Full length chimeric antibodies and VH / VL nucleotide and amino acid sequences [Table 5]

[0140] 3. Construction of Positive Reference Antibody Expression Vector GC5B596 was selected as a reference antibody (monoclonal antibody, source WO2018017786A2), and the nucleotide sequences of the antibody VH (SEQ ID NO: 44) and VL (SEQ ID NO: 45) were obtained using chemical synthesis and linked to the nucleotide sequences of the human IgG1 heavy chain constant region and the kappa light chain constant region, respectively, and an expression vector expressing the positive reference antibody was constructed using the pcDNA3.1(+) vector, which was used for transfection and expression to prepare the positive reference antibody. The positive reference antibody (monoclonal antibody) is referred to herein as BM-mAb.

[0141] 4. Preparation of anti-hGPRC5D chimeric antibody and positive reference antibody According to the operation manual of the expiCHO expression system (Gibco, catalog number: A29129), the anti-hGPRC5D antibody was transiently transfected and expressed, and after transfection, the expiCHO cells were shaken and cultured for 7 days under conditions of 37°C and 8% CO2. The cell culture supernatant was collected, the clarified culture supernatant was loaded onto a Protein A column (GE Healthcare, catalog number: 17-5474), 10 column volumes of PBS buffer were added to wash the Protein A column, and acetate buffer (300 mM acetate, pH 3.6) was added to elute and collect the IgG antibody bound to the Protein A column. The collected IgG antibody component was replaced with PBS buffer using an ultrafiltration device (molecular weight cutoff 30 kDa, Millipore, catalog number: UFC903024) to obtain an anti-hGPRC5D antibody solution.

[0142] Example 4: Measurement of binding activity of anti-hGPRC5D antibody to cells stably expressing human GPRC5D 1. Binding characteristics of anti-hGPRC5D chimeric antibody to stable high-expressing human GPRC5D cells Cell concentration 3×10 5 Cells / mL CHO-K1-hGPRC5D hi The cell suspension (GPRC5D expression level is approximately 2E+06-3E+06 antigens / cell) was plated at 100 μL / well on a 96-well U-bottom plate, gradient-diluted (final concentration range: 4.1-3000 ng / mL, 3-fold gradient dilution) anti-hGPRC5D antibody was added, mixed uniformly, and incubated at 4°C for 1 hour. After washing the cells with PBS containing 2% (volume percent) FBS, PE-labeled goat anti-human IgG Fcγ antibody (Jackson Immuno Research, catalog number: 109-116-170) was added and incubated at 4°C for 1 hour. The cells were washed and resuspended in PBS containing 2% (volume percent) FBS, and the fluorescent signal was detected using a flow cytometer (Sartorius, IQue3) to detect the anti-hGPRC5D antibody and CHO-K1-hGPRC5D. hiBinding to the antibody was analyzed by the mean fluorescence intensity (MFI) of staining. EC calculated by Graphpad Prism. 50 The analysis results are shown in Figure 2. The chimeric antibodies Chi-89, Chi-105, Chi-128, Chi-131, Chi-164, Chi-169, and Chi-180 all exhibited concentration gradient-dependent binding activity to cells highly expressing hGPRC5D, and had higher maximum antigen-binding ability than BM-mAb.

[0143] 2. Binding characteristics of anti-hGPRC5D chimeric antibody to stable low-expressing human GPRC5D cells Transfect the plasmid pcDNA3.1-hGPRC5D into CHO-K1 cells according to the instruction manual of Lipofectamine 3000 transfection reagent (Thermo, catalog number: L3000015), creating CHO-K1-hGPRC5D. low A stable low-expressing cell line (GPRC5D expression level was approximately 2000-2500 antigens / cell) was obtained. The cell concentration was 3×10 5 Cells / mL CHO-K1-hGPRC5D low The cell suspension was plated at 100 μL / well in a 96-well U-bottom plate, gradient-diluted (final concentration range: 4.1-3000 ng / mL, 3-fold gradient dilution) anti-hGPRC5D antibody was added, mixed uniformly, and incubated at 4°C for 1 hour. After washing the cells with PBS containing 2% (volume percent) FBS, PE-labeled goat anti-human IgG Fcγ antibody (Jackson Immuno Research, Catalog No.: 109-116-170) was added and incubated at 4°C for 1 hour. The cells were washed and resuspended in PBS containing 2% (volume percent) FBS, and the fluorescent signals were detected using a flow cytometer (Sartorius, IQue3) to detect the binding between the anti-hGPRC5D antibody and CHO-K1-hGPRC5D. lowBinding to hGPRC5D was analyzed by the mean fluorescence intensity (MFI) of staining. As shown in Figure 3, in cells with low hGPRC5D expression, the chimeric antibodies Chi-89, Chi-105, Chi-164, and Chi-180 show binding levels comparable to BM-mAb, while Chi-128, Chi-131, and Chi-169 show strong target binding ability.

[0144] Example 5: Measurement of binding activity of anti-hGPRC5D antibody to tumor cells Cell concentration 3×10 5 H929 and MM1S human myeloma cell suspensions at 100 μL / mL were plated in 96-well U-bottom plates, gradient-diluted (final concentration range 4.1-1000 ng / mL, 3-fold gradient dilution) anti-hGPRC5D antibody was added, mixed uniformly, and incubated at 4°C for 1 hour. After washing the cells with PBS containing 2% (volume percent) FBS, PE-labeled goat anti-human IgG Fcγ antibody (Jackson Immuno Research, Catalog No.: 109-116-170) was added and incubated at 4°C for 1 hour. The cells were washed and resuspended in PBS containing 2% (volume percent) FBS, and the fluorescent signal was detected using a flow cytometer (Sartorius, IQue3), and the binding of anti-hGPRC5D antibody to human myeloma cells was analyzed by the mean fluorescence intensity (MFI) of staining and plotted using Graphpad Prism. As can be seen from Figures 4A and 4B, the chimeric antibodies Chi-89, Chi-105, Chi-128, Chi-131, and Chi-169 all exhibit concentration gradient-dependent binding activity to H929 cells and MM1S cells.

[0145] Example 6: Cross-species activity assay of anti-hGPRC5D antibodies Cell concentration 5×10 5Cell suspensions of CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D at 100 μL / well were plated on a 96-well U-bottom plate, and gradient-diluted (final concentration range: 4.6-10000 ng / mL, 3-fold gradient dilution) anti-hGPRC5D antibody was added, mixed uniformly, and incubated at 4°C for 1 hour. After washing with PBS containing 2% (volume percent) FBS, PE-labeled goat anti-human IgG Fcγ antibody (Jackson Immuno Research, catalog number: 109-116-170) was added and incubated at 4°C for 1 hour. The cells were washed and resuspended in PBS containing 2% (volume percent) FBS, and the fluorescent signals were detected using a flow cytometer (Sartorius, IQue3), and the binding of anti-hGPRC5D antibodies to CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D cells was analyzed by the mean fluorescence intensity (MFI) of staining. As shown in Figures 5A and 5B, the chimeric antibodies Chi-89, Chi-128, Chi-131, and Chi-169 all exhibited concentration gradient-dependent binding activity to CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D cells. This indicates that, compared to BM-mAb, these anti-hGPRC5D antibodies have cross-binding activity with monkey and mouse GPRC5D, whereas the chimeric antibody Chi-105 has cross-binding activity only with monkey GPRC5D.

[0146] Example 7: Verification of specificity of anti-hGPRC5D antibody 1. Verification of binding of anti-hGPRC5D chimeric antibody to the same family protein at the cellular level In addition to GPRC5D, known GPRC family proteins also include GPRC5A (RAIG1), GPRC5B (RAIG2), and GPRC5C (RAIG3). The cDNAs encoding full-length GPRC5A (SEQ ID NO: 46), GPRC5B (SEQ ID NO: 47), and GPRC5C (SEQ ID NO: 48) were obtained by gene synthesis and subcloned into vector pcDNA3.1(+) to obtain pcDNA3.1-GPRC5A-GFP, pcDNA3.1-GPRC5B-GFP, and pcDNA3.1-GPRC5C-GFP recombinant plasmids. The recombinant plasmid DNAs were transfected into CHO-K1 cells, respectively, according to the instruction manual of Lipofectamine 3000 transfection reagent (Thermo, Cat. No.: L3000015), to obtain stable cell lines of CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C.

[0147] Cell concentration 5×10 5Cell suspensions of CHO-K1-GPRC5A, CHO-K1-GPRC5B and CHO-K1-GPRC5C at 100 μL / well were plated on a 96-well U-bottom plate, gradient-diluted (final concentration range: 4.1-3000 ng / mL, 3-fold gradient dilution) anti-hGPRC5D antibody was added, mixed uniformly and incubated at 4°C for 1 hour. After washing with PBS containing 2% (volume percent) FBS, APC-labeled goat anti-human IgG Fcγ antibody (Jackson Immuno Research, catalog number: 109-135-098) was added and incubated at 4°C for 1 hour. The cells were washed and resuspended in PBS containing 2% (volume percent) FBS, and the fluorescent signals were detected using a flow cytometer (Sartorius, IQue3), and the binding of anti-hGPRC5D antibodies to CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C cells was analyzed by the mean fluorescence intensity (MFI) of staining. As shown in Figures 6A, 6B, and 6C, the chimeric antibodies Chi-89, Chi-105, Chi-128, Chi-131, and Chi-169 all did not bind to CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C cells within the concentration range of 4.1 to 3000 ng / mL, and BM-mAb shows nonspecific binding to CHO-K1-GPRC5A cells at concentrations above 1000 ng / mL.

[0148] 2. Verification of binding of anti-hGPRC5D chimeric antibody to CHO-K1 cells First, biotin-conjugated anti-hGPRC5D antibodies were prepared according to the instructions for EZ-linkNHS-Biotin biotin reagent (Thermo, catalog number: 20217), and named Biotin-BM-mAb, Biotin-Chi-89, Biotin-Chi-128, and Biotin-Chi-131, respectively. Here, Biotin means biotin.

[0149] Cell concentration 3×10 5CHO-K1 cell suspensions at 100 μL / well were plated in 96-well U-bottom plates, gradient-diluted (3-fold gradient dilution, final concentration range 4.6-10000 ng / mL) biotin-conjugated anti-hGPRC5D antibodies were added, mixed uniformly, and incubated at 4°C for 1 hour. After washing with PBS containing 2% (volume percent) FBS, PE-labeled streptavidin (BD, Catalog No.: 554061) was added and incubated at 4°C for 1 hour. The cells were washed and resuspended in PBS containing 2% (volume percent) FBS, and the fluorescent signal was detected using a flow cytometer (Sartorius, IQue3), and the mean fluorescence intensity (MFI) of staining was used to analyze whether the anti-hGPRC5D antibodies, whose MFI signal was amplified after biotin conjugation, nonspecifically bound to CHO-K1 cells. The results are shown in FIG. 7, in which the biotin-conjugated chimeric antibodies Chi-89, Chi-128, and Chi-131 all produced non-binding signals with CHO-K1 cells.

[0150] Example 8: Internalization activity of anti-hGPRC5D antibody MM1S cells were resuspended in RPMI 1640 medium (Gibco, Catalog No.: 22400071) containing 10% (volume percent) FBS to a cell concentration of 2 × 10 6The cells / mL were adjusted and 20 μL / well was plated in a 96-well V-bottom plate. Antibody internalization reagent (Sartorius, Catalog No.: 90564) was added to anti-hGPRC5D antibodies BM-mAb, Chi-89, Chi-128, and Chi-131, and isotype control antibody hIgG1 (Biointron, Catalog No.: B117901), respectively, and incubated at 37°C in the dark for 15 min. Then, gradient dilutions of the above antibodies were performed (final concentration range was 0.46-1000ng / mL, 3-fold gradient dilution) using RPMI1640 medium (Gibco, Catalog No.: 22400071) containing 10% (volume percent) FBS, and 20 μL / well was pipetted, mixed evenly with the cell suspension, and incubated at 37°C for 2 h. After incubation, the cells were washed and resuspended in PBS containing 2% (volume percent) FBS, and the fluorescent signals were detected using a flow cytometer (Sartorius, IQue3), and the internalization activity of anti-hGPRC5D antibodies was analyzed by the mean fluorescence intensity (MFI) of staining. The results are shown in Figure 8, and the internalization of target GPRC5D mediated by chimeric antibodies Chi-89, Chi-128, and Chi-131 was at a level comparable to that of BM-mAb, resulting in only a weak internalization effect.

Claims

1. HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, 9, 17, 25 or 33; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, 10, 18, 26 or 34, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, 11, 19, 27 or 35 a heavy chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, 12, 20, 28 or 36; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, 13, 21, 29 or 37, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, 14, 22, 30 or 38 a light chain variable region comprising An isolated anti-GPRC5D antibody, or an antigen-binding fragment thereof, comprising:

2. The anti-GPRC5D antibody (i) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:3 a heavy chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:6 a light chain variable region comprising (ii) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 11 a heavy chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 14 a light chain variable region comprising (iii) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19 a heavy chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 22 a light chain variable region comprising (iv) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:27 a heavy chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30 or a light chain variable region comprising (v) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35 a heavy chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38 a light chain variable region comprising The isolated anti-GPRC5D antibody or antigen-binding fragment thereof of claim 1, comprising:

3. An isolated anti-GPRC5D antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having the variable region sequences set forth in SEQ ID NO: 7, 15, 23, 31, or 39, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having the variable region sequences set forth in SEQ ID NO: 8, 16, 24, 32, or 40.

4. a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO:7, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO:8; a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO:15, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO:16; a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO:23, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO:

24.

4. The isolated anti-GPRC5D antibody or antigen-binding fragment thereof according to claim 3, comprising: a light chain variable region comprising HCDR1, HCDR2, and LCDR3; a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO: 31, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO: 32; or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 comprising the variable region sequence set forth in SEQ ID NO: 39, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 comprising the variable region sequence set forth in SEQ ID NO:

40.

5. the heavy chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 7, 15, 23, 31, or 39, and / or The isolated anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 8, 16, 24, 32 or 40.

6. The heavy chain variable region and the light chain variable region are (i) the heavy chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:8; (ii) the heavy chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 15, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 16; (iii) the heavy chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:23, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:24; (iv) the heavy chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 31, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 32; or (v) the heavy chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 39, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:

40. The isolated anti-GPRC5D antibody or antigen-binding fragment thereof according to claim 5, which is any one of the following:

7. The antigen-binding fragment may be a Fab fragment, a Fab' fragment, a F(ab') ２ fragment, Fd fragment, Fv fragment, isolated CDR region or scFv, Optionally, the anti-GPRC5D antibody is a chimeric antibody, a humanized antibody, or a fully human antibody; The isolated anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the anti-GPRC5D antibody is optionally of the IgG1, IgG2, IgG3, or IgG4 isotype.

8. The anti-GPRC5D antibody or antigen fragment thereof binds to the human GPRC5D antigen and optionally also has any one or more of the following properties: (1) binding to monkey GPRC5D antigen; (2) binding to murine GPRC5D antigen, or (3) An isolated anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which does not bind to one or more of GPRC5A, GPRC5B, and GPRC5C.

9. A fusion protein comprising the anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

10. A pharmaceutical composition comprising the anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and further comprising a pharmaceutically acceptable carrier.

11. An isolated nucleic acid encoding the anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

12. A vector comprising the nucleic acid of claim 11.

13. A host cell comprising the vector of claim 12.

14. A method for preparing the anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising culturing a host cell containing a nucleic acid encoding the GPRC5D antibody or antigen-binding fragment thereof, and recovering the anti-GPRC5D antibody or antigen-binding fragment thereof from the host cell or host cell culture medium.

15. Use of an anti-GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in the preparation of a medicament for treating a disease, reducing a tumor, or inhibiting tumor cell growth, wherein preferably the disease is cancer or an autoimmune disease, preferably the cancer is multiple myeloma.

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