Antibodies binding to gprc5d and uses thereof

Abstract

Provided are antibodies binding to GPRC5D and uses thereof. Specifically provided are anti-GPRC5D antibodies and antigen-binding fragments thereof, nucleic acids encoding the same, vectors comprising the nucleic acids, cells comprising the vectors, pharmaceutical compositions comprising the same, and uses thereof in reducing or inhibiting growth of tumor cells, treating diseases, etc.

Description

Technical Field

[0001] This disclosure relates to antibodies, and more particularly to antibodies that bind to GPRC5D, their preparation methods, and their uses. Background Technology

[0002] Multiple myeloma (MM) is a malignant tumor of plasma cells characterized by the abnormal proliferation of plasma cells derived from the bone marrow. This leads to a variety of disease-related symptoms in MM patients, including osteonecrosis, bone marrow infiltration, kidney failure, and immunodeficiency. Current treatment options for multiple myeloma include proteasome inhibitors, immunomodulatory drugs, monoclonal antibodies, and stem cell transplantation.

[0003] GPRC5D (G protein-coupled receptor class C group 5member D) belongs to the retinoic acid-induced orphan G protein-coupled receptor (RAIG) family. It is a seven-transmembrane protein composed of 345 amino acid residues. Its normal physiological function is related to duracin structure, but its specific biological function or ligands remain unclear. Some studies have shown that GPRC5D has an expression threshold exceeding 50% in plasma cells derived from malignant bone marrow in 65% of multiple myeloma patients, and its expression is independent of BCMA (B cell maturation antigen). Other studies have shown that GPRC5D overexpression is associated with tumor burden and poor prognosis in multiple myeloma patients. In normal tissues, GPRC5D is not expressed or is expressed at very low levels, except in hair follicles of the skin and plasma cells derived from bone marrow. GPRC5D may provide new treatment options for multiple myeloma patients, highlighting its clinical value.

[0004] As a potential therapeutic target, several antibodies targeting GPRC5D are under development, but the options are still limited and more are needed. Summary of the Invention

[0005] This disclosure provides antibodies that bind to GPRC5D, as well as related nucleic acids, vectors, cells, compositions, preparation methods, and uses that can encode the provided antibodies.

[0006] On one hand, this disclosure provides an isolated anti-GPRC5D antibody or its antigen-binding fragment, wherein the anti-GPRC5D antibody comprises:

[0007] Heavy chain variable region, which contains:

[0008] HCDR1, which contains the amino acid sequence shown in SEQ ID NO: 1, 9, 17, 25 or 33,

[0009] HCDR2, comprising the amino acid sequence shown in SEQ ID NO: 2, 10, 18, 26 or 34, and

[0010] HCDR3, comprising the amino acid sequence shown in SEQ ID NO: 3, 11, 19, 27 or 35; and

[0011] The light chain variable region, which includes:

[0012] LCDR1, which contains the amino acid sequence shown in SEQ ID NO: 4, 12, 20, 28 or 36,

[0013] LCDR2, comprising the amino acid sequence shown in SEQ ID NO: 5, 13, 21, 29 or 37, and

[0014] LCDR3 contains the amino acid sequence shown in SEQ ID NO: 6, 14, 22, 30 or 38.

[0015] In one embodiment of this disclosure, the anti-GPRC5D antibody comprises:

[0016] (i) The heavy chain variable region, which includes:

[0017] HCDR1 contains the amino acid sequence shown in SEQ ID NO: 1.

[0018] HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and

[0019] HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3; and

[0020] The light chain variable region, which includes:

[0021] LCDR1 contains the amino acid sequence shown in SEQ ID NO: 4.

[0022] LCDR2, containing the amino acid sequence shown in SEQ ID NO: 5, and

[0023] LCDR3 contains the amino acid sequence shown in SEQ ID NO: 6;

[0024] (ii) Heavy chain variable region, which includes:

[0025] HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9.

[0026] HCDR2 containing the amino acid sequence shown in SEQ ID NO: 10, and

[0027] HCDR3 containing the amino acid sequence shown in SEQ ID NO: 11; and

[0028] The light chain variable region, which includes:

[0029] LCDR1 contains the amino acid sequence shown in SEQ ID NO: 12.

[0030] LCDR2, containing the amino acid sequence shown in SEQ ID NO: 13, and

[0031] LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14;

[0032] (iii) Heavy chain variable region, which includes:

[0033] HCDR1 contains the amino acid sequence shown in SEQ ID NO: 17.

[0034] HCDR2 containing the amino acid sequence shown in SEQ ID NO: 18, and

[0035] HCDR3 containing the amino acid sequence shown in SEQ ID NO: 19; and

[0036] The light chain variable region, which includes:

[0037] LCDR1 contains the amino acid sequence shown in SEQ ID NO: 20.

[0038] LCDR2, containing the amino acid sequence shown in SEQ ID NO: 21, and

[0039] LCDR3 contains the amino acid sequence shown in SEQ ID NO: 22;

[0040] (iv) Heavy chain variable region, which includes:

[0041] HCDR1 contains the amino acid sequence shown in SEQ ID NO: 25.

[0042] HCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, and

[0043] HCDR3 containing the amino acid sequence shown in SEQ ID NO: 27; and

[0044] The light chain variable region, which includes:

[0045] LCDR1 contains the amino acid sequence shown in SEQ ID NO: 28.

[0046] LCDR2, which contains the amino acid sequence shown in SEQ ID NO: 29, and

[0047] LCDR3 containing the amino acid sequence shown in SEQ ID NO: 30; or

[0048] (v) Heavy chain variable region, which includes:

[0049] HCDR1 contains the amino acid sequence shown in SEQ ID NO: 33.

[0050] HCDR2 containing the amino acid sequence shown in SEQ ID NO: 34, and

[0051] HCDR3 containing the amino acid sequence shown in SEQ ID NO: 35; and

[0052] The light chain variable region, which includes:

[0053] LCDR1 contains the amino acid sequence shown in SEQ ID NO: 36.

[0054] LCDR2, containing the amino acid sequence shown in SEQ ID NO: 37, and

[0055] LCDR3 contains the amino acid sequence shown in SEQ ID NO: 38.

[0056] On one hand, this disclosure provides an isolated anti-GPRC5D antibody or its antigen-binding fragment, wherein the anti-GPRC5D antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the variable region sequence shown in SEQ ID NO: 7, 15, 23, 31 or 39, and the light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the variable region sequence shown in SEQ ID NO: 8, 16, 24, 32 or 40.

[0057] On the one hand, this disclosure provides a fusion protein comprising the anti-GPRC5D antibody or its antigen-binding fragment described herein.

[0058] On the one hand, this disclosure provides a pharmaceutical composition comprising an anti-GPRC5D antibody or an antigen-binding fragment thereof as described herein, or the fusion protein described herein, and further comprising a pharmaceutically acceptable carrier.

[0059] On the one hand, this disclosure provides an isolated nucleic acid that encodes the anti-GPRC5D antibody or its antigen-binding fragment described herein.

[0060] On the one hand, this disclosure provides a carrier containing the nucleic acid described herein.

[0061] On the one hand, this disclosure provides a host cell that contains the vector described herein or has the nucleic acid described herein integrated into its genome.

[0062] On one hand, this disclosure provides a method for preparing the anti-GPRC5D antibody or its antigen-binding fragment as described herein, comprising: culturing the host cells, and recovering the anti-GPRC5D antibody or its antigen-binding fragment from the host cells or host cell culture medium.

[0063] On the one hand, this disclosure provides the use of the aforementioned anti-GPRC5D antibody or its antigen-binding fragment, and the aforementioned fusion protein, in the preparation of medicaments for treating diseases, preferably cancer or autoimmune diseases.

[0064] On one hand, this disclosure provides a method for reducing tumors or inhibiting tumor cell growth in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of the anti-GPRC5D antibody or its antigen-binding fragment as described herein, the fusion protein, or the pharmaceutical composition thereof.

[0065] On one hand, this disclosure provides a method for treating a disease in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-GPRC5D antibody or its antigen-binding fragment as described herein, the fusion protein, or the pharmaceutical composition thereof, preferably the disease being cancer or an autoimmune disease.

[0066] On the one hand, this disclosure provides a kit comprising an anti-GPRC5D antibody or its antigen-binding fragment as described herein, the immunoconjugate described herein, or the pharmaceutical composition described herein. Attached Figure Description

[0067] Figure 1 For some mice, anti-hGPRC5D antibody and CHO-K1-hGPRC5D hi OD of CHO-K1-cynoGPRC5D (CHO-K1-monkey GPRC5D) and CHO-K1-murineGPRC5D (CHO-K1-mouse GPRC5D) by cell ELISA 450 ;

[0068] Figure 2 For FACS detection of MFI and EC by chimeric antibody binding to cells expressing high levels of hGPRC5D 50 ;

[0069] Figure 3 For FACS detection of MFI of chimeric antibody binding to cells with low hGPRC5D expression;

[0070] Figure 4A For FACS detection of MFI of anti-GPRC5D antibody binding to H929 cells;

[0071] Figure 4B MFI for FACS detection of anti-GPRC5D antibody binding to MM1S cells;

[0072] Figure 5A MFI for FACS detection of anti-GPRC5D antibody binding to CHO-K1-cynoGPRC5D cells;

[0073] Figure 5B MFI for FACS detection of anti-GPRC5D antibody binding to CHO-K1-munneGPRC5D cells;

[0074] Figure 6A MFI for FACS detection of anti-GPRC5D antibody binding to CHO-K1-GPRC5A cells;

[0075] Figure 6B MFI for FACS detection of anti-GPRC5D antibody binding to CHO-K1-GPRC5B cells;

[0076] Figure 6C MFI for FACS detection of anti-GPRC5D antibody binding to CHO-K1-GPRC5C cells;

[0077] Figure 7 MFI for FACS detection of anti-GPRC5D antibody binding to CHO-K1 cells;

[0078] Figure 8 This study aims to determine the internalization activity of the anti-GPRC5D antibody. Detailed Implementation

[0079] This document describes exemplary embodiments of the present disclosure. However, those skilled in the art will understand that the scope of protection of the present disclosure is not limited thereto. Various modifications, alterations, or changes can be made based on the spirit and concept of the present disclosure, and the modified, altered, or changed content still falls within the scope of the present disclosure.

[0080] the term

[0081] The term "antibody" is used in its broadest sense and encompasses a wide range of antibody structures, including both natural and artificial antibodies, such as monoclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), and single-chain antibodies, as long as they exhibit the desired antigen-binding activity. An antigen-binding fragment refers to a molecule other than the intact antibody that contains a portion of the 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.

[0082] The term "isotype" refers to the type of antibody encoded by the heavy chain constant region gene. In one embodiment, the anti-GPRC5D antibody disclosed herein is an IgG1 or IgG4 isotype. The anti-GPRC5D antibody and its antigen-binding fragment herein can be derived from any species, including but not limited to mice, rats, rabbits, primates, llamas, and humans. The anti-GPRC5D antibody can be a chimeric antibody, a humanized antibody, or a fully human antibody. In one particular embodiment, the anti-GPRC5D antibody provided herein is humanized. In one particular embodiment, the anti-GPRC5D antibody provided herein is chimeric.

[0083] The term "chimeric antibody" refers to an antibody having 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 may comprise a murine variable region and a human constant region.

[0084] "Humanized antibody" is an antibody that contains a complementarity-determining region (CDR) derived from a non-human antibody, and a frame region and a constant region derived from a human antibody. For example, the humanized antibody that binds GPRC5D provided herein may contain a CDR derived from one or more murine antibodies, as well as a human frame region and a constant region.

[0085] The term "monoclonal antibody" ("mAb") refers to an antibody molecule of a single-molecule composition. A monoclonal antibody composition exhibits single binding specificity and affinity for a specific epitope, or, in the case of a bispecific monoclonal antibody, exhibits dual binding specificity for two different epitopes. mAb is an example of an isolated antibody. mAbs can be produced using hybridoma techniques, recombinant techniques, transgenic techniques, or other techniques known to those skilled in the art.

[0086] The term "variable domain" or "variable region" refers to a domain of an antibody involved in the binding of the antibody to an antigen. For example, natural four-chain antibodies (e.g., derived from humans, mice, etc.) have a heavy chain variable region (VH) and a light chain variable region (VL), while antibodies derived from animals such as camels or sharks have only a heavy chain variable domain. In most cases, each variable domain of a natural antibody consists essentially of four "frame regions" and three "complementarity-determining regions." The four frame regions are referred to as frame region 1 (FR1), frame region 2 (FR2), frame region 3 (FR3), and frame region 4 (FR4); these frame regions are separated by complementarity-determining regions (CDRs), as referred to in the art and hereinafter. Therefore, the general structure of a variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. As a specific example, the general structure of a heavy chain variable region can be represented as: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. As a specific example, the general structure of the variable region of the light chain can be represented as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4.

[0087] The "CDR" (Complementarity-Determining Region), also known as the "Hypervariant Region (HVR)", is a key component in antibodies. For example, naturally occurring four-chain antibodies typically contain six CDRs: three in the heavy chain variable region (HCDR1, HCDR2, and HCDR3) and three in the light chain variable region (LCDR1, LCDR2, and LCDR3). Heavy chain-only antibodies or antibodies with a single variable domain typically have three CDRs (CDR1, CDR2, and CDR3).

[0088] Currently, there are many methods for defining CDRs. The Kabat definition, based on sequence variability, is the most commonly used (Elvin A. Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); while the Chothia 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 the AbM antibody modeling software from Oxford Molecular. The "contact" definition of CDRs is based on the analysis of available complex crystal structures. However, it should be noted that the boundaries of CDRs for the same antibody variable region obtained by different methods of definition may differ; that is, the CDR sequences for the same antibody variable region defined by different methods may differ. Therefore, when referring to antibodies defined by specific CDR sequences according to certain divisions of this disclosure, the scope of said antibodies also includes antibodies defined by CDR sequences converted to any other arbitrary definition (e.g., a combination of one or more of the definitions of IMGT, Chothia, AbM, etc.). Although the CDRs claimed in this disclosure are based on the sequences shown in Tables S1 and S2, the amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this disclosure. For example, under another definition, the amino acid sequence of the chimeric antibody Chi-131 heavy chain CDR1 is NYVMH (SEQ ID NO: 71).

[0089] The term “frame region” (FR) refers to amino acid residues with variable domains other than the CDR residues defined herein.

[0090] The term "isolated" refers to a target compound (e.g., an antibody or nucleic acid) that has been isolated from its natural environment.

[0091] As used in this article, the term "EC" 50 "IC50" refers to the effective concentration, the 50% maximum response of the antibody. As used herein, the term "IC50" is... 50 "This refers to the inhibitory concentration, which represents the 50% maximum response of the antibody." (EC) 50 and IC 50Both can be measured by ELISA or FACS analysis or any other method known in the art.

[0092] The term "K" D "In this context, it refers to the equilibrium dissociation constant, expressed as molar concentration (M). The K of the antibody..." D The value can be determined using methods known in the art. A preferred assay antibody K D The method is to use surface plasmon resonance, and more preferably to use biosensor systems, such as the Biacore system.

[0093] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Preferably, the subject according to this disclosure is a human. Unless otherwise stated, the terms "patient" or "subject" may be used interchangeably.

[0094] As used herein, “cancer” refers to a physiological condition in mammals characterized by uncontrolled cell growth. Uncontrolled cell division and growth can lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. “Cancer” or “cancer tissue” may include tumors.

[0095] The term "treatment" refers to an attempt to alter the natural course of disease in an individual and can be a clinical intervention performed for prevention or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving the prognosis.

[0096] As used herein, the term “therapeutic effective amount” refers to the amount of a compound, composition, or combination of drugs necessary to provide a therapeutic benefit to a subject.

[0097] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0098] "A pharmaceutical composition is a composition that contains an active ingredient and a pharmaceutically acceptable carrier."

[0099] The "percentage of identity (%)" for an amino acid sequence refers to the percentage of amino acid residues in the sequence to be aligned that are identical to those in the specific amino acid sequence shown herein, after aligning the sequence to be aligned with the specific amino acid sequence shown herein, and if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, without any conserved substitutions as part of the sequence identity. Amino acid sequence identity alignment can be performed using various methods within the art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the aligned sequences, including any algorithm required to achieve maximum alignment across the full length of the compared sequences.

[0100] The terms “Xn” and “Xaa” are equivalent and refer to unspecified amino acids, the scope of which is specified by subsequent definitions in the relevant descriptions.

[0101] The terms “comprising,” “containing,” or “including,” and variations thereof, shall be understood as “including but not limited to,” meaning that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be covered.

[0102] In this document, unless the context clearly indicates otherwise, singular terms encompass plural referents, and vice versa.

[0103] For purposes of description and disclosure, all patents, patent applications, and other identified publications are expressly incorporated herein by reference. These publications are provided solely because their publications predate the filing date of this disclosure. All statements regarding the dates of these documents or representations of their contents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of the dates or contents of these documents. Furthermore, in any country, any reference to these publications herein does not constitute an endorsement that such publications are part of the general knowledge in the art. Various aspects of this disclosure will be further described in detail in the following sections.

[0104] I. Anti-GPRC5D antibody and its antigen-binding fragment

[0105] In this article, the terms “human GPRC5D” and “hGPRC5D” are used interchangeably; “antibody that binds to GPRC5D” and “anti-GPRC5D antibody” are used interchangeably.

[0106] This disclosure provides novel antibodies that bind to GPRC5D. Anti-GPRC5D antibodies exhibit a number of other desirable properties for therapeutic and / or diagnostic applications. For example, in some embodiments, the anti-GPRC5D antibody exhibits good specificity for GPRC5D. In some embodiments, the anti-GPRC5D antibody can bind to monkey GPRC5D and / or mouse GPRC5D, thereby facilitating pharmacological and / or safety evaluations during antibody development. In some embodiments, the anti-GPRC5D antibody provided herein exhibits better antigen-binding performance compared to some known antibodies, such as better binding ability to cells with low GPRC5D expression. In some embodiments, the anti-GPRC5D antibody exhibits good killing properties against tumor cells, such as multiple myeloma cells.

[0107] On one hand, this disclosure provides an isolated anti-GPRC5D antibody or its antigen-binding fragment, wherein the anti-GPRC5D antibody comprises:

[0108] Heavy chain variable region, which contains:

[0109] HCDR1, which contains the amino acid sequence shown in SEQ ID NO: 1, 9, 17, 25 or 33,

[0110] HCDR2, comprising the amino acid sequence shown in SEQ ID NO: 2, 10, 18, 26 or 34, and

[0111] HCDR3, comprising the amino acid sequence shown in SEQ ID NO: 3, 11, 19, 27 or 35; and

[0112] The light chain variable region, which includes:

[0113] LCDR1, which contains the amino acid sequence shown in SEQ ID NO: 4, 12, 20, 28 or 36,

[0114] LCDR2, comprising the amino acid sequence shown in SEQ ID NO: 5, 13, 21, 29 or 37, and

[0115] LCDR3 contains the amino acid sequence shown in SEQ ID NO: 6, 14, 22, 30 or 38.

[0116] In some embodiments, the anti-GPRC5D antibody comprises:

[0117] The heavy chain variable region comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3; and

[0118] The light chain variable region comprises: LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0119] In some embodiments, the anti-GPRC5D antibody comprises:

[0120] The heavy chain variable region comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 9, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 11; and

[0121] The light chain variable region comprises: LCDR1 containing the amino acid sequence shown in SEQ ID NO: 12, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 14.

[0122] In some embodiments, the anti-GPRC5D antibody comprises:

[0123] The heavy chain variable region comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 17, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 18, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 19; and

[0124] The light chain variable region comprises: LCDR1 containing the amino acid sequence shown in SEQ ID NO: 20, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 22.

[0125] In some embodiments, the anti-GPRC5D antibody comprises:

[0126] The heavy chain variable region comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 27; and

[0127] The light chain variable region comprises: LCDR1 containing the amino acid sequence shown in SEQ ID NO: 28, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 30.

[0128] In some embodiments, the anti-GPRC5D antibody comprises:

[0129] The heavy chain variable region comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 33, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 34, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 35; and

[0130] The light chain variable region comprises: LCDR1 containing the amino acid sequence shown in SEQ ID NO: 36, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38.

[0131] In some embodiments, an isolated anti-GPRC5D antibody or its antigen-binding fragment is provided, wherein the anti-GPRC5D antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of the variable region sequence shown in SEQ ID NO: 7, 15, 23, 31, or 39, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 of the variable region sequence shown in SEQ ID NO: 8, 16, 24, 32, or 40. In some specific embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the variable region sequence shown in SEQ ID NO: 7, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the variable region sequence shown in SEQ ID NO: 8. In some specific embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the variable region sequence shown in SEQ ID NO: 15, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the variable region sequence shown in SEQ ID NO: 16. In some specific embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the variable region sequence shown in SEQ ID NO: 23, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the variable region sequence shown in SEQ ID NO: 24. In some specific embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the variable region sequence shown in SEQ ID NO: 31, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the variable region sequence shown in SEQ ID NO: 32. In some specific embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the variable region sequence shown in SEQ ID NO: 39, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the variable region sequence shown in SEQ ID NO: 40. In some embodiments, the CDR regions of the antibody may be defined according to kabat, Chothia, IMGT, or other definitions.

[0132] 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 with the sequence shown in SEQ ID NO: 7, 15, 23, 31, or 39. In some specific embodiments, the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO: 7, 15, 23, 31, or 39.

[0133] 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 with the sequence shown in SEQ ID NO: 8, 16, 24, 32, or 40. In some specific embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, 16, 24, 32, or 40.

[0134] 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 with 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%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 8, 16, 24, 32, or 40. In some specific embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, 15, 23, 31 or 39, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, 16, 24, 32 or 40.

[0135] 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 with the sequence shown 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 with the sequence shown in SEQ ID NO: 8. Furthermore, in some such embodiments, the anti-GPRC5D antibody further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0136] 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 with the sequence shown 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 with the sequence shown in SEQ ID NO: 16. Furthermore, in some such embodiments, the anti-GPRC5D antibody further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 9, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 10, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 11, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 12, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 14.

[0137] 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 with the sequence shown in SEQ ID NO: 23, 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 with the sequence shown in SEQ ID NO: 24. Furthermore, in some such embodiments, the anti-GPRC5D antibody further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 17, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 18, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 20, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 22.

[0138] 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 with the sequence shown in SEQ ID NO: 31, 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 with the sequence shown in SEQ ID NO: 32. Furthermore, in some such embodiments, the anti-GPRC5D antibody further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 27, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 28, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 30.

[0139] 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 with the sequence shown in SEQ ID NO: 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 with the sequence shown in SEQ ID NO: 40. Furthermore, in some such embodiments, the anti-GPRC5D antibody further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 33, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 34, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 35, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 36, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38.

[0140] In some specific embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 8.

[0141] In some specific embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 16.

[0142] In some specific embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 24.

[0143] In some specific embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 31, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 32.

[0144] In some specific embodiments, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 39, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 40.

[0145] In some embodiments, the anti-GPRC5D antibody provided herein comprises a heavy chain and a light chain, which, in addition to the corresponding heavy chain variable region and light chain variable region described herein, also include a constant region. From the N-terminus to the 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.

[0146] In some embodiments, the light chain constant region of the anti-GPRC5D antibody is the human κ chain constant region. In some embodiments, the light chain constant region of the anti-GPRC5D antibody is the human λ chain constant region.

[0147] 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.

[0148] In some embodiments, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 69, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 70.

[0149] 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 with the sequence shown 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 with the sequence shown in SEQ ID NO: 51. In one specific embodiment, the anti-GPRC5D antibody comprises a heavy chain of the amino acid sequence shown in SEQ ID NO: 49 and a light chain of the amino acid sequence shown in SEQ ID NO: 51. In other 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 with the sequence shown 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 with the sequence shown in SEQ ID NO: 55. In one specific embodiment, the anti-GPRC5D antibody comprises a heavy chain of the amino acid sequence shown in SEQ ID NO: 53 and a light chain of the amino acid sequence shown in SEQ ID NO: 55. In other 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 with the sequence shown 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 with the sequence shown in SEQ ID NO: 59. In one specific embodiment, the anti-GPRC5D antibody comprises a heavy chain of the amino acid sequence shown in SEQ ID NO: 57 and a light chain of the amino acid sequence shown in SEQ ID NO: 59.In other 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 with the sequence shown 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 with the sequence shown in SEQ ID NO: 63. In one specific embodiment, the anti-GPRC5D antibody comprises a heavy chain of the amino acid sequence shown in SEQ ID NO: 61 and a light chain of the amino acid sequence shown in SEQ ID NO: 63. In other 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 with the sequence shown 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 with the sequence shown in SEQ ID NO: 67. In one specific embodiment, the anti-GPRC5D antibody comprises a heavy chain of the amino acid sequence shown in SEQ ID NO: 65 and a light chain of the amino acid sequence shown in SEQ ID NO: 67.

[0150] In some embodiments, the anti-GPRC5D antibody cross-reacts with monkey GPRC5D antigen and / or mouse GPRC5D antigen.

[0151] In some embodiments, the anti-GPRC5D antibody or its antigen fragment binds to the human GPRC5D antigen, and optionally also has any one or more of the following properties:

[0152] (1) Binding to monkey GPRC5D antigen;

[0153] (2) Binding to mouse GPRC5D antigen; or

[0154] (3) Not combined with one or more of GPRC5A, GPRC5B and GPRC5C.

[0155] In some embodiments, the anti-GPRC5D antibody or its antigen fragment binds to the human GPRC5D antigen, also binds to the monkey GPRC5D antigen and / or the mouse GPRC5D antigen, and further does not bind to GPRC5A, GPRC5B and GPRC5C.

[0156] In some implementations, the anti-GPRC5D antibody is a monoclonal antibody.

[0157] In some implementations, the anti-GPRC5D antibody is a monospecific antibody.

[0158] In some embodiments, the anti-GPRC5D antibody is a multispecific antibody, such as a bispecific antibody or a trispecific antibody.

[0159] On the other hand, this document provides anti-GPRC5D antibodies or antigen-binding fragments thereof that bind to the same epitope on GPRC5D as any of the exemplary antibodies provided herein, such as those binding to the same epitope on Chi-131, Chi-169, Chi-89, etc. On the other hand, this document provides anti-GPRC5D antibodies or antigen-binding fragments thereof that compete with any of the exemplary antibodies provided herein, such as those competing with Chi-131 for GPRC5D binding, or Chi-169 for GPRC5D binding. Binding to GPRC5D can be measured by ELISA, flow cytometry, surface plasmon resonance (SPR) assay, or any other method known in the art.

[0160] This document also provides exemplary monoclonal antibodies binding 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 some of the 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.

[0161] Table S1: Heavy Chain CDR Sequences

[0162]

[0163] Table S2: Light Chain CDR Sequences

[0164]

[0165] Table S3: Partial Antibody Variable Region Sequence

[0166]

[0167]

[0168] II. Antibody variants

[0169] In some embodiments, variants of the anti-GPRC5D antibody or antigen-binding fragments thereof are provided. Amino acid sequence variants 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, deleting amino acid residues from the amino acid sequence of the antibody, inserting amino acid residues into the amino acid sequence, and / or substituting amino acid residues in the amino acid sequence. Any combination of deletions, insertions, and substitutions can be produced to obtain the final construct, provided that the final variant possesses the desired characteristics, such as antigen-binding activity, tumor-killing efficacy, etc.

[0170] III. Fusion Protein

[0171] In some embodiments, this disclosure provides a fusion protein comprising the anti-GPRC5D antibody described herein or an antigen-binding fragment thereof.

[0172] IV. Pharmaceutical Compositions

[0173] This disclosure provides pharmaceutical compositions comprising the described anti-GPRC5D antibody or its antigen-binding fragment, or the described fusion protein, and further comprising a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated from any one or more of the Chi-89, Chi-105, Chi-128, Chi-131, Chi-164, Chi-169, and Chi-180 antibodies, along with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other reagents.

[0174] V. Isolated nucleic acids

[0175] This disclosure provides isolated nucleic acids encoding the anti-GPRC5D antibody or its antigen-binding fragment described herein. In some embodiments, the nucleic acid encodes an antigen-binding fragment such as Chi-89, Chi-105, Chi-128, Chi-131, Chi-164, Chi-169, or Chi-180. In some embodiments, the nucleic acid encodes an antibody such as Chi-89, Chi-105, Chi-128, Chi-131, Chi-164, Chi-169, and Chi-180. Exemplary examples of nucleic acid sequences of anti-GPRC5D antibodies or their antigen-binding fragments are listed in the sequence listing.

[0176] VI. Carrier

[0177] This disclosure provides vectors comprising the isolated nucleic acids described herein. In some embodiments, the vector is a cloning vector; in other embodiments, the vector is an expression vector. The expression vector may be any expression vector capable of expressing the anti-GPRC5D antibody described herein or its antigen-binding fragment; a specific example is pcDNA3.1.

[0178] VII. Host cell

[0179] In some embodiments, this disclosure provides a host cell that contains the vector described herein or has the nucleic acid integrated into its genome. The host cell is a suitable host cell for cloning or expressing an anti-GPRC5D antibody or its antigen-binding fragment. 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 yeast cells, mammalian cells, or other cells suitable for preparing an anti-GPRC5D antibody or its antigen-binding fragment. Mammalian cells, for example, are Chinese hamster ovary (CHO) cells or CHO-S cells.

[0180] VIII. Methods for preparing anti-GPRC5D antibodies and their antigen-binding fragments

[0181] In some embodiments, this disclosure provides a method for preparing the anti-GPRC5D antibody or its antigen-binding fragment thereof, the method comprising: culturing the host cells described herein, and recovering the anti-GPRC5D antibody or its antigen-binding fragment from the host cells or a host cell culture medium.

[0182] To generate the aforementioned anti-GPRC5D antibody or its antigen-binding fragment, a nucleic acid encoding the anti-GPRC5D antibody or its antigen-binding fragment is inserted into a vector for further cloning and / or expression in host cells. The nucleic acid can be obtained using various methods well-known in the art, such as gene splicing and chemical synthesis. Recovery can be performed using steps such as centrifugation and affinity chromatography.

[0183] IX. Uses

[0184] In some implementations, the anti-GPRC5D antibody or its antigen-binding fragment provided herein, and the fusion protein therein, can be used to treat cancer, reduce tumors, or inhibit tumor cell growth.

[0185] This disclosure provides the use of the described anti-GPRC5D antibody or its antigen-binding fragment, or the described fusion protein, in the preparation of pharmaceuticals. 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 tumor cell growth.

[0186] This disclosure provides a method for reducing or inhibiting tumor cell growth in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of the antiGPRC5D antibody or its antigen-binding fragment, the fusion protein, or the pharmaceutical composition.

[0187] This disclosure provides a method of treating a disease in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of the said anti-GPRC5D antibody or its antigen-binding fragment, the said fusion protein, or the said pharmaceutical composition. Subjects requiring treatment include those who already have a disease or condition, and those who may develop a disease or condition and whose aim is to prevent, delay, or reduce the disease or condition.

[0188] The aforementioned diseases are either cancer or autoimmune diseases. In some embodiments, the autoimmune disease is, for example, systemic lupus erythematosus and / or rheumatoid arthritis. In some embodiments, the cancer is multiple myeloma.

[0189] In some embodiments, a method for detecting or measuring GPRC5D in a sample is provided, which includes contacting the sample with an anti-GPRC5D antibody or an antigen-binding fragment thereof as described herein and detecting or measuring the binding complex.

[0190] X. Reagent kit

[0191] This disclosure provides a kit comprising the anti-GPRC5D antibody or its antigen-binding fragment described herein, the fusion protein described herein, or the pharmaceutical composition described herein.

[0192] This document describes a kit comprising the described anti-GPRC5D antibody or its antigen-binding fragment, the described fusion protein, or the described pharmaceutical composition. The kit may be used to implement the intended use of the anti-GPRC5D antibody or its antigen-binding fragment, the described fusion protein, or the described pharmaceutical composition, or for other uses. In some embodiments, the kit may include the described anti-GPRC5D antibody or its antigen-binding fragment, the described fusion protein, or the described pharmaceutical composition, as well as reagents 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 required from a commercial and user perspective, such as additional buffers, diluents, needles, syringes, etc.

[0193] This disclosure also provides the following specific implementation schemes, but the scope of protection of this disclosure is not limited thereto:

[0194] Implementation Scheme 1. An isolated anti-GPRC5D antibody or an antigen-binding fragment thereof, wherein the anti-GPRC5D antibody comprises:

[0195] Heavy chain variable region, which contains:

[0196] HCDR1, which contains the amino acid sequence shown in SEQ ID NO: 1, 9, 17, 25 or 33,

[0197] HCDR2, comprising the amino acid sequence shown in SEQ ID NO: 2, 10, 18, 26 or 34, and

[0198] HCDR3, comprising the amino acid sequence shown in SEQ ID NO: 3, 11, 19, 27 or 35; and

[0199] The light chain variable region, which includes:

[0200] LCDR1, which contains the amino acid sequence shown in SEQ ID NO: 4, 12, 20, 28 or 36,

[0201] LCDR2, comprising the amino acid sequence shown in SEQ ID NO: 5, 13, 21, 29 or 37, and

[0202] LCDR3 contains the amino acid sequence shown in SEQ ID NO: 6, 14, 22, 30 or 38.

[0203] Implementation Scheme 2. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to Implementation Scheme 1, wherein the anti-GPRC5D antibody comprises:

[0204] (i) The heavy chain variable region, which includes:

[0205] HCDR1 contains the amino acid sequence shown in SEQ ID NO: 1.

[0206] HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and

[0207] HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3; and

[0208] The light chain variable region, which includes:

[0209] LCDR1 contains the amino acid sequence shown in SEQ ID NO: 4.

[0210] LCDR2, containing the amino acid sequence shown in SEQ ID NO: 5, and

[0211] LCDR3 contains the amino acid sequence shown in SEQ ID NO: 6;

[0212] (ii) Heavy chain variable region, which includes:

[0213] HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9.

[0214] HCDR2 containing the amino acid sequence shown in SEQ ID NO: 10, and

[0215] HCDR3 containing the amino acid sequence shown in SEQ ID NO: 11; and

[0216] The light chain variable region, which includes:

[0217] LCDR1 contains the amino acid sequence shown in SEQ ID NO: 12.

[0218] LCDR2, containing the amino acid sequence shown in SEQ ID NO: 13, and

[0219] LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14;

[0220] (iii) Heavy chain variable region, which includes:

[0221] HCDR1 contains the amino acid sequence shown in SEQ ID NO: 17.

[0222] HCDR2 containing the amino acid sequence shown in SEQ ID NO: 18, and

[0223] HCDR3 containing the amino acid sequence shown in SEQ ID NO: 19; and

[0224] The light chain variable region, which includes:

[0225] LCDR1 contains the amino acid sequence shown in SEQ ID NO: 20.

[0226] LCDR2, containing the amino acid sequence shown in SEQ ID NO: 21, and

[0227] LCDR3 contains the amino acid sequence shown in SEQ ID NO: 22;

[0228] (iv) Heavy chain variable region, which includes:

[0229] HCDR1 contains the amino acid sequence shown in SEQ ID NO: 25.

[0230] HCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, and

[0231] HCDR3 containing the amino acid sequence shown in SEQ ID NO: 27; and

[0232] The light chain variable region, which includes:

[0233] LCDR1 contains the amino acid sequence shown in SEQ ID NO: 28.

[0234] LCDR2, which contains the amino acid sequence shown in SEQ ID NO: 29, and

[0235] LCDR3 containing the amino acid sequence shown in SEQ ID NO: 30; or

[0236] (v) Heavy chain variable region, which includes:

[0237] HCDR1 contains the amino acid sequence shown in SEQ ID NO: 33.

[0238] HCDR2 containing the amino acid sequence shown in SEQ ID NO: 34, and

[0239] HCDR3 containing the amino acid sequence shown in SEQ ID NO: 35; and

[0240] The light chain variable region, which includes:

[0241] LCDR1 contains the amino acid sequence shown in SEQ ID NO: 36.

[0242] LCDR2, containing the amino acid sequence shown in SEQ ID NO: 37, and

[0243] LCDR3 contains the amino acid sequence shown in SEQ ID NO: 38.

[0244] Implementation Scheme 3. An isolated anti-GPRC5D antibody or an antigen-binding fragment thereof, wherein the anti-GPRC5D antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the variable region sequence shown in SEQ ID NO: 7, 15, 23, 31 or 39, and the light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the variable region sequence shown in SEQ ID NO: 8, 16, 24, 32 or 40.

[0245] Implementation Scheme 4. The anti-GPRC5D antibody or its antigen-binding fragment according to Implementation Scheme 3, wherein the anti-GPRC5D antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the variable region sequence shown in SEQ ID NO: 7, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the variable region sequence shown in SEQ ID NO: 8; wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the variable region sequence shown in SEQ ID NO: 15, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the variable region sequence shown in SEQ ID NO: 16; wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the variable region sequence shown in SEQ ID NO: 23, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the variable region sequence shown in SEQ ID NO: 24; wherein the heavy chain variable region comprises HCDRI, HCDR2, and HCDR3 of the variable region sequence shown in SEQ ID NO: 31, and the light chain variable region comprises SEQ ID NO: 8. The variable region sequences shown in SEQ ID NO: 32 are LCDR1, LCDR2, and LCDR3; or, the heavy chain variable region includes the variable region sequences shown in SEQ ID NO: 39, HCDR1, HCDR2, and HCDR3, and the light chain variable region includes the variable region sequences shown in SEQ ID NO: 40, LCDR1, LCDR2, and LCDR3.

[0246] Implementation Scheme 5. An isolated anti-GPRC5D antibody or its antigen-binding fragment according to any one of Implementation Schemes 1-4, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 7, 15, 23, 31 or 39.

[0247] Implementation Scheme 6. An isolated anti-GPRC5D antibody or its antigen-binding fragment according to any one of Implementation Schemes 1-5, wherein the light chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 8, 16, 24, 32 or 40.

[0248] Implementation Scheme 7. An isolated anti-GPRC5D antibody or its antigen-binding fragment according to any one of Implementation Schemes 1-6, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% identity with 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 with the sequence shown in SEQ ID NO: 8, 16, 24, 32 or 40.

[0249] Implementation Scheme 8. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to Implementation Scheme 7, wherein the heavy chain variable region and the light chain variable region are selected from any one of the following:

[0250] (i) The heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 8;

[0251] (ii) The heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 15, and the light chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 16;

[0252] (iii) The heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 23, and the light chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 24;

[0253] (iv) The heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 31, and the light chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 32; or,

[0254] (v) The heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 39, and the light chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 40.

[0255] Implementation Scheme 9. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to Implementation Scheme 7, wherein the heavy chain variable region and the light chain variable region are selected from any one of the following:

[0256] (i) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8;

[0257] (ii) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16;

[0258] (iii) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24;

[0259] (iv) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 32; or,

[0260] (v) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 39, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 40.

[0261] Implementation Scheme 10. An isolated anti-GPRC5D antibody or its antigen-binding fragment according to any one of Implementation Schemes 1-9, wherein the antigen-binding fragment is a Fab fragment, a Fab′ fragment, an F(ab′)2 fragment, an Fd fragment, an Fv fragment, an isolated CDR region, or a scFv.

[0262] Implementation Scheme 11. An isolated anti-GPRC5D antibody or its antigen-binding fragment according to any one of Implementation Schemes 1-10, wherein the anti-GPRC5D antibody is chimeric, humanized or fully human.

[0263] Implementation Scheme 12. An isolated anti-GPRC5D antibody or its antigen-binding fragment according to any one of Implementation Schemes 1-11, wherein the anti-GPRC5D antibody is an IgG1, IgG2, IgG3 or IgG4 isotype.

[0264] Implementation Scheme 13. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to any one of Implementation Schemes 1-12, wherein the anti-GPRC5D antibody or its antigen fragment binds to the human GPRC5D antigen, and optionally further possesses any one or more of the following properties:

[0265] (1) Binding to monkey GPRC5D antigen;

[0266] (2) Binding to mouse GPRC5D antigen;

[0267] (3) Not combined with one or more of GPRC5A, GPRC5B and GPRC5C.

[0268] Implementation Scheme 14. An isolated anti-GPRC5D antibody or its antigen-binding fragment according to any one of Implementation Schemes 1-13, wherein the anti-GPRC5D antibody is a monospecific antibody or a multispecific antibody.

[0269] Implementation Scheme 15. A fusion protein comprising an anti-GPRC5D antibody or an antigen-binding fragment thereof according to any one of Implementation Schemes 1-14.

[0270] Implementation Scheme 16. A pharmaceutical composition comprising an anti-GPRC5D antibody or an antigen-binding fragment thereof according to any one of Implementation Schemes 1-14, or an immunoconjugate according to Implementation Scheme 15, further comprising a pharmaceutically acceptable carrier.

[0271] Implementation Scheme 17. An isolated nucleic acid encoding an anti-GPRC5D antibody or an antigen-binding fragment thereof according to any one of Implementation Schemes 1-14.

[0272] Implementation Scheme 18. A vector comprising the nucleic acid described in Implementation Scheme 17.

[0273] Implementation Scheme 19. A host cell comprising the vector according to Implementation Scheme 18 or having the nucleic acid according to Implementation Scheme 17 integrated into its genome.

[0274] Implementation Scheme 20. A method for preparing an anti-GPRC5D antibody or an antigen-binding fragment thereof according to any one of Implementation Schemes 1-14, comprising: culturing the host cells of Implementation Scheme 19, and recovering the anti-GPRC5D antibody or an antigen-binding fragment thereof from the host cells or a host cell culture medium.

[0275] Implementation Scheme 21. An anti-GPRC5D antibody or its antigen-binding fragment according to any one of Implementation Schemes 1-14, a fusion protein according to Implementation Scheme 15, or a pharmaceutical composition according to Implementation Scheme 16, for treating a disease, or reducing a tumor or inhibiting tumor cell growth; preferably, the disease is cancer or an autoimmune disease.

[0276] Implementation Scheme 22. Use of the anti-GPRC5D antibody or its antigen-binding fragment according to any one of Implementation Schemes 1-14, or the fusion protein according to Implementation Scheme 15, in the preparation of a medicament for treating a disease or for reducing tumors or inhibiting tumor cell growth, preferably the disease being cancer or an autoimmune disease.

[0277] Implementation Scheme 23. A method for reducing tumor or inhibiting tumor cell growth in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-GPRC5D antibody or an antigen-binding fragment thereof according to any one of Implementation Schemes 1-14, a fusion protein according to Implementation Scheme 15, or a pharmaceutical composition according to Implementation Scheme 16.

[0278] Implementation Scheme 24. A method of treating a disease in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-GPRC5D antibody or an antigen-binding fragment thereof according to any one of Implementation Schemes 1-14, a fusion protein according to Implementation Scheme 15, or a pharmaceutical composition according to Implementation Scheme 16, preferably the disease being cancer or an autoimmune disease.

[0279] Implementation Scheme 25. The scheme according to Implementation Scheme 21, 22 or 24, wherein the cancer is multiple myeloma.

[0280] Implementation Scheme 26. A kit comprising an anti-GPRC5D antibody or an antigen-binding fragment thereof according to any one of Implementation Schemes 1-14, a fusion protein according to Implementation Scheme 15, or a pharmaceutical composition according to Implementation Scheme 16.

[0281] Example

[0282] Example 1: Antigen Immunization

[0283] 1. DNA antigen immunization

[0284] The cDNA sequence encoding the human GPRC5D protein (amino acid sequence as shown in SEQ ID NO: 41) was obtained through gene synthesis and subcloned into the expression vector pcDNA3.1(+) to construct the plasmid pcDNA3.1-hGPRC5D. Large-scale plasmid preparation was performed according to the instructions of the endotoxin-free large-scale extraction kit (QIAGEN, Cat: 12391).

[0285] Using plasmid pcDNA3.1-hGPRC5D as the antigen, A / J mice (Nanjing University Model Animal Institute), BALB / c mice (Shanghai Lingchang), and SJL mice (Beijing Vital River) were immunized, respectively. First, hyaluronidase (Sigma, Cat: H4272) was pre-injected into the muscles of the left and right hind limbs of each mouse for pretreatment. Then, CpG (InvivoGen, Cat: tlrl-1826) and plasmid pcDNA3.1-hGPRC5D were mixed at a 1:1 mass ratio, and the mixed antigen complex was injected into the pre-treated muscle sites of the mice using a live gene delivery system (Shanghai Tarissa Biotechnology Co., Ltd., Type II). The same immunization was repeated every 2-3 weeks, for a total of 4 immunizations. After immunization, serum was collected from each mouse.

[0286] 2. Evaluation of mouse serum titers

[0287] To evaluate the immune response to anti-human GPRC5D antibodies produced in mice immunized with the DNA antigen, antibody titers in mouse serum were detected using FACS. Mice with higher serum titers were selected for subsequent pre-splenic cell fusion pulse immunization.

[0288] 3. Cell antigen immunity

[0289] Following the instructions of the Lipofectamin 3000 transfection reagent (Thermo, Cat: L3000015), plasmid pcDNA3.1-hGPRC5D was transfected into CHO-K1 cells to obtain a stable, high-expression hGPRC5D cell line, CHO-K1-hGPRC5D. h i. Using the above CHO-K1-hGPRC5D hihi Cells were used as antigens for shock immunization in mice: 3-4 days before spleen cell fusion, the final cell concentration was 5 × 10⁻⁶. 7 CHO-K1-hGPRC5D per mL hi The cell suspension was mixed with CpG (InvivoGen, Cat: tlrl-1826) at a final concentration of 0.5 mg / mL and injected into mice intraperitoneally at a dose of 100 μL / mouse.

[0290] Example 2: Screening of mouse anti-human GPRC5D antibodies

[0291] 1. Preparation of hybridoma cells

[0292] On the day of fusion, mouse spleens were aseptically harvested, ground, and then washed with erythrocyte lysis buffer (Sigma, Cat: R7757) and PBS. The spleen cells were resuspended in electrofusion buffer (BTX, Cat: 47-0001) and then mixed with SP2 / 0 mouse myeloma cells at a 2:1 ratio. Spleen cell fusion was then performed using an electrofusion apparatus (BTX). The fused cells were diluted with hybridoma medium (Gibco, Cat: 12045-076) containing 1×HAT (Gibco, Cat: 21060-017) and cultured at 37°C and 5% CO2 for 7-10 days to prepare hybridoma cells. The culture supernatant of the hybridoma cells was collected for screening of mouse anti-human GPRC5D antibodies.

[0293] 2. Construction of stable cell lines

[0294] To evaluate the species-cross-binding activity of mouse anti-human GPRC5D antibodies at the cellular level, the following cell lines were constructed: cDNA encoding full-length monkey GPRC5D (amino acid sequence as shown in SEQ ID NO: 42) and mouse GPRC5D (amino acid sequence as shown in SEQ ID NO: 43) were synthesized and then subcloned into the vector pcDNA3.1(+) to obtain recombinant plasmids pcDNA3.1-cynoGPRC5D and pcDNA3.1-murineGPRC5D. Following the instructions of the Lipofectamin 3000 transfection reagent (Thermo, Cat: L3000015), plasmids pcDNA3.1-cynoGPRC5D and pcDNA3.1-murineGPRC5D were transfected into CHO K1 cells to obtain stable CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D cell lines, respectively.

[0295] 3. Initial screening using cell ELISA

[0296] Collect CHO-K1-hGPRC5D in the logarithmic growth phase hi Cells were resuspended in DMEM / F-12 (Gibco, Cat: 11320033) medium containing 10% FBS and the cell concentration was adjusted to 1×10⁻⁶. 6 Cells / mL were seeded at 100 μL / well in 96-well plates and incubated overnight at 37°C and 5% CO2. The next day, the plates were washed with PBS, and 4% (v / v) paraformaldehyde (Beyotime, Cat: P0099) was added for fixation at room temperature for 30 minutes. After washing with PBS, 200 μL / well of PBS containing 3% (v / v) BSA was added and the plates were blocked at room temperature for 2 hours. After blocking, 100 μL / well of hybridoma 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, Cat: 115-035-068) was added and incubated at room temperature for 1 hour. After washing with PBS, 100 μL / well of TMB reaction solution (Solepro, Cat: RP1200) was added and incubated at room temperature in the dark for 5 minutes. The reaction was terminated with 0.5 M H2SO4. The OD450 absorbance was read using a Bio-rad iMark microplate reader. Hybridoma cells corresponding to culture supernatant with absorbance greater than 1.0 were selected as positive clones for subsequent FACS screening.

[0297] 4. Flow cytometry (FACS) for antibody screening

[0298] The cell concentration was 5 × 105 Cell suspension of CHO-K1-cynoGPRC5D cells at a density of 100 μL / well was seeded into 96-well U-plates. 100 μL of culture supernatant from positive clones was added to each well, and the mixture was incubated at 4°C for 1 hour. After washing with PBS containing 2% (v / v) FBS, Alexa Fluor 488-conjugated goat anti-mouse IgG+IgM (H+L) antibody (Jackson Immuno Research, Cat: 115-545-044) was added, and the cells were incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, IQue3), and the binding of anti-hGPRC5D antibody to CHO-K1-cynoGPRC5D cells was analyzed by mean fluorescence intensity (MFI).

[0299] 5. Subcloning preparation

[0300] Selected cells after ELISA and FACS screening, and then compared with CHO-K1-hGPRC5D hi Hybridoma cells that were positive for both CHO-K1-cynoGPRC5D were expanded and cultured. The expanded cells were mixed with semi-solid medium D (Stemcell, Cat: 3810) at a volume ratio of 1:10 and incubated at 37°C and 5% CO2 for 5 days. Single cell clusters were aspirated under a 4x microscope and transferred to 96-well plates coated with hybridoma medium (Gibco, Cat: 12045076), and cultured for another 2 days at 37°C and 5% CO2. The supernatant from each subclonal cell culture was collected for further subclonal screening.

[0301] 6. Subcloning screening

[0302] (1) FACS screening of anti-hGPRC5D antibodies

[0303] The cell concentration was 3×10 5H929 (NCI H929) cell suspension was seeded at 100 μL / well in 96-well U-type plates, and 100 μL / well of subclonal cell culture supernatant or an equal volume of medium was added. After mixing, the plates were incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS, and then Alexa Fluor 488 conjugated goat anti-mouse IgG+IgM (H+L) antibody (Jackson Immuno Research, Cat: 115-545-044) was added. The plates were incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, IQue3). The binding of mouse anti-hGPRC5D antibody to H929 cells was analyzed by the mean fluorescence intensity (MFI). Some results are shown in Table 1.

[0304] Table 1. Assay results of FACS binding of mouse anti-hGPRC5D antibody to H929 cells.

[0305] mu-7 53151 mu-74.3 18078 mu-131 12826 mu-9 10704 mu-89 11919 mu-153 46867 mu-45 21606 mu-105 13248 mu-164 14184 mu-38.1 75956 mu-125 11670 mu-169 59318 mu-35 54719 mu-126 10271 nu-180 26579 mu-51 95531 mu-128 15661 mu-183 27008 mu-58 464150 mu-129 7904 Medium 8242

[0306] (2) Cellular ELISA screening of anti-hGPRC5D antibody

[0307] Collect CHO-K1-hGPRC5D in the logarithmic growth phase. hi Cells, CHO-K1-cynoGPRC5D cells, CHO-K1-murineGPRC5D cells, and CHO-K1 cells were resuspended in DMEM / F-12 (Gibco, Cat: 11320033) medium containing 10% (v / v) FBS, and the cell concentration was adjusted to 1×10⁻⁶. 6Cells / mL were seeded at 100 μL / well in 96-well plates and incubated overnight at 37°C and 5% CO2. The next day, the plates were washed with PBS, and 4% (v / v) paraformaldehyde (Beyotime, Cat: P0099) was added for fixation at room temperature for 30 minutes. After washing with PBS, 200 μL / well of PBS containing 3% (v / v) BSA was added and the plates were blocked at room temperature for 2 hours. After blocking, 50 μL / well of subclonal cell culture supernatant was added to each well of 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, Cat: 115-035-068) was added and incubated at room temperature for 1 hour. After washing with PBS, 100 μL / well of TMB reaction solution (Solepro, Cat: RP1200) was added and incubated at room temperature in the dark for 5 minutes. The reaction was terminated with 0.5 M H2SO4. The OD450 absorbance was read using a Bio-rad iMark microplate reader. A bar chart of subclonal absorbance was plotted using Graphpad Prism, as shown below. Figure 1 Typical mouse anti-hGPRC5D antibodies (e.g., mu-89, mu-105, mu-126, mu-128, mu-131, mu-153, mu-164, mu-169, mn-180, and mu-183) are shown, targeting CHO-K1-hGPRC5D. hi The absorbance values ​​of the cells, CHO-K1-cynoGPRC5D cells and / or CHO-K1-murineGPRC5D cells, were at least 2-fold different from those of the control cells CHO-K1.

[0308] Example 3: Preparation of anti-hGPRC5D chimeric antibody

[0309] 1. Sequencing of the variable region encoding mouse anti-hGPRC5D antibody

[0310] Total RNA was isolated from selected hybridoma cells according to the instructions of the RNA extraction kit (Takara, Cat: 9767), and first-strand cDNA was synthesized using a reverse transcription kit (Thermo, Cat: K1652). Using the first-strand cDNA as a template, primers for the mouse heavy and light chain constant regions were mixed, and the variable region sequence of the mouse anti-hGPRC5D antibody was obtained by cloning and sequencing using polymerase chain reaction (PCR).

[0311] 2. Construction of chimeric antibody expression vector

[0312] Using a chemical synthesis method, 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 heavy chain constant region and Kappa light chain constant region, respectively. Recombinant human-mouse chimeric antibody expression vectors were constructed using the pcDNA3.1(+) vector and transfected for expression to prepare chimeric antibodies. The full length of each chimeric antibody, as well as the nucleotide and amino acid sequences of VH / VL, are detailed in Table 2.

[0313] Table 2. Full-length chimeric antibody and nucleotide and amino acid sequences of VH / VL.

[0314]

[0315] 3. Construction of positive control antibody expression vector

[0316] GC5B596 was selected as the reference antibody (monoclonal antibody, source WO2018017786A2). The nucleotide sequences of antibodies VH (SEQ ID NO: 44) and VL (SEQ ID NO: 45) were obtained through chemical synthesis and ligated to the nucleotide sequences of the human IgG1 heavy chain constant region and the Kappa light chain constant region, respectively. An expression vector for expressing the positive reference antibody was constructed using the pcDNA3.1(+) vector and transfected for expression to prepare the positive reference antibody. The positive reference antibody (monoclonal antibody) was named BM-mAb in this paper.

[0317] 4. Preparation of anti-hGPRC5D chimeric antibody and positive reference antibody

[0318] Transient transfection expression of anti-hGPRC5D antibody was performed according to the operating manual of the expiCHO expression system (Gibco, Cat: A29129). After transfection, expiCHO cells were cultured at 37°C with 8% CO2 for 7 days with shaking. The cell culture supernatant was collected, and the clarified culture supernatant was loaded onto a protein A column (GE Healthcare, Cat: 17-5474). The protein A column was washed with 10 column volumes of PBS buffer, and then eluted and collected with acetate buffer (300mM acetic acid, pH 3.6). The collected IgG antibody fraction was replaced with PBS buffer through an ultrafiltration device (molecular weight cutoff 30kDa, Millipore, Cat: UFC903024) to obtain the anti-hGPRC5D antibody solution.

[0319] Example 4: Assay of the binding activity of anti-hGPRC5D antibody to cells stably expressing human GPRC5D

[0320] 1. Binding properties of anti-hGPRC5D chimeric antibody to cells stably overexpressing human GPRC5D

[0321] The cell concentration was 3×10 5 CHO-K1-hGPRC5D per mL hi (GPRC5D expression abundance is approximately 2E+06-3E+06 antigens / cell) Cell suspension was seeded at 100 μL / well in 96-well U-type plates. Serially diluted anti-hGPRC5D antibody (final concentration range 4.1-3000 ng / mL, 3-fold serial dilution) was added, mixed, and incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS, and then PE-conjugated goat anti-human IgG Fcγ antibody (Jackson Immuno Research, Cat: 109-116-170) was added and incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, IQue3). The mean fluorescence intensity (MFI) of the staining was used to analyze the interaction between anti-hGPRC5D antibody and CHO-K1-hGPRC5D. hi Cell binding. EC calculated using GraphpadPrism. 50 The analysis results are as follows Figure 2 As shown, 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 on hGPRC5D cells with high expression, and had a higher maximum antigen binding amount than BM-mAb.

[0322] 2. Binding properties of anti-hGPRC5D chimeric antibody to cells with stable low expression of human GPRC5D

[0323] Following the instructions of the Lipofectamin 3000 transfection reagent (Thermo, Cat: L3000015), plasmid pcDNA3.1-hGPRC5D was transfected into CHO-K1 cells to obtain CHO-K1-hGPRC5D. low Stable low-expression cell lines (GPRC5D expression abundance of approximately 2000-2500 antigens / cell). Cell concentration was set at 3 × 10⁻⁶ cells / cell. 5 CHO-K1-hGPRC5D per mL lowCell suspension was seeded at 100 μL / well in 96-well U-type plates. Serially diluted anti-hGPRC5D antibody (final concentration range 4.1-3000 ng / mL, 3-fold serial dilution) was added, and the mixture was incubated at 4°C for 1 hour. After washing cells with PBS containing 2% (v / v) FBS, PE-conjugated goat anti-human IgG Fcγ antibody (Jackson Immuno Research, Cat: 109-116-170) was added, and the cells were incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, IQue3). The mean fluorescence intensity (MFI) of the staining was used to analyze the interaction between anti-hGPRC5D antibody and CHO-K1-hGPRC5D. low Cellular binding. For example... Figure 3 As shown, on cells with low hGPRC5D expression, the chimeric antibodies Chi-89, Chi-105, Chi-164, and Chi-180 had comparable binding levels to BM-mAb, while Chi-128, Chi-131, and Chi-169 exhibited stronger target binding ability.

[0324] Example 5: Assay of the binding activity of anti-hGPRC5D antibody to tumor cells

[0325] Cell concentrations of 3×10⁻⁶ were respectively... 5 H929 and MM1S human myeloma cell suspensions were seeded at 100 μL / well in 96-well U-type plates. Serially diluted anti-hGPRC5D antibody (final concentration range 4.1-1000 ng / mL, 3-fold serial dilution) was added, mixed, and incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS, and then PE-conjugated goat anti-human IgG Fcγ antibody (Jackson Immuno Research, Cat: 109-116-170) was added and incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, IQue3). The binding of anti-hGPRC5D antibody to human myeloma cells was analyzed by mean fluorescence intensity (MFI) of the stained cells. Graphpad Prism assay was used. Figure 4A and Figure 4B It was found that the chimeric antibodies Chi-89, Chi-105, Chi-128, Chi-131 and Chi-169 all exhibited concentration-gradient-dependent binding activity on H929 and MM1S cells.

[0326] Example 6: Determination of cross-species activity of anti-hGPRC5D antibody

[0327] Cell concentrations of 5×10⁻⁶ were respectively... 5 CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D cell suspensions were seeded at 100 μL / well in 96-well U-plates. Serially diluted anti-hGPRC5D antibody (final concentration range 4.6-10000 ng / mL, 3-fold serial dilution) was added, mixed, and incubated at 4°C for 1 hour. After washing with PBS containing 2% (v / v) FBS, PE-conjugated goat anti-human IgG Fcγ antibody (Jackson Inmuno Research, Cat: 109116170) was added, and incubated at 4°C for 1 hour. Cells were washed and resuspended with PBS containing 2% (v / v) FBS, and fluorescence signals were detected using flow cytometry (Sartorius, IQue3). The binding of anti-hGPRC5D antibody to CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D cells was analyzed by mean fluorescence intensity (MFI). Figure 5A and 5B As shown, chimeric antibodies Chi-89, Chi-128, Chi-131, and Chi-169 exhibited concentration-gradient binding activity in both CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D cells, indicating that these anti-hGPRC5D antibodies, compared to BM-mAb, possess cross-binding activity with both monkey and mouse GPRC5D. In contrast, chimeric antibody Chi-105 only exhibits cross-binding activity with monkey GPRC5D.

[0328] Example 7: Specificity verification of anti-hGPRC5D antibody

[0329] 1. Verification of the binding of anti-hGPRC5D chimeric antibody to its family of proteins at the cellular level.

[0330] Besides GPRC5D, the GPRC family of proteins includes GPRC5A (RAIG1), GPRC5B (RAIG2), and GPRC5C (RAIG3). cDNAs encoding full-length GPRC5A (SEQ ID NO: 46), GPRC5B (SEQ ID NO: 47), and GPRC5C (SEQ ID NO: 48) were synthesized and subcloned into the vector pcDNA3.1(+) to obtain recombinant plasmids pcDNA3.1-GPRC5A-GFP, pcDNA3.1-GPRC5B-GFP, and pcDNA3.1-GPRC5C-GFP. Following the instructions of the Lipofectamin 3000 transfection reagent (Thermo, Cat: L3000015), the recombinant plasmid DNA was transfected into CHO-K1 cells to obtain stable cell lines CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C.

[0331] Cell concentrations of 5×10⁻⁶ were respectively... 5 CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C cell suspensions were seeded at 100 μL / well in 96-well U-plates. Serially diluted anti-hGPRC5D antibody (final concentration range 4.1-3000 ng / mL, 3-fold serial dilution) was added, mixed, and incubated at 4°C for 1 hour. After washing with PBS containing 2% (v / v) FBS, APC-conjugated goat anti-human IgG Fcγ antibody (Jackson Immuno Research, Cat: 109-135-098) was added, and incubated at 4°C for 1 hour. Cells were washed and resuspended in PBS containing 2% (v / v) FBS. Fluorescence signals were detected using a flow cytometer (Sartorius, IQue3). The binding of anti-hGPRC5D antibody to CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C cells was analyzed by mean fluorescence intensity (MFI) of the staining. Figure 6A , 6B As shown in Figure 6C, the chimeric antibodies Chi 89, Chi 105, Chi 128, Chi 131, and Chi-169 did not bind to CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C cells at concentrations ranging from 4.13000 ng / mL, while BM-mAb showed nonspecific binding to CHO-K1-GPRC5A cells at concentrations greater than 1000 ng / mL.

[0332] 2. Validation of the binding of anti-hGPRC5D chimeric antibody to CHO-K1 cells

[0333] First, following the instructions of the EZ-link NHS-Biotin reagent (Thermo, Cat: 20217), biotin-conjugated anti-hGPRC5D antibodies were prepared and named Biotin-BM-mAb, Biotin-Chi-89, Biotin-Chi-128, and Biotin-Chi-131, respectively, where Biotin represents biotin.

[0334] The cell concentration was 3×10 5 CHO-K1 cell suspension (cells / mL) was seeded at 100 μL / well in 96-well U-type plates. Serially diluted biotin-conjugated anti-hGPRC5D antibody (3-fold serial dilution, final concentration range 4.6-10000 ng / mL) was added, mixed, and incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS, and then PE-conjugated streptavidin (BD, Cat: 554061) was added, followed by incubation at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS and resuspended. Fluorescence signals were detected using flow cytometry (Sartorius, IQue3). The mean fluorescence intensity (MFI) was used to analyze whether the biotin-conjugated anti-hGPRC5D antibody, with its amplified MFI signal, exhibited non-specific binding to CHO-K1 cells. Results are as follows: Figure 7 As shown, the biotin-conjugated chimeric antibodies Chi-89, Chi-128, and Chi-131 all produced non-binding signals with CHO-K1 cells.

[0335] Example 8: Internalization activity of anti-hGPRC5D antibody

[0336] MM1S cells were resuspended in RPMI 1640 medium (Gibco, Cat: 22400071) containing 10% (v / v) FBS and the cell concentration was adjusted to 2 × 10⁻⁶ cells / mL. 6Cells were seeded at a rate of 20 μL / well in 96-well V plates. Antibody internalization reagent (Sartorius, Cat: 90564) was added to anti-hGPRC5D antibody BM-mAb, Chi-89, Chi-128, and Chi-131, as well as isotype control antibody hIgG1 (Biointron, Cat: B117901), and the plates were incubated at 37°C in the dark for 15 minutes. Then, the antibodies were serially diluted (final concentration range: 0.46-1000 ng / mL, 3-fold serial dilution) in RPMI 1640 medium (Gibco, Cat: 22400071) containing 10% (v / v) FBS. 20 μL of each diluted antibody was added to each well and mixed with the cell suspension, then incubated at 37°C for 2 hours. After incubation, cells were washed with PBS containing 2% (v / v) FBS and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, IQue3), and the internalization activity of the anti-hGPRC5D antibody was analyzed by the mean fluorescence intensity (MFI) of the staining. Results are as follows: Figure 8 As shown, the internalization of the target GPRC5D mediated by chimeric antibodies Chi-89, Chi-128, and Chi-131 was comparable to that of BM-mAb, producing only a weak internalization effect.

Claims

1. An isolated anti-GPRC5D antibody or an antigen-binding fragment thereof, wherein the anti-GPRC5D antibody comprises: Heavy chain variable region, which contains: HCDR1, as shown in the amino acid sequence of SEQ ID NO: 1, HCDR2, as shown in the amino acid sequence of SEQ ID NO: 2, and HCDR3, as shown in the amino acid sequence of SEQ ID NO: 3; and The light chain variable region, which includes: LCDR1, as shown in the amino acid sequence of SEQ ID NO: 4, LCDR2, as shown in the amino acid sequence of SEQ ID NO: 5, and LCDR3, as shown in the amino acid sequence of SEQ ID NO:

6.

2. An isolated anti-GPRC5D antibody or its antigen-binding fragment, wherein the anti-GPRC5D antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of the variable region sequence shown in SEQ ID NO: 7, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 of the variable region sequence shown in SEQ ID NO: 8; wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined using the same definition, and the definition is a Kabat definition, a Chothia definition, or an IMGT definition.

3. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO:

7.

4. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:

7.

5. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence having at least 87% identity with the sequence shown in SEQ ID NO:

7.

6. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence having at least 89% identity with the sequence shown in SEQ ID NO:

7.

7. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the light chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO:

8.

8. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the light chain variable region comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:

8.

9. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the light chain variable region comprises an amino acid sequence having at least 87% identity with the sequence shown in SEQ ID NO:

8.

10. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the light chain variable region comprises an amino acid sequence having at least 89% identity with the sequence shown in SEQ ID NO:

8.

11. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO:

8.

12. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:

8.

13. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence having at least 87% identity with the sequence shown in SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 87% identity with the sequence shown in SEQ ID NO:

8.

14. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence having at least 89% identity with the sequence shown in SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence having at least 89% identity with the sequence shown in SEQ ID NO:

8.

15. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or an Fv fragment.

16. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the antigen-binding fragment is scFv.

17. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the anti-GPRC5D antibody is chimeric, humanized, or fully human.

18. The isolated anti-GPRC5D antibody or its antigen-binding fragment according to claim 1 or 2, wherein the anti-GPRC5D antibody is an IgG1, IgG2, IgG3 or IgG4 isotype.

19. A pharmaceutical composition comprising an anti-GPRC5D antibody or an antigen-binding fragment thereof according to any one of claims 1-18, further comprising a pharmaceutically acceptable carrier.

20. An isolated nucleic acid encoding an anti-GPRC5D antibody or an antigen-binding fragment thereof according to any one of claims 1-18.

21. A vector comprising the nucleic acid according to claim 20.

22. A host cell comprising the vector of claim 21 or having the nucleic acid of claim 20 integrated into its genome.

23. A method of making an anti-GPRC5D antibody or antigen binding fragment thereof of any one of claims 1-18, comprising: Culture the host cells of claim 22, and recover the anti-GPRC5D antibody or its antigen-binding fragment from the host cells or the host cell culture medium.

24. Use of the anti-GPRC5D antibody or its antigen-binding fragment according to any one of claims 1-18 or the pharmaceutical composition according to claim 19 in the preparation of a medicament for treating a disease, said disease being multiple myeloma.

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