Novel anti-GPRC5D antibody

Abstract

The present invention provides anti-GPRC5D antibodies or antigen-binding fragments thereof, isolated polynucleotides encoding same, pharmaceutical compositions containing same, and uses thereof.

Description

[Technical Field]

[0001] This application relates generally to novel anti-GPRC5D antibodies and uses thereof. [Background technology]

[0002] G-protein-coupled receptor family C group 5 member D (GPRC5D) is a seven-transmembrane protein and an orphan receptor. Overexpression of GPRC5D has been reported in patients with multiple myeloma. High expression is significantly correlated with poor disease and treatment outcomes. Given the specific high expression of GPRC5D protein in tumor cells, GPRC5D is likely to be another promising candidate target for the treatment of multiple myeloma.

[0003] Therefore, there is a strong need in the art for the development of novel anti-GPRC5D antibodies. Summary of the Invention

[0004] Throughout this application, the articles "a" and "an" and "said" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antibody" means one antibody or multiple antibodies.

[0005] The present application provides anti-GPRC5D antibodies (eg, anti-human GPRC5D) or antigen-binding fragments thereof, isolated polynucleotides encoding same, pharmaceutical compositions containing same, and uses thereof.

[0006] In one aspect, the present application provides an anti-GPRC5D antibody or antigen-binding fragment thereof, comprising three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the heavy chain complementarity determining regions are the heavy chain variable region (V) shown in SEQ ID NO: 13. H) and the light chain complementarity determining region is the same as the light chain variable region (V L The present invention provides an antibody or antigen-binding fragment thereof, wherein the three light chain complementarity-determining regions are identical to those contained within the three light chain complementarity-determining regions.

[0007] In some embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions, HCDR1, HCDR2, and HCDR3, and three light chain complementarity determining regions, LCDR1, LCDR2, and LCDR3, wherein a) the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 or a variant thereof having three, two, or one or less amino acid substitutions, or SEQ ID NO: 9 or a variant thereof having three, two, or one or less amino acid substitutions; b) the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 or a variant thereof having three, two, or one or less amino acid substitutions, or SEQ ID NO: 10 or a variant thereof having three, two, or one or less amino acid substitutions; and c) the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 or a variant thereof having three, two, or one or less amino acid substitutions. d) the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or a variant thereof with three, two or one amino acid substitutions, or SEQ ID NO: 11 or a variant thereof with three, two or one or less amino acid substitutions; d) the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or a variant thereof with three, two or one or less amino acid substitutions, or SEQ ID NO: 7 or a variant thereof with three, two or one or less amino acid substitutions; e) the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or a variant thereof with three, two or one or less amino acid substitutions, or SEQ ID NO: 8 or a variant thereof with three, two or one or less amino acid substitutions; and f) the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3 or a variant thereof with three, two or one or less amino acid substitutions.

[0008] In some embodiments, a) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof having three, two or one or less amino acid substitutions, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof having three, two or one or less amino acid substitutions, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof having three, two or one or less amino acid substitutions, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof having three, two or one or less amino acid substitutions, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof having three, two or one or less amino acid substitutions, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof having three, two or one or less amino acid substitutions. or the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 8 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof having 3, 2 or 1 or less amino acid substitutions.

[0009] In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13, or a variant thereof with no more than three, two, or one amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12, or a variant thereof with no more than three, two, or one amino acid substitutions.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof is humanized.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and / or light chain variable region (V L ), wherein a) the heavy chain variable region comprises an amino acid sequence selected from the group of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, or a variant thereof with no more than 3, 2, or 1 amino acid substitutions; and b) the light chain variable region is selected from the amino acid sequence of the group of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or a variant thereof with no more than 3, 2, or 1 amino acid substitutions.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and / or light chain variable region (V L ), wherein a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof having 3, 2, or no more than 1 amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14 or a variant thereof having 3, 2, or no more than 1 amino acid substitutions, b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17 or a variant thereof having 3, 2, or no more than 1 amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof having 3, 2, or no more than 1 amino acid substitutions, and c) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, wherein the variant still retains specific binding affinity to GPRC5D.

[0013] In some embodiments, the amino acid substitutions are not within the CDR regions.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), or scFv dimer (bivalent diabody).

[0015] In some embodiments, the antibody or antigen-binding fragment thereof further comprises an immunoglobulin constant region, optionally comprising a human immunoglobulin constant region, or optionally comprising a human IgG constant region.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof is a humanized monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment thereof is bispecific or multispecific.

[0017] In some embodiments, the IgG constant region is derived from IgG1, IgG2, IgG3, or IgG4.

[0018] In some embodiments, the constant region comprises one or more amino acid residue substitutions or modifications, which result in increased CDC or ADCC relative to the wild-type constant region.

[0019] In another aspect, the present application provides a nucleic acid comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof described in the present application.

[0020] In another aspect, the present application provides a vector comprising a nucleic acid described herein.

[0021] In another aspect, the present application provides a host cell comprising a nucleic acid described herein or a vector described herein.

[0022] In another aspect, the present application provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an immune cell signaling domain, wherein the antigen-binding domain specifically binds to GPRC5D and comprises an antigen-binding fragment of the GPRC5D antibody provided in the present application.

[0023] In another aspect, the present application provides a nucleic acid encoding a chimeric antigen receptor (CAR) described herein.

[0024] In another aspect, the present application provides a cell comprising a nucleic acid sequence provided herein that encodes a chimeric antigen receptor (CAR) described herein.

[0025] In another aspect, the present application provides a genetically modified cell that expresses a chimeric antigen receptor (CAR) described herein.

[0026] In another aspect, the present application provides a conjugate comprising an antibody or antigen-binding fragment thereof described herein and a payload conjugated thereto, wherein the payload is selected from the group consisting of a radioactive label, a fluorescent label, an enzyme substrate label, an affinity purification tag, a tracking molecule, an anti-cancer drug, and a cytotoxic molecule.

[0027] In another aspect, the present application provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described in the present application, a nucleic acid described in the present application, a vector described in the present application, a conjugate described in the present application, a nucleic acid described in the present application comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) described in the present application, a cell described in the present application expressing a chimeric antigen receptor (CAR) described in the present application, and a pharmaceutically acceptable carrier.

[0028] In another aspect, the present application provides a method for treating or preventing a disease, condition, or symptom, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in the present application, a pharmaceutical composition described in the present application, a conjugate described in the present application, or a composition described in the present application.

[0029] In one embodiment, the disease, condition or symptom is selected from the group of cancer, immune disorders and inflammation.

[0030] In another aspect, the present application provides a method of stimulating an immune cell (such as a T cell or NK cell)-mediated immune response in a mammal against cells or tissues expressing GPRC5D, the method comprising administering to the mammal an effective amount of genetically modified cells to express a CAR described in the present application.

[0031] In another aspect, the present application provides a method for treating a mammal having a GPRC5D-associated disease or condition, the method comprising administering to the mammal an effective amount of a cell provided in the present application, thereby treating the mammal.

[0032] In one embodiment, the cells are autologous T cells or autologous NK cells. [Brief explanation of the drawings]

[0033] [Figure 1] 1 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to HEK293 cells expressing hGPRC5D in a FACS experiment. [Figure 2] 1 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to CHOS cells expressing hGPRC5D in a FACS experiment. [Figure 3] 1 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to MM.1R cells that naturally express GPRC5D in a FACS experiment. [Figure 4]1 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to NCI-H929 cells that naturally express GPRC5D in a FACS experiment. [Figure 5] 1 shows the binding of GPRC5D antibodies (ch-72C7 and GC5B596) to RPMI-8226 cells that naturally express GPRC5D in a FACS experiment. [Figure 6] 1 shows the cytotoxic effects of GPRC5D antibodies (ch-72C7 and GC5B596) on NCI-H929 cells that naturally express GPRC5D in an ADCC effect evaluation experiment. [Figure 7] 1 shows the cytotoxic effects of GPRC5D antibodies (ch-72C7 and GC5B596) on MM.1R cells that naturally express GPRC5D in an ADCC effect evaluation experiment. DETAILED DESCRIPTION OF THE INVENTION

[0034] The following description of the present application is intended to merely illustrate various embodiments of the present application. Therefore, the specific modifications discussed should not be construed as limiting the scope of the present application. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present application, and it is understood that such equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0035] definition As used herein, the term "antibody" includes any immunoglobulin, monoclonal, polyclonal, multivalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. A naturally occurring, intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and each heavy chain contains a variable region (V H ) and the first, second, third, and optionally fourth constant regions (C H1 , C H2 , CH3 , C H4 Mammalian light chains are classified as lambda or kappa, and each light chain consists of a variable region (V L) and constant regions. Antibodies are "Y" shaped, with the tail of the Y consisting of the second and third constant regions of two heavy chains joined together via disulfide bonds. Each arm of the Y contains the variable region and first constant region of a single heavy chain joined to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain CDRs include LCDR1, LCDR2, and LCDR3, and heavy chain CDRs include HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified according to the rules of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol., December 5, 186(3):651-63 (1985); Chothia, C. and Lesk, A.M., J. Molecular Biology, 196, 901 (1987); Chothia, C. et al., Nature, December 21-28, 342(6252):877-83 (1989); Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 1999). Interest), 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003). Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005). Marie-Paule Lefranc, Molecular Biology of B cells (2nd ed.), Chapter 26, 481-514 (2015).The three CDRs are interposed between adjacent sections known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified into classes based on the amino acid sequence of the constant regions of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0036] As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), diabodies, multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies. An antigen-binding fragment is capable of binding to the same antigen as the parent antibody.

[0037] "Fab," with respect to an antibody, refers to the portion of an antibody consisting of a single light chain (variable and constant regions) linked by disulfide bonds to the variable region and first constant region of a single heavy chain.

[0038] "Fab'" refers to a Fab fragment that includes part of the hinge region.

[0039] "F(ab')2" refers to a dimer of Fab'. With respect to antibodies, "Fv" refers to the minimum antibody fragment containing a complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0040] "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, a "(dsFv)2" or "(dsFv-dsFv')" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. H The moieties are linked by a peptide linker (e.g., a long flexible linker) and each of the two V L In some embodiments, the dsFv-dsFv' is bispecific, with each disulfide paired heavy and light chain having a different antigen specificity.

[0041] A "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to each other either directly or via a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0042] "Fc," with respect to an antibody (e.g., an antibody of the IgG, IgA, or IgD isotype), refers to the portion of the antibody consisting of the second and third constant domains of a first heavy chain linked via disulfide bonds to the second and third constant domains of a second heavy chain. For antibodies of the IgM and IgE isotypes, Fc further comprises a fourth constant domain. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0043] "Single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv connected to the Fc region of an antibody.

[0044] A "diabody" or "dAb" comprises a small antibody fragment with two antigen-binding sites, wherein these fragments are V or V2 on the same polypeptide chain. L V connected to the domain H Domain (V H -V L or V L -V H ) (see, e.g., Holliger P. et al., Proceedings of the National Academy of Sciences, July 15, 90(14):6444-8 (1993); EP404097; WO93 / 11161). By using a linker that is too short to allow pairing of the two domains on the same chain, these domains are forced to pair with complementary domains on another chain, thus generating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes). In certain embodiments, an "scFv dimer" is a diabody that targets two different V H -V L V dimerized with the moiety H -V L (linked by a peptide linker), whereby the V of one part is a bivalent diabody or bispecific scFv (BsFv). H is another part of V L to form two binding sites that may target the same antigen (or epitope) or different antigens (or epitopes). In another embodiment, an "scFv dimer" is a dimer of V L1 -V H2 (linked by a peptide linker) and associated V H1 -V L2 (linked by a peptide linker), whereby V H1 and V L1 , and V H2 and V L2 are coordinated, and each coordinated pair has a different antigen specificity.

[0045] A "domain antibody" refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. H The domains are covalently linked with peptide linkers to generate bivalent or multivalent domain antibodies. H The domains may target the same or different antigens.

[0046] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment in which a portion of the heavy and / or light chain is derived from one species and the remaining portion of the heavy and / or light chain is derived from another species. In an illustrative example, a chimeric antibody can contain a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0047] As used herein, the term "humanized" means that the antibody or antigen-binding fragment contains CDRs derived from a non-human animal, FR regions derived from a human, and, if applicable, constant regions derived from a human.

[0048] As used herein, a "bispecific" antibody is an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes, which may be on the same antigen or on two different antigens.

[0049] As used herein, a "multispecific" antibody is an artificial antibody that has fragments derived from two or more different monoclonal antibodies and is capable of binding to two or more different epitopes, which may be on the same antigen or on different antigens.

[0050] As used herein, "GPRC5D" refers to G protein-coupled receptor family C group 5 member D derived from primates (e.g., humans, monkeys), etc. In certain embodiments, GPRC5D is human GPRC5D. An exemplary sequence of human GPRC5D includes the human GPRC5D protein (UniProt number Q9NZD1). GPRC5D is a relatively new target for multiple myeloma immunotherapy. It is an orphan G protein-coupled receptor of unknown function that is highly expressed on malignant bone marrow plasma cells and hard keratinous structures, including hair shafts, nails, and the central region of the tongue (see Smith EL et al., Sci Transl Med 2019, 11:eaau7746; Pillarisetti K et al., Blood 2020, 135:1232-43; and Inoue S et al., J Invest Dermatol 2004, 122:565-73). High expression of GPRC5D is associated with poor prognosis in multiple myeloma (see Atamaniuk J et al., Eur J Clin Invest 2012, 42:953-60). GPRC5D has been used as a target for CAR-T therapy in multiple myeloma, with promising results in preclinical studies (see de Larrea CF et al., Blood Cancer Discov 2020, 1:146). It is currently the target of the bispecific antibody JNJ-64407564 (talquetamab) in four phase I clinical trials.

[0051] The term "anti-GPRC5D antibody" refers to an antibody capable of specifically binding to GPRC5D (eg, human GPRC5D). The term "anti-human GPRC5D antibody" refers to an antibody capable of specifically binding to human GPRC5D.

[0052] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, for example, between an antibody and an antigen. Specific binding can be characterized by binding affinity, e.g., K D value, i.e., the ratio of the dissociation rate to the association rate (k off / k on ) is expressed as K D can be determined by any conventional method known in the art, including, but not limited to, surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (such as FACS). -6 M (e.g., ≦5×10 -7 M, ≤ 2 × 10 -7 M, ≤10 -7 M, ≤ 5 × 10 -8 M, ≤ 2 × 10 -8 M, ≤10 -8 M, ≤ 5 × 10 -9 M, ≤ 4 × 10 -9 M, ≤ 3 × 10 -9 M, ≤ 3 × 1 -9 M or ≦10 -9 M)'s K D The value can indicate specific binding between the antibody or antigen-binding fragment thereof and GPRC5D (eg, human GPRC5D).

[0053] With respect to amino acid sequences, a "conservative substitution" refers to the replacement of an amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues having hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), acidic side chains (e.g., Asp, Glu), basic side chains (e.g., His, Lys, and Arg), or aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions generally do not significantly alter the conformational structure of a protein, thereby preserving the biological activity of the protein.

[0054] As used herein, the term "homologous" refers to a nucleic acid sequence (or its complementary strand) or amino acid sequence that has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to another sequence when optimally aligned.

[0055] "Percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in the candidate sequence that are identical with the amino acid (or nucleic acid) residues in the reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignments for purposes of determining percent amino acid (or nucleic acid) sequence identity can be performed using, for example, BLASTN, BLASTp (available at the website of the National Center for Biotechnology Information (NCBI) in the United States; see also Altschul SF et al., Journal of Molecular Biology 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res. 25:3389-3402 (1997)), ClustalW2 (available at the website of the European Bioinformatics Institute in the United States; see also Higgins DG et al., Methods in Enzymology 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, UK ... Altschul SF et al., Journal of Molecular Biology 215:403-410 (1990); This can be achieved by publicly available tools such as the ALIGN or Megalign (DNASTAR) software, see, for example, "The Journal of Molecular Biology and Biosciences, Vol. 1, No. 1, pp. 23(21):2947-8 (2007)," and by those skilled in the art who use the default parameters provided by these tools or customize the parameters for the alignment, e.g., by selecting a suitable algorithm.

[0056] As used herein, "effector function" refers to a biological activity resulting from the binding of the Fc region of an antibody to an effector such as the C1 complex and an Fc receptor. Exemplary effector functions include complement-dependent cytotoxicity (CDC), which is mediated by the interaction of an antibody with C1q on the C1 complex, antibody-dependent cellular cytotoxicity (ADCC), which is mediated by the binding of the Fc region of an antibody to an Fc receptor on an effector cell, and phagocytosis. Effector function can be assessed by various assays (such as Fc receptor binding assays, C1q binding assays, and cytolytic assays).

[0057] An "isolated" material has been altered by artifical means from its natural state. When an "isolated" composition or material occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living animal is not "isolated," but the polynucleotide or polypeptide would be "isolated" if it is sufficiently free from the coexisting materials of its natural state so that it exists in a substantially pure state. An "isolated nucleic acid sequence" refers to the sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that is at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure as measured by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, or capillary electrophoresis) or chromatographic methods (such as ion exchange chromatography or reverse-phase HPLC).

[0058] As used herein, the term "vector" refers to a vehicle into which a genetic element can be operably inserted and expressed to produce the protein, RNA, or DNA encoded by the genetic element or to copy the genetic element. A vector can be used to transform, transduce, or transfect a host cell to express the genetic element carried by the vector within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as λ phage or M13 phage, and animal viruses. A vector can contain various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Additionally, a vector can contain an origin of replication. A vector can also contain materials to aid in cell entry, including, but not limited to, viral particles, liposomes, or protein coatings. The vector may be an expression vector or a cloning vector. The present application provides vectors (e.g., expression vectors) containing a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0059] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide and / or vector can be introduced or has been introduced.

[0060] As used herein, "treating" a condition includes alleviating the condition, delaying the onset or rate of progression of the condition, reducing the risk of developing the condition, delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0061] As used herein, a "GPRC5D-associated" disease or condition refers to any disease or condition caused, exacerbated, or otherwise associated with increased or decreased expression or activity of GPRC5D. In some embodiments, the GPRC5D-associated disease or condition is cancer, such as myeloma. In certain embodiments, the GPRC5D-associated disease or condition is characterized by overexpression of the GPRC5D gene. In one embodiment, the GPRC5D-associated disease or condition includes, but is not limited to, GPRC5D-positive breast cancer, multiple myeloma, Waldenstrom's macroglobulinemia, endometrial cancer, ovarian cancer, lung cancer, gastric cancer, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, colorectal cancer, esophageal cancer, bladder cancer, cervical cancer, blood cancer, lymphoma, or malignant melanoma.

[0062] The term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other ingredients that make up the formulation, and physiologically compatible with the recipient thereof.

[0063] Anti-GPRC5D antibody The present application provides anti-GPRC5D antibodies or antigen-binding fragments thereof. The anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein can specifically bind to GPRC5D.

[0064] In certain embodiments, the antibodies and fragments thereof provided herein exhibit a chromatin concentration of 8×10 -8 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 8×10 -9 M or less, 5×10 -9 M or less, 2×10 -9 M or less, or 10 -9 K below M DBiacore analysis is based on surface plasmon resonance technology, see for example Murphy, M. et al., Current protocols in protein science, Chapter 19, Unit 19.14, 2006.

[0065] The binding of antibodies to human GPRC5D was measured at the "half maximal effective concentration" (EC 50 ) value. 50 The EC value can be measured by binding assays known in the art, e.g., sandwich assays such as enzyme-linked immunosorbent assays (ELISAs), flow cytometry assays, and other binding assays. In certain embodiments, the antibodies and fragments thereof provided herein have an EC value of 1 μg / ml or less, 2 μg / ml or less, 3 μg / ml or less, 4 μg / ml or less, 5 μg / ml or less, or 10 μg / ml or less, as measured by, for example, a flow cytometry assay. 50 At this value (i.e., 50% binding concentration), the antibody specifically binds to cells expressing human GPRC5D.

[0066] As used herein, "binding ability" refers to the ability of a molecule (e.g., an antibody) to bind to another molecule (e.g., an antigen). This ability can be measured using any suitable binding assay known in the art, e.g., based on binding activity to an antigen of interest. For example, the antibody of interest may be labeled so that its binding activity to the antigen can be directly quantified. Alternatively, the binding activity of an antibody of interest (i.e., a primary antibody) to its antigen may be detected using a labeled secondary antibody (e.g., an anti-species antibody), which detects the complex of the primary antibody and its antigen by binding to the primary antibody in the complex, thereby indirectly quantifying the binding activity. Labeled antibodies can be detected by, for example, enzyme-linked immunosorbent assay (ELISA, e.g., the label is an enzyme), flow cytometry (e.g., the label is fluorescent), Western blotting (e.g., the label is a fluorescent or radioligand), colorimetric analysis, chemiluminescence-based methods, and the like.

[0067] In certain embodiments, the present application provides an anti-GPRC5D monoclonal antibody ch-72C7.

[0068] As used herein, "ch-72C7" refers to a monoclonal antibody having a light chain variable region containing the sequence of SEQ ID NO: 12 and a heavy chain variable region containing the sequence of SEQ ID NO: 13. ch-72C7 is a mouse-derived antibody, and its CDR sequences can be classified by methods known in the art, including, but not limited to, CDR classification according to the IMGT system or CDR classification based on the Kabat numbering system.

[0069] Table 1 below shows the CDR sequences of antibody ch-72C7 classified according to the IMGT numbering system. Table 2 below shows the CDR sequences of antibody ch-72C7 classified according to the Kabat numbering system. Table 3 below shows the amino acid sequences of the heavy and light chain variable regions of GPRC5D. [Table 1] [Table 2] [Table 3]

[0070] In certain embodiments, the application provides anti-GPRC5D antibodies and antigen-binding fragments thereof that comprise one or more (eg, 1, 2, 3, 4, 5, or 6) CDR sequences of antibody ch-72C7.

[0071] In a specific embodiment, the present application provides an anti-GPRC5D antibody (e.g., an anti-human GPRC5D antibody) and antigen-binding fragments thereof, comprising three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the three heavy chain complementarity determining regions are the heavy chain variable region (V) shown in SEQ ID NO: 13. H) and the three light chain complementarity determining regions are the same as the light chain variable region (V) shown in SEQ ID NO: 12. L The present invention provides antibodies and antigen-binding fragments thereof, wherein the three light chain complementarity-determining regions are identical to those contained within the three light chain complementarity-determining regions.

[0072] In certain embodiments, the present application provides anti-GPRC5D antibodies (e.g., anti-human GPRC5D antibodies) and antigen-binding fragments thereof comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs, wherein the one or more CDRs comprise an array selected from the group consisting of SSVSF (SEQ ID NO: 1), DTT (SEQ ID NO: 2), QQWNSHPLT (SEQ ID NO: 3), GYPFTNYW (SEQ ID NO: 4), INPSNGRT (SEQ ID NO: 5), and ARGFAY (SEQ ID NO: 6).

[0073] In certain embodiments, the present application provides an anti-GPRC5D antibody (e.g., an anti-human GPRC5D antibody) or antigen-binding fragment thereof comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs, wherein the one or more CDRs comprise an sequence selected from the group consisting of SASSSVSFMH (SEQ ID NO: 7), DTTKLAS (SEQ ID NO: 8), QQWNSHPLT (SEQ ID NO: 3), NYWMH (SEQ ID NO: 9), EINPSNGRTNYNEKFKS (SEQ ID NO: 10), and GFAY (SEQ ID NO: 11).

[0074] In certain embodiments, the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 or a variant thereof with 3, 2 or 1 or less amino acid substitutions, or SEQ ID NO: 9 or a variant thereof with 3, 2 or 1 or less amino acid substitutions, the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 or a variant thereof with 3, 2 or 1 or less amino acid substitutions, or SEQ ID NO: 10 or a variant thereof with 3, 2 or 1 or less amino acid substitutions, and the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 or a variant thereof with 3, 2 or 1 or less amino acid substitutions, or SEQ ID NO: 11 or a variant thereof with 3, 2 or 1 or less amino acid substitutions. and variants thereof, wherein the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or a variant thereof having 3, 2 or no more than 1 amino acid substitutions, or SEQ ID NO: 7 or a variant thereof having 3, 2 or no more than 1 amino acid substitutions, the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or a variant thereof having 3, 2 or no more than 1 amino acid substitutions, or SEQ ID NO: 8 or a variant thereof having 3, 2 or no more than 1 amino acid substitutions, and the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3 or a variant thereof having 3, 2 or no more than 1 amino acid substitutions.

[0075] In certain embodiments, in the anti-GPRC5D antibody or antigen-binding fragment thereof provided in the present application, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof having three, two or one or less amino acid substitutions, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof having three, two or one or less amino acid substitutions, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof having three, two or one or less amino acid substitutions, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof having three, two or one or less amino acid substitutions, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof having three, two or one or less amino acid substitutions, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof having three, two or one or less amino acid substitutions.

[0076] In certain embodiments, in the anti-GPRC5D antibody or antigen-binding fragment thereof provided in the present application, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9 or a variant thereof having three, two, or one or less amino acid substitutions, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10 or a variant thereof having three, two, or one or less amino acid substitutions, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11 or a variant thereof having three, two, or one or less amino acid substitutions, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof having three, two, or one or less amino acid substitutions, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 8 or a variant thereof having three, two, or one or less amino acid substitutions, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof having three, two, or one or less amino acid substitutions.

[0077] In certain embodiments, the amino acid substitutions are conservative substitutions. In certain embodiments, the amino acid substitutions are not within the CDR regions. In certain embodiments, the variants still retain specific binding affinity to GPRC5D (e.g., human GPRC5D).

[0078] In certain embodiments, the present application provides an anti-GPRC5D antibody or an antigen-binding fragment thereof comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 1, an LCDR2 having the sequence set forth in SEQ ID NO: 2, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 3, and / or an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 4, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0079] In certain embodiments, the present application provides an anti-GPRC5D antibody or an antigen-binding fragment thereof comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 7, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 8, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 3, and / or an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 9, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 10, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 11.

[0080] CDRs are known to be responsible for antigen binding. However, it has been found that not all six CDRs are essential or inalterable. In other words, one or more CDRs in the anti-GPRC5D antibody ch-72C7 may be replaced, altered, or modified while substantially retaining the specific binding affinity to GPRC5D.

[0081] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise the heavy chain CDR3 sequence of antibody ch-72C7. In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise the heavy chain CDR3 sequence of SEQ ID NO: 6, wherein the CDR3 is numbered according to the IMGT numbering system. In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise the heavy chain CDR3 sequence of SEQ ID NO: 11, wherein the CDR3 is numbered according to the Kabat numbering system.

[0082] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise any suitable framework region (FR) sequence, as long as the antibodies and antigen-binding fragments thereof are capable of specifically binding to GPRC5D. The CDR sequences provided in Table 1 or Table 2 above are obtained from mouse antibodies, but can be grafted to suitable FR sequences of any suitable species, such as mouse, human, rat, or rabbit, by suitable methods known in the art, such as recombinant techniques.

[0083] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein are humanized. Humanized antibodies or antigen-binding fragments thereof are desirable due to their reduced immunogenicity in humans. Humanized antibodies have chimeric variable regions in which non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can essentially be achieved by replacing the corresponding human CDR genes in a human immunoglobulin gene with non-human (e.g., murine) CDR genes (see, e.g., Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0084] Suitable human heavy and light chain variable domains may be selected to achieve this goal by methods known in the art. In an illustrative example, a "best-fit" approach may be used, in which a non-human (e.g., rodent) antibody variable domain sequence is screened or BLASTed against a database of known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as a human scaffold for grafting the non-human CDR sequences (see, e.g., Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Molecular Biology 196:901). Alternatively, a framework derived from the consensus sequence of all human antibodies may be used for grafting the non-human CDRs (see, e.g., Carter et al. (1992) Proceedings of the National Academy of Sciences of the United States of America 89:4285; Presta et al. (1993) J. Immunol. 151:2623).

[0085] In certain embodiments, the humanized antibodies or antigen-binding fragments thereof provided herein are composed substantially entirely of human sequences, except for the CDR sequences, which are non-human. In some embodiments, the variable region FR and constant region, if present, are derived completely or substantially from human immunoglobulin sequences. The human FR sequences and human constant region sequences may be derived from different human immunoglobulin genes, e.g., the FR sequences are derived from one human antibody and the constant region is derived from another human antibody. In some embodiments, the humanized antibody or antigen-binding fragment comprises human heavy chain HFR1-4 and / or light chain LFR1-4.

[0086] In some embodiments, a human-derived FR region can contain the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues in the human FR are substituted with the corresponding residue from the parent non-human antibody. In certain embodiments, it may be necessary to make the humanized antibody or fragment thereof closely resemble the non-human parent antibody structure. In certain embodiments, the humanized antibody or antigen-binding fragment provided herein contains no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each of the human FR sequences, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all FRs of the heavy or light chain variable domain. In some embodiments, such changes in amino acid residues can occur only in the heavy chain FR region, only in the light chain FR region, or in both chains.

[0087] In certain embodiments, one or more amino acid residues are remutated to the corresponding residue found, for example, in the non-human parent antibody from which the CDR sequence was derived (e.g., in a murine framework region). One skilled in the art can select appropriate mutation positions according to principles known in the art. For example, positions for mutation can be selected where: 1) residues in the human germline sequence framework are rare (e.g., less than 20% or less than 10% in human variable region sequences); 2) the position is adjacent to one or more of the three CDRs in the primary sequence of the human germline chain, such that it may interact with residues in the CDRs; or 3) the position is close to the CDRs in a three-dimensional model, such that it is likely to interact with amino acids in the CDRs. The residues at the selected positions may be remutated to the corresponding residue in the parent antibody, or may be mutated to a residue typical of a human sequence, i.e., a residue that occurs more frequently at that position in known human sequences belonging to the same subgroup as the human germline sequence (see U.S. Pat. No. 5,693,762).

[0088] In certain embodiments, the humanized light and heavy chains described in the present application are substantially non-immunogenic in humans and retain substantially the same, and even higher, affinity for GPRC5D compared to the parent antibody.

[0089] This application is 1) "22Mono5JO4" comprising a heavy chain variable region (22Mono5JO4-VH) represented by the amino acid sequence of SEQ ID NO: 15 and a light chain variable region (22Mono5JO4-VL) represented by the amino acid sequence of SEQ ID NO: 14; 2) "22Mono3L7F" including a heavy chain variable region (22Mono3L7F-VH) shown in the amino acid sequence of SEQ ID NO: 17 and a light chain variable region (22Mono3L7F-VL) shown in the amino acid sequence of SEQ ID NO: 16; 3) "22Mono4DN4" comprising a heavy chain variable region (22Mono4DN4-VH) shown in the amino acid sequence of SEQ ID NO: 19 and a light chain variable region (22Mono4DN4-VL) shown in the amino acid sequence of SEQ ID NO: 18; 4) "22Mono5UQY" comprising a heavy chain variable region (22Mono5UQY-VH) represented by the amino acid sequence of SEQ ID NO: 21 and a light chain variable region (22Mono5UQY-VL) represented by the amino acid sequence of SEQ ID NO: 20; Further provided are exemplary humanized antibodies of ch-72C7, including: [Table 4]

[0090] In certain embodiments, the present application further provides a humanized anti-GPRC5D antibody or antigen-binding fragment thereof, which comprises an HFR1, HFR2, HFR3, and / or HFR4 sequence contained in a heavy chain variable region selected from the group consisting of 22Mono5JO4-VH (SEQ ID NO: 15), 22Mono3L7F-VH (SEQ ID NO: 17), 22Mono4DN4-VH (SEQ ID NO: 19), and 22Mono5UQY-VH (SEQ ID NO: 21).

[0091] In certain embodiments, the present application further provides a humanized anti-GPRC5D antibody or antigen-binding fragment thereof comprising an LFR1, LFR2, LFR3 and / or LFR4 sequence contained in a light chain variable region, wherein the light chain variable region is selected from the group consisting of 22Mono5JO4-VL (SEQ ID NO: 14), 22Mono3L7F-VL (SEQ ID NO: 16), 22Mono4DN4-VL (SEQ ID NO: 18) and 22Mono5UQY-VL (SEQ ID NO: 20).

[0092] In certain embodiments, the humanized anti-GPRC5D antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 21, and / or a light chain variable region sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20.

[0093] These exemplary humanized anti-GPRC5D antibodies retain specific binding ability or affinity to GPRC5D and are at least equivalent to, and even superior to, the parent murine antibody ch-72C7 in these respects. Exemplary data are provided in Examples 4 and 5.

[0094] In some embodiments, the anti-GPRC5D antibodies and antigen-binding fragments provided herein comprise all or a portion of a heavy chain variable region and / or all or a portion of a light chain variable region. In one embodiment, the anti-GPRC5D antibodies and antigen-binding fragments provided herein are single-domain antibodies consisting of all or a portion of a heavy chain variable region provided herein. Details of such single-domain antibodies are available in the art (see, for example, U.S. Patent No. 6,248,516).

[0095] In certain embodiments, the anti-GPRC5D antibodies and fragments thereof provided herein further comprise an immunoglobulin (Ig) constant region, and optionally further comprise heavy and / or light chain constant regions. In certain embodiments, the heavy chain constant region comprises a CH1, hinge, and / or CH2-CH3 region (or optionally a CH2-CH3-CH4 region). In certain embodiments, the anti-GPRC5D antibodies and fragments thereof provided herein comprise a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the light chain constant region comprises Cκ or Cλ. The constant regions of the anti-GPRC5D antibodies and fragments thereof provided herein may be identical to the wild-type constant region sequence or may differ by one or more mutations.

[0096] In some embodiments, the CH1 region of the anti-GPRC5D antibody or antigen-binding fragment thereof of the present application comprises the sequence set forth in SEQ ID NO:24. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 24)

[0097] In some embodiments, the CL region of the anti-GPRC5D antibody or antigen-binding fragment thereof of the present application comprises the sequence shown in SEQ ID NO:25. RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25)

[0098] In certain embodiments, the heavy chain constant region comprises an Fc region. The Fc region is known to mediate effector functions, such as ADCC and CDC of antibodies. The Fc regions of different Ig isotypes have different abilities to induce effector functions. For example, it is recognized that the Fc regions of IgG1 and IgG3 induce ADCC and CDC more effectively than the Fc regions of IgG2 and IgG4. In certain embodiments, the anti-GPRC5D antibodies and fragments thereof provided herein further comprise a human IgG1, IgG2, IgG3, or IgG4 constant region. In certain embodiments, the anti-GPRC5D antibodies and antigen-binding fragments thereof provided herein comprise an Fc region of an IgG1 or IgG3 isotype capable of inducing ADCC or CDC, or a constant region of an IgG4 or IgG2 isotype with reduced or depleted effector function.

[0099] Further provided herein are anti-GPRC5D antigen-binding fragments. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-GPRC5D antibodies provided herein, including, for example, the exemplary antibodies whose CDR sequences and variable sequences are shown in Tables 1 and 2, and their different variants (affinity variants, glycosylation variants, Fc variants, cysteine-engineered variants, etc., as described in subsequent sections of this application).

[0100] In certain embodiments, the anti-GPRC5D antigen-binding fragment provided herein is a diabody, a single-chain Fv fragment (scFv), an scFv dimer, a BsFv, a disulfide bond stabilized Fv (dsFv), (dsFv)2, dsFv-dsFv', an Fv fragment, Fab, Fab', F(ab')2, a bispecific antibody, a ds diabody, a domain antibody, a single domain antibody, or a bivalent domain antibody.

[0101] Various techniques can be used to produce such antigen-binding fragments. Exemplary methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)), recombinant expression (e.g., of Fab, Fv, and ScFv antibody fragments) in host cells (e.g., E. coli), screening (e.g., of ScFv) from phage display libraries as discussed above, and chemical coupling of two Fab'-SH fragments to form an F(ab')2 fragment (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0102] In certain embodiments, the anti-GPRC5D antigen-binding fragments provided herein are disulfide-stabilized Fvs. Analysis of antibody crystal structures revealed that cysteine ​​mutations can be introduced into relatively conserved sequences at the VL-VH interface to form disulfide bonds between the VL and VH, thereby covalently connecting them. The covalent bond between the VL and VH significantly improved the stability of the antibody. The first dsFvs (disulfide Fvs) were constructed by introducing disulfide bonds at the VH-VL interface through covalent interactions between cysteine ​​residues in the CDRs of each fragment (see Glockshuber, R. et al., Comparison of Strategies for Stabilizing Immunoglobulin Fv Fragments (1990) Biochemistry, 291362-1367). Although this method did not affect the antibody activity, it is difficult to make this method a universal solution for constructing various antibodies because detailed structural information of the CDRs of the original antibody is required for "customized" design to avoid interference with the antigen recognition / binding ability of the CDRs. To ensure the wide application of this method, amino acids at selected sites in the conserved FRs must be involved in the construction of dsFvs.

[0103] Since 1993, several VH-VL pairing sites suitable for covalent bond formation have been discovered, such as VH44-VL100, VH105-VL43, VH100b-VL49, VH100-VL150, and VH101-VL46, where the numbering is according to Kabat numbering (Reiter, Y. et al., Stabilization of Fv fragments of recombinant immunotoxins by disulfide bonds engineered into conserved framework regions (1994) Biochemistry, 335451-5459; Jung, J. et al., J. Immunotoxin Fv fragment stabilization by disulfide bonds engineered into conserved framework regions (1994) Biochemistry, 335451-5459; (See, for example, SH et al., Design of Interchain Disulfide Bonds in the Fv Fragment Framework Region of Monoclonal Antibody B3 (1994) Proteins, Structure, Function, Genes, 19, 35-47; Glockshuber, R. et al., Comparison of Strategies for Stabilizing Immunoglobulin Fv Fragments (1990) Biochemistry, 291362-1367; and Zhu, Z. et al., Remodeling of Domain Interfaces to Promote Heterodimer Formation (1997) Protein Science, 6, 781-788.) Among these, VH44-VL100 and VH105-VL43 are more widely used because they are superior to the others in many aspects (protein expression level, monolayer rate, Tm, affinity, etc.), although to different degrees.

[0104] In certain embodiments, the anti-GPRC5D antigen-binding fragments provided herein are scFvs. The generation of scFvs is described, for example, in WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. scFvs can be fused at the amino or carboxyl terminus to effector proteins to provide fusion proteins (see, for example, "Antibody Engineering," edited by Borrebaeck).

[0105] In certain embodiments, the antibodies and fragments thereof provided herein have sufficient specific binding affinity for human GPRC5D to provide diagnostic and / or therapeutic uses.

[0106] Antibody variants The antibodies or antigen-binding fragments thereof provided herein further encompass various antibody variants thereof.

[0107] In certain embodiments, antibody variants comprise one or more modifications or substitutions in one or more CDR sequences provided in Tables 1 and 2 above, one or more variable region sequences (but not in the CDR sequences) provided in Tables 3 and 4 above, and / or in the constant region (e.g., Fc region). Such variants retain the binding specificity of the parent antibody to the antigen GPRC5D, while having one or more desired properties conferred by one or more modifications or one or more substitutions. By way of example, antibody variants may have improved antigen binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamination, reduced or eliminated one or more effector functions, improved FcRn receptor binding, and increased pharmacokinetic half-life, pH sensitivity, and / or conjugation suitability (e.g., one or more introduced cysteine ​​residues).

[0108] To identify suitable or preferred residues to modify or replace, parent antibody sequences may be screened by methods known in the art, such as "alanine scanning mutagenesis" (see, e.g., Cunningham and Wells (1989) Science 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) may be identified, and the target residues may be replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and the modified antibodies may be produced and screened for properties of interest. If substitutions at particular amino acid positions exhibit functional changes of interest, the positions may be identified as potential residues for modification or replacement. Potential residues may be further evaluated by substituting them with different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0109] Affinity variants Affinity variants may contain modifications or substitutions in one or more of the CDR sequences provided in Table 1 or Table 2 above, one or more of the FR sequences provided herein, or the heavy or light chain variable region sequences provided in Table 3 or Table 4 above. FR sequences can be easily identified by those skilled in the art based on the CDR sequences in Table 1 or Table 2 above and the variable region sequences in Table 3 or Table 4 above, since it is known in the art that a CDR region is sandwiched between two FR regions in the variable region. Affinity variants retain the specific binding affinity of the parent antibody to the antigen GPRC5D, and thus have improved specific binding affinity to the antigen GPRC5D over the parent antibody. In certain embodiments, the substitutions in at least one (or all) of the CDR sequences, FR sequences, or variable region sequences comprise conservative substitutions.

[0110] Those skilled in the art will understand that one or more amino acid residues in the CDR sequences and variable region sequences provided in Tables 1 to 4 above can be substituted, while the resulting antibody or antigen-binding fragment will still retain its binding affinity or ability to the antigen GPRC5D, and thus may have improved binding affinity or ability. Various methods known in the art can be used to achieve this goal. For example, phage display technology can be used to generate and express a library of antibody variants (e.g., Fab or scFv variants), which can then be screened for binding affinity to the corresponding antigen (e.g., GPRC5D). As another example, computer software can be used to virtually simulate the binding of an antibody to the antigen GPRC5D to identify amino acid residues on the antibody that form the binding interface. Such residues may be avoided from substitution to prevent a decrease in binding affinity, or may be targeted for substitution to provide stronger binding.

[0111] In certain embodiments, the humanized antibodies or antigen-binding fragments provided herein comprise one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences, hi certain embodiments, affinity variants comprise a total of no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the CDR and / or FR sequences.

[0112] In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof comprises one, two, or three CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with a sequence listed in Table 1 or Table 2 above (or their sequences), and retains binding affinity to GPRC5D at a level similar to, or even higher than, that of the parent antibody.

[0113] In certain embodiments, anti-GPRC5D antibodies and antigen-binding fragments thereof comprise one or more variable region sequences that share at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with a sequence listed in Table 3 or Table 4 above (or with such sequences), and retain a similar, or even higher, level of binding affinity to GPRC5D than the parent antibody. In some embodiments, a total of 1 to 10 amino acid substitutions, insertions, or deletions are present in the variable region sequences listed in Table 3 above. In some embodiments, the substitutions, insertions, or deletions occur within regions outside the CDRs (e.g., within the FRs).

[0114] Glycosylation variants The anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein also encompass glycosylation variants that can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment.

[0115] An antibody or antigen-binding fragment thereof may contain one or more modifications that introduce or remove glycosylation sites. Glycosylation sites are amino acid residues whose side chains can be attached to a carbohydrate moiety (e.g., an oligosaccharide structure). Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue (e.g., an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline). O-linked glycosylation refers to the attachment of one of N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine. Removal of native glycosylation sites can be conveniently achieved, for example, by altering the amino acid sequence so that one of the above-mentioned tripeptide sequences (for N-linked glycosylation sites) or a serine or threonine residue (for O-linked glycosylation sites) present in the sequence is substituted. Similarly, new glycosylation sites can be created by introducing such tripeptide sequences or serine or threonine residues.

[0116] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein comprise a mutation at N297 (eg, N297A, N297Q, or N297G) to remove a glycosylation site.

[0117] Cysteine ​​engineered variants The anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein also include cysteine ​​engineered variants that contain one or more introduced free cysteine ​​amino acid residues.

[0118] A free cysteine ​​residue is one that is not part of a disulfide bridge. Cysteine ​​engineered variants are useful for conjugation at the engineered cysteine ​​site, e.g., via maleimide or haloacetyl, particularly with, for example, cytotoxic and / or imaging compounds, labels, or radioisotopes. Methods for engineering antibodies or antigen-binding fragments thereof to introduce free cysteine ​​residues are known in the art; see, e.g., WO2006 / 034488.

[0119] Fc variants The anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein also encompass Fc variants comprising one or more amino acid residue modifications or substitutions in the Fc region and / or hinge region to provide altered effector functions, such as ADCC and CDC. Methods for altering ADCC activity by antibody engineering have been described in the art, e.g., Shields R L et al., J Biol Chem. 2001, 276(9):6591-604; Idusogie E E et al., J Immunol. 2000, 164(8):4178-84; Steurer W et al., J Immunol. 1995, 155(3):1165-74; Idusogie E E et al., J Immunol. 2001, 166(4):2571-5; Lazar G A et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS) 2006, 103(11):4005-4010; Ryan M C et al., Mol. Cancer Ther. 2007, 6:3009-3018; Richards See JO, et al., Molecular Cancer Therapeutics 2008, 7(8):2517-27; Shields RL et al., J. Biol. Chem 2002, 277:26733-26740; Shinkawa T. et al., J. Biol. Chem 2003, 278:3466-3473.

[0120] The CDC activity of the antibodies provided herein may also be altered, for example, by increasing or decreasing Clq binding and / or CDC (see, e.g., WO 99 / 51642; Duncan and Winter, Nature, 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants). One or more amino acids selected from amino acid residues 329, 331, and 322 of the Fc region may be replaced with another amino acid residue to alter Clq binding and / or reduce or eliminate complement-dependent cytotoxicity (CDC) (see U.S. Pat. No. 6,194,551 by Idusogie et al.). One or more amino acid substitutions may be introduced to alter the ability of the antibody to fix complement (see PCT Publication WO 94 / 29351 by Bodmer et al.).

[0121] In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein has reduced effector function and comprises one or more amino acid substitutions in IgG1 at positions selected from the group consisting of 234, 235, 237, 238, 268, 297, 309, 330, and 331, where amino acid positions are numbered according to the EU numbering system. In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof provided herein has an IgG1 isotype and comprises one or more amino acid substitutions selected from the group consisting of N297A, N297Q, N297G, L235E, L234A, L235A, L234F, P331S, and any combination thereof. In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein have an IgG2 isotype and contain one or more amino acid substitutions selected from the group consisting of H268Q, V309L, A330S, P331S, V234A, G237A, P238S, H268A, and any combination thereof (e.g., H268Q / V309L / A330S / P331S, V234A / G237A / P238S / H268A / V309L / A330S / P331S). In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein have an IgG4 isotype and contain one or more amino acid substitutions selected from the group consisting of N297A, N297Q, N297G, L235E, L234A, L235A, and any combination thereof. In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein have an IgG2 / IgG4 cross-isotype, examples of which are described in Rother RP et al., Nat Biotechnol 25:1256-1264 (2007).

[0122] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein have an IgG1 isotype and comprise one or more amino acid substitutions at one or more positions 234, 235, and 331. In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein have an IgG1 isotype and comprise the triple mutation L234F / L235E / P331S in the Fc region.

[0123] In certain embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof provided herein have increased ADCC and / or increased affinity for Fcγ receptors, and , 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439 (see WO 00 / 42072 by Presta). Particular mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. Furthermore, it is shown that combination mutants such as T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A can improve FcγRIII binding.

[0124] In certain embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof contains one or more amino acid substitutions that improve pH-dependent binding to neonatal Fc receptor (FcRn). By binding to FcRn at acidic pH, such variants avoid lysosomal degradation and are subsequently translocated and released extracellularly, which may result in an extended pharmacokinetic half-life. Methods for engineering antibodies and antigen-binding fragments thereof to improve their binding affinity to FcRn are well known in the art, see, for example, Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Vol. 1, Chapter 27: Engineering of the Fc region for improved PK, Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277 (2010); and Hinton, P. et al., Journal of Immunology, 176:346-356 (2006).

[0125] The antibodies or antigen-binding fragments thereof provided herein can be monoclonal, polyclonal, humanized, chimeric, recombinant, bispecific, multispecific, bivalent, or anti-idiotypic antibodies. Recombinant antibodies are antibodies prepared in vitro by recombinant methods and not in an animal.

[0126] Conjugates In some embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof further comprises a conjugate moiety. The conjugate moiety is capable of linking to the antibody or antigen-binding fragment thereof. The conjugate moiety is a moiety capable of linking to the antibody or antigen-binding fragment thereof. Taking into consideration, a variety of conjugate moieties can be linked to the antibodies or antigen-binding fragments thereof provided herein (see, e.g., "Conjugate Vaccines," in Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate moieties can be linked to the antibody or antigen-binding fragment thereof by covalent bonding, affinity bonding, intercalation, coordinate bonding, complexation, association, blending, or addition, as well as other methods.

[0127] In certain embodiments, the antibodies and antigen-binding fragments disclosed herein can be engineered to contain specific sites outside the epitope-conjugating moiety that can be utilized for binding to one or more conjugating moieties. By way of example, such sites can contain one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent attachment to the conjugating moiety.

[0128] In certain embodiments, an antibody may be linked to a conjugate moiety indirectly or through another conjugate moiety. For example, an antibody or antigen-binding fragment thereof may be conjugated to biotin and then indirectly conjugated to a second conjugate conjugated to avidin. The conjugate may be a clearance modifier, a toxin (e.g., a chemotherapeutic agent), a detectable label (e.g., a radioisotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label), or a purification moiety.

[0129] A "toxin" can be any agent that is harmful to or capable of damaging or killing cells. Examples of toxins include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, and the like. D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin CC) and cis-dichlorodiamineplatinum(II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), antimitotic agents (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin-binding agents.

[0130] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, glucose oxidase, or β-D-galactosidase), radioisotopes (e.g., 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi and 32 P, other lanthanides), luminescent labels, plastid moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0131] In certain embodiments, the conjugate moiety may be a clearance modifier, which serves to extend the half-life of the antibody. Illustrative examples include water-soluble polymers such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and ethylene glycol / propylene glycol copolymers. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when two or more polymers are attached, they may be the same or different molecules.

[0132] In certain embodiments, the conjugated moiety may be a purification moiety such as a magnetic bead.

[0133] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are used as the basis for conjugates.

[0134] Chimeric Antigen Receptor (CAR) Compositions The present application further provides a chimeric antigen receptor (CAR) comprising the anti-GPRC5D antigen-binding domain provided herein and an immune cell activation domain. A chimeric antigen receptor (CAR) is an engineered chimeric receptor that combines the antigen-binding domain of an antibody with one or more signaling domains for immune cell activation. Suitable immune cells may include, but are not limited to, T cells and natural killer (NK) cells. Immune cells such as T cells and natural killer (NK) cells may be genetically engineered to express a CAR. T cells expressing a CAR are called CAR-T cells. NK cells expressing a CAR are called CAR-NK cells. CARs can mediate antigen-specific cellular immune activity in T cells or NK cells, thereby enabling CAR-T or CAR-NK cells to eliminate cells expressing the target antigen (e.g., tumor cells). In one embodiment, binding of the CAR-T or CAR-NK cells provided herein to GPRC5D expressed, for example, on cancer cells, causes the CAR-T or CAR-NK cells to proliferate and / or be activated, wherein the activated CAR-T or CAR-NK cells can release cytotoxic factors such as perforin, granzymes, and granulysin to initiate cytolysis and / or apoptosis of cancer cells.

[0135] In certain embodiments, the CAR comprises an anti-GPRC5D antigen-binding domain, a transmembrane domain, and an immune cell activation domain, wherein the antigen-binding domain specifically binds to GPRC5D and comprises an antigen-binding fragment of an antibody provided herein. In certain embodiments, the CAR further comprises a costimulatory signaling region, optionally linked to the transmembrane domain and the immune cell activation domain, respectively.

[0136] In some embodiments, the anti-GPRC5D antigen-binding domain of the CAR comprises one or more CDR sequences provided herein, one or more heavy chain variable domains or light chain variable domains provided herein, or one or more antigen-binding fragments derived from any of the anti-GPRC5D antibodies provided herein.

[0137] In some embodiments, it is beneficial for the antigen-binding domain to be derived from the same species, where the CAR will ultimately be used in that same species. For example, when used in humans, it may be beneficial for the antigen-binding domain used in the CAR to be derived from a human antibody or a humanized antibody. In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain may exist in a variety of other forms, including, for example, Fv, Fab, and (Fab')2, and bifunctional (i.e., bispecific) hybrid antibody fragments (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In certain embodiments, the antigen-binding domain comprises a Fab or scFv.

[0138] In certain embodiments, CAR comprises a transmembrane domain fused with the extracellular antigen binding domain of CAR.When expressed in cells, the anti-GPRC5D antigen binding domain is extracellular, and the immune cell activation domain is intracellular.In one embodiment, the transmembrane domain can be selected to naturally associate with one domain in CAR.

[0139] The transmembrane domain of the CARs provided herein may be derived from the transmembrane domain of any transmembrane protein, such as the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD 154. In some embodiments, the transmembrane domain of the CAR may use various human hinges, such as a human Ig (immunoglobulin) hinge.

[0140] Additionally, the transmembrane domain of the CAR provided herein may be synthetic, for example, containing primarily hydrophobic residues (e.g., leucine and valine). In one embodiment, the synthetic transmembrane domain contains a triplet of phenylalanine, tryptophan, and valine at each end. Optionally, a short oligopeptide or polypeptide linker, 2-10 amino acids in length, can connect the transmembrane domain and the intracellular signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0141] The costimulatory signaling region in the CARs provided herein may act antigen-independently to mediate T cell or NK cell activation and may be derived from costimulatory molecules required for an effective lymphocyte response to antigen. Exemplary costimulatory molecules include those derived from ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, among others.

[0142] The immune cell activation domain of the CARs provided herein can be a T cell activation domain. The T cell activation domain can be linked to a transmembrane domain or to a costimulatory signaling region. The T cell activation domain can include a TCR signaling domain. The TCR signaling domain can activate a T cell expressing the CAR to exert at least one normal TCR effector function of a T cell, for example, accessory activity including cytolytic activity or cytokine secretion. The TCR signaling domain can be a full-length naturally occurring intracellular signaling domain or a fragment thereof sufficient to signal select TCR effector functions. Exemplary intracellular signaling domains that can be used in the CARs provided herein include the cytoplasmic sequences of a T cell receptor (TCR) or co-receptor, and any derivative or variant of these sequences, as well as any synthetic sequence with the same functional capability, which co-initiate signaling following CAR activation.

[0143] The stimulatory TCR signaling domain can comprise a signaling motif, referred to as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAMs comprising TCR signaling domains that can be used in the CARs provided herein include ITAMs derived from CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In certain embodiments, the TCR signaling domain comprises a cytoplasmic signaling sequence derived from CD3-ζ.

[0144] In another aspect, the present application provides a nucleic acid encoding a chimeric antigen receptor (CAR) described herein. In another aspect, the present application provides a cell comprising a nucleic acid sequence provided herein that encodes a chimeric antigen receptor (CAR) described herein. In another aspect, the present application provides a genetically modified cell that expresses a chimeric antigen receptor (CAR) described herein.

[0145] In one embodiment, the cells are autologous T cells or autologous NK cells. As used herein, "autologous" refers to any material derived from the same individual that is subsequently reintroduced into that individual. In one embodiment, the cells are allogeneic T cells or allogeneic NK cells.

[0146] Polynucleotides and Recombinant Methods The present application provides isolated polynucleotides encoding anti-GPRC5D antibodies and antigen-binding fragments thereof. In another aspect, the present application provides nucleic acids encoding the chimeric antigen receptors (CARs) described herein. The terms "nucleic acid" or "polynucleotide," as used herein, refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. In certain embodiments, the isolated polynucleotides encode the variable regions of the exemplary antibodies provided herein. Unless otherwise indicated, a particular polynucleotide sequence implicitly encompasses not only the sequence explicitly indicated, but also conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more (or all) selected codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acids Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0147] DNA encoding a monoclonal antibody is readily isolated and sequenced by conventional procedures (e.g., oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains). The encoding DNA may also be obtained by synthetic methods.

[0148] Isolated polynucleotides encoding anti-GPRC5D antibodies and antigen-binding fragments thereof (e.g., including the sequences shown in Tables 1-4) or CARs may be inserted into vectors for further cloning (amplification of the DNA) or expression by recombinant techniques known in the art. Many vectors are available. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0149] The present application provides an expression vector comprising an isolated polynucleotide provided herein. In certain embodiments, the polynucleotide provided herein encodes an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker. Exemplary vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage, and M13 phage, as well as the plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, and pCI. , pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, and the like.

[0150] A vector containing a polynucleotide sequence encoding an antibody or antigen-binding fragment thereof may be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria such as Gram-negative or Gram-positive organisms, for example Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella such as Salmonella typhimurium, Serratia such as Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas aeruginosa (P. aeruginosa, and Streptomyces.

[0151] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, numerous other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe, e.g., Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 56,500), Kluyveromyces spp. ... 36,906), Kluyveromyces hosts such as Kluyveromyces thermotolerans and Kluyveromyces marxianus, Yarrowia (EP 402,226), Pichia pastoris (EP 183,070), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa, e.g., Schwanniomyces occidentalis, Schwanniomyces hosts include Schwanniomyces, such as A. occidentalis, and filamentous fungi, such as Neurospora, Penicillium, and Tolypocladium, as well as Aspergillus hosts, such as A. nidulans and A. niger.

[0152] Suitable host cells for expressing the glycosylated antibodies or antigen fragments provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 virus strain of Bombyx mori NPV, are publicly available, and such viruses can be used as viruses herein in accordance with the present invention, particularly for transfection of Spodoptera fallopian tuberculosis cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0153] However, vertebrate cells have received the most attention, and propagation of vertebrate cells in culture (tissue culture) has become common practice. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7, ATCC CRL 1651), human embryonic kidney (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proceedings of the National Academy of Sciences of the United States of America 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals of the New York Academy of Sciences, 383:44-68 (1982)), MRC5 cells, FS4 cells, and a human hepatocellular carcinoma line (Hep G2). In some preferred embodiments, the host cells are mammalian cultured cell lines such as CHO, BHK, NS0, 293, and their derivatives.

[0154] Host cells are transformed with an expression or cloning vector for producing the anti-GPRC5D antibody or antigen-binding fragment thereof described above and cultured in conventional nutrient media modified as necessary for inducing promoters, selecting transformants, or amplifying the gene encoding the desired sequence. In another embodiment, antibodies may be produced by homologous recombination, as known in the art. In certain embodiments, host cells are capable of producing the antibodies or antigen-binding fragments thereof provided herein.

[0155] Host cells for producing the antibodies or antigen-binding fragments thereof provided herein can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma are suitable for culturing host cells. Additionally, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Pat. Reissue No. 30,985 can be used as a culture medium for the host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those of skill in the art. Culture conditions, such as temperature and pH, will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0156] When using recombinant techniques, antibodies may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, particulate cell debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992), describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0157] Anti-GPRC5D antibodies and antigen-binding fragments thereof prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being preferred.

[0158] In certain embodiments, solid-phase-immobilized protein A is used for immunoaffinity purification of antibodies and their antigen-binding fragments. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices can also be used. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Depending on the antibody recovered, other protein purification techniques can also be used, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0159] Following any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer with a pH of about 2.5 to 4.5, preferably at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0160] Pharmaceutical Composition The present application further provides pharmaceutical compositions comprising the anti-GPRC5D antibody or antigen-binding fragment thereof of the present application, or the polynucleotide of the present application, the vector described in the present application, the conjugate described in the present application, the nucleic acid described in the present application comprising a nucleic acid sequence encoding the chimeric antigen receptor (CAR) described in the present application, or the cell described in the present application expressing the chimeric antigen receptor (CAR) described in the present application, and one or more pharmaceutically acceptable carriers.

[0161] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering / chelating agents, diluents, adjuvants, excipients, or other non-toxic auxiliary substances, or other ingredients known in the art, or various combinations thereof.

[0162] Suitable ingredients may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisol, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in compositions comprising the antibodies or antigen-binding fragments and conjugates provided herein reduces oxidation of the antibodies or antigen-binding fragments. This oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Thus, in certain embodiments, compositions are provided comprising one or more of the antibodies or antigen-binding fragments thereof disclosed herein and one or more antioxidants, such as methionine. Further provided are methods for preventing oxidation of, extending the shelf life of, and / or improving the efficacy of, the antibodies or antigen-binding fragments provided herein by combining the antibodies or antigen-binding fragments with one or more antioxidants, such as methionine.

[0163] As further described, pharmaceutically acceptable carriers include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; procaine hydrochloride; hydrochloride), suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN®-80), chelating agents such as ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antibacterial agents utilized as carriers may be added to pharmaceutical compositions in multidose containers and include phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable nontoxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0164] The pharmaceutical compositions may be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained-release formulations, or powders. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0165] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition can be prepared in any conventional form, such as a liquid solution, suspension, emulsion, or solid form suitable for preparing a liquid solution, suspension, or emulsion. Preparations for injection can include sterile and / or non-pyrogenic solutions prepared for injection, sterile dry soluble preparations such as lyophilized powders prepared to be combined with a solvent immediately before use, including hypodermic tablets, sterile suspensions prepared for injection, sterile dry insoluble preparations prepared to be combined with a vehicle immediately before use, and sterile and / or non-pyrogenic emulsions. The solution can be aqueous or non-aqueous.

[0166] In certain embodiments, unit dose parenteral preparations are packaged in ampoules, vials, or syringes with needles. All preparations for parenteral administration shall be sterile and non-pyrogenic, as known and practiced in the art.

[0167] In certain embodiments, a sterile, lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent may contain an excipient that improves the stability or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer such as citric acid, sodium phosphate, or potassium phosphate, or other such buffer known to those of skill in the art. In one embodiment, the buffer has a pH of about neutral. The desired formulation is then obtained by sterile filtration of the solution under standard conditions known to those of skill in the art, followed by lyophilization. In one embodiment, the resulting solution is apportioned into vials for lyophilization. Each vial may contain a single dose or multiple doses of an anti-GPRC5D antibody or antigen-binding fragment thereof, or a composition thereof. Overfilling the vial with a small amount (e.g., about 10%) beyond that required for a dose or set of doses may be acceptable to facilitate accurate sample draws and accurate dosing. The lyophilized powder may be stored under appropriate conditions, such as at about 4°C to room temperature.

[0168] Reconstitution of lyophilized powder with water for injection provides a formulation for use in parenteral administration.In one embodiment, sterile and / or non-pyrogenic water or other suitable liquid carrier is added to lyophilized powder for reconstitution.The exact amount depends on the given selected therapy and can be empirically determined.

[0169] How to use The present application further provides a method for treating a GPRC5D-related disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein or a pharmaceutical composition provided herein.

[0170] In another aspect, the present application provides a method of stimulating an immune cell (such as a T cell or NK cell)-mediated immune response in a mammal against cells or tissues expressing GPRC5D, the method comprising administering to the mammal an effective amount of genetically modified cells to express a CAR described in the present application.

[0171] In another aspect, the present application provides a method for treating a mammal having a GPRC5D-associated disease or condition, the method comprising administering to the mammal an effective amount of a cell provided in the present application, thereby treating the mammal.

[0172] In some embodiments, the GPRC5D-associated disease or condition is characterized by expression or overexpression of GPRC5D. Overexpression of GPRC5D has been documented in several autoimmune diseases, including myeloma.

[0173] In certain embodiments, GPRC5D-associated diseases or conditions include, but are not limited to, cancer and other hyperproliferative diseases, immune disorders, and inflammation.

[0174] In some embodiments, the cancer and other hyperproliferative disorders include benign or malignant tumors, leukemias, and lymphoid malignancies. Depending on the type of cell having the cancer or hyperproliferative disorder, examples include neurons, glial cells, astrocytes, hypothalamus, gland cells, macrophages, epithelial cells, endothelial cells, stromal malignancies, etc. Depending on the organ / site having the cancer or hyperproliferative disorder, examples include the head, neck, eye, oral cavity, larynx, esophagus, breast, skin, bone, lung, colon, rectum, stomach, spleen, kidney, skeletal muscle, subcutaneous tissue, metastatic melanoma, endometrium, prostate, breast, ovary, testis, thyroid, blood, lymph nodes, kidney, liver, pancreas, brain, or central nervous system.

[0175] In some embodiments, the immune disease and / or inflammation is selected from the group consisting of alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, adrenal autoimmune disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, Sjogren's syndrome, psoriasis, atherosclerosis, diabetic and other retinopathies, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasia (including Graves' disease), corneal and other tissue transplants, and chronic inflammation, sepsis, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusion injury, septicemia, endotoxic shock, Cystic fibrosis, endocarditis, psoriasis, arthritis (e.g., psoriatic arthritis), anaphylactic shock, organ ischemia, reperfusion injury, spinal cord injury and allograft rejection, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue and immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Guillain-Barré syndrome Barre), Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, hypertrichosis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary non-cancer Inflammatory diseases include inflammatory bowel disease, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteoarthritis, and chronic inflammation resulting from chronic viral or bacterial infections.

[0176] In one embodiment, the GPRC5D-associated disease or condition is cancer, particularly multiple myeloma, hi one embodiment, the GPRC5D-associated disease or condition is an autoimmune disease such as systemic lupus erythematosus and / or rheumatoid arthritis.

[0177] In certain embodiments, the GPRC5D-related disease or condition is a GPRC5D-expressing cancer. As used herein, "GPRC5D-expressing cancer" refers to any cancer or tumor in which GPRC5D is expressed on the surface of cancer cells. In certain embodiments, the expression level of GPRC5D on GPRC5D-expressing cancer cells is significantly higher than the expression level of GPRC5D on normal cells.

[0178] In certain embodiments, the subject is identified as having cancer cells that express GPRC5D. The presence and / or expression level of GPRC5D on cancer cells can be determined by various methods known in the art. A biological sample containing or suspected to contain cancer cells can be obtained from the subject. In some embodiments, the biological sample may be derived from cancer cells or cancer tissue. In certain embodiments, the biological sample may be further processed to isolate analytes, such as nucleic acids or proteins. The presence and / or expression level of GPRC5D can be determined by, for example, quantitative fluorescence cytometry, immunohistochemistry (IHC), or nucleic acid-based methods. For example, a biological sample from the subject can be exposed to an anti-GPRC5D antibody or an antigen-binding fragment thereof, which binds to and detects expressed GPRC5D protein. Alternatively, GPRC5D can be detected at the nucleic acid expression level by methods such as qPCR, reverse transcriptase PCR, microarray, SAGE, and FISH.

[0179] In one embodiment, the GPRC5D-related disease or condition includes, but is not limited to, GPRC5D-expressing breast cancer, multiple myeloma, Waldenstrom's macroglobulinemia, endometrial cancer, ovarian cancer, lung cancer, gastric cancer, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, colorectal cancer, esophageal cancer, bladder cancer, cervical cancer, blood cancer, lymphoma, or malignant melanoma.

[0180] The therapeutically effective amount of an antibody or antigen-binding fragment or pharmaceutical composition provided herein will depend on various factors known in the art, such as the subject's weight, age, past medical history, current drug treatment, health condition and potential cross-reactivity, allergies, hypersensitivity and side effects, as well as the route of administration and the extent of disease development. Doses can be proportionally increased or decreased by one skilled in the art (e.g., a physician or veterinarian) depending on these and other circumstances or requirements.

[0181] In certain embodiments, the antibodies or antigen-binding fragments or pharmaceutical compositions provided herein may be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the administered dose may vary over the course of treatment. For example, in certain embodiments, an initial administered dose may be higher than subsequent administered doses. In certain embodiments, the administered dose may vary over the course of treatment depending on the subject's response.

[0182] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered or several divided doses may be administered over time.

[0183] The antibodies and antigen-binding fragments disclosed herein can be administered by any route known in the art, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous (including intravenous infusion), intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0184] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein may be administered alone or in combination with one or more additional therapeutic procedures or agents. For example, the antibodies or antigen-binding fragments disclosed herein can be administered in combination with one or more treatments for a second therapeutic agent (e.g., a chemotherapeutic agent, an anti-cancer drug, a radiation therapy agent, an immunotherapy agent, an anti-angiogenic agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, or a palliative treatment), surgery for the treatment of cancer (e.g., tumor resection), or a chemotherapy-related complication.

[0185] The term "immunotherapy," as used herein, refers to a type of therapy that stimulates or generally strengthens the immune system to fight diseases such as cancer. Immunotherapy includes passive immunotherapy (e.g., antibody therapy or CAR-T cell therapy) by delivering drugs (e.g., effector cells) with confirmed tumor immune reactivity, which can directly or indirectly mediate anti-tumor effects and do not necessarily rely on an intact host immune system. Immunotherapy may also include active immunotherapy, in which treatment relies on in vivo stimulation of the endogenous host immune system to combat diseased cells by administration of immune response modifiers.

[0186] In certain of these embodiments, an antibody or antigen-binding fragment disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments, the antibody or antigen-binding fragment and the additional therapeutic agents may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment that is administered "in combination with" another therapeutic agent is not necessarily administered simultaneously with or in the same composition as that agent. An antibody or antigen-binding fragment that is administered before or after another agent is considered to be administered "in combination with" that agent, as that term is used herein, even if the antibody or antigen-binding fragment and the second agent are administered via different routes. When possible, additional therapeutic agents administered in combination with an antibody or antigen-binding fragment thereof disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent or according to Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition, Medical Economics Company, ISBN: 1563634457, 57th Edition (November 2002)), or protocols well known in the art.

[0187] The present application further provides a method for inhibiting the growth of cells expressing GPRC5D in vivo or in vitro, the method comprising contacting cells expressing GPRC5D with an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the present application provides a method for regulating GPRC5D activity in cells expressing GPRC5D, the method comprising exposing cells expressing GPRC5D to an antibody or antigen-binding fragment thereof provided herein.

[0188] In some embodiments, the present application provides a method for detecting the presence or level of GPRC5D in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the method further comprises determining the presence or content of GPRC5D in the sample. In certain embodiments, the biological sample contains cancer cells.

[0189] In some embodiments, the present application provides a method for diagnosing a GPRC5D-related disease or condition in a subject, the method comprising: a) contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein; b) measuring the presence or content of GPRC5D in the sample; and c) correlating the presence or content of GPRC5D with the presence or status of a GPRC5D-related disease or condition in the subject.

[0190] In some embodiments, the present application provides detection or treatment kits comprising an antibody or antigen-binding fragment thereof provided herein and instructions for use, optionally in combination with a detectable moiety. In some embodiments, the present application provides kits comprising an antibody or antigen-binding fragment thereof provided herein, optionally conjugated to a detectable moiety. The kits can be applied to the detection of GPRC5D or the treatment of GPRC5D-related diseases or conditions.

[0191] In some embodiments, the present application provides kits comprising an antibody or antigen-binding fragment thereof provided herein and a second therapeutic agent, which can be used to treat, prevent, and / or ameliorate a GPRC5D-associated disorder.

[0192] In some embodiments, the present application further provides the use of an antibody or antigen-binding fragment thereof provided herein for the manufacture of a medicament for treating a GPRC5D-related disease or condition in a subject, or for the manufacture of a diagnostic reagent for diagnosing a GPRC5D-related disease or condition.

[0193] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, are within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but are merely intended to illustrate specific embodiments within the scope of the invention. Those skilled in the art will be able to develop equivalent compositions, materials, and methods without inventive effort and without departing from the scope of the invention. It will be understood that many variations can be made to the procedures described herein within the scope of the invention. It is the intention of the inventors that such variations are included within the scope of the invention.

[0194] Example Example 1 Preparation of GPRC5D monoclonal antibody In this example, monoclonal antibodies were prepared using mice immunized with a tumor cell line expressing GPRC5D.

[0195] 1.1 Construction of 293T-GPRC5D and CHOS-GPRC5D cell lines Human GPRC5D (hGPRC5D) was overexpressed in HEK293 cells (ATCC) and CHOS cells (Invitrogen) by lentiviral infection (MOI = 3-10, 5 μg / ml polybrene). Seventy-two hours after cell infection, the corresponding antibiotic was added and the cells were cultured for 2-4 weeks. The cells were then expanded and cryopreserved to obtain two overexpressing cell lines, HEK293-hGPRC5D and CHOS-hGPRC5D, for subsequent immunological experiments.

[0196] 1.2 Construction of control antibody GC5B596 The HC and LC plasmids for the GC5B596 antibody were constructed using the following sequence constructions, and then transiently transfected into CHO cells and cultured. The supernatant was then purified by affinity chromatography to obtain the control antibody GC5B596. [Table 5]

[0197] 1.3 Mouse immunization / hybridoma fusion To generate anti-human GPRC5D antibodies, Balb / c mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., Category 216) were immunized with the constructed HEK293-GPRC5D cells overexpressing human GPRC5D. The primary adjuvant was complete Freund's adjuvant CFA (InvivoGen, Product Code: vac-cfa-60), followed by IFA (InvivoGen, Product Code: vac-ifa-60). Multiple immunizations were performed subcutaneously. After multiple immunizations, spleen cells from the immunized mice were fused with mouse myeloma cells SP2 / 0 using polyethylene glycol fusion and cultured in HAT selection medium to obtain hybridoma cells capable of expressing antibodies and growing indefinitely in vitro. The hybridoma cells were then plated into 96-well cell culture plates for further culture.

[0198] 1.4 Hybridoma cloning and screening The binding ability of antibodies secreted by hybridoma cells to GPRC5D was detected at the cellular level in a 96-well cell culture plate. GPRC5D-highly expressing cells were cultured in DMEM medium containing 10% FBS, digested with TrypLE trypsin, centrifuged, and resuspended in DPBS solution containing 2% BSA (FACS buffer, 4°C). 5 × 10 5Cells were added to a U-bottom 96-well plate at 50 μl per well and placed in a round-bottom low-binding 96-well plate. 50 μl of mouse hybridoma supernatant was added and incubated at 4°C for 1 hour. The supernatant was removed by centrifugation and washed twice with FACS buffer. A secondary antibody (DyLight488 goat anti-human IgG, Abcam, ab97003) was added to each well and incubated at 4°C for 0.5 hours. The supernatant was removed by centrifugation and washed twice with FACS buffer. The cells were then resuspended in FACS buffer and fluorescence measurements were performed on the cells in the experimental plate using a flow cytometer (BD, model number Canto II) to determine the binding status of the hybridoma supernatant to the cells. At the same time, a similar binding assay was performed using HEK293 cells as the background for constructing GPRC5D-overexpressing cells. Using positive HEK293-hGPRC5D binding and negative HEK293 cell binding as the standard, clones that were positive for GPRC5D binding were selected and subjected to two to three rounds of subcloning.

[0199] 1.5 Hybridoma sequencing / recombinant expression vector construction The Ch-72C7 clone was obtained by screening. Selected hybridoma clones were subjected to hybridoma sequencing according to standard hybridoma sequencing methods to obtain the heavy and light chain variable regions (VH and VL) of the selected clones. VH and VL were synthesized by total gene synthesis and linked to human IgG1 and kappa chain constant regions. The heavy and light chain sequences were ligated into the pcDNA3.4 vector and transiently expressed in the 293 system followed by protein A / G purification. The resulting chimeric recombinant antibody was ultrafiltered and the buffer was replaced with PBS solution. The sequencing results for the Ch-72C7 clone are shown in Table 6. [Table 6]

[0200] Example 2 Antigen Binding FACS Experiment HEK293-hCD22GPRC5D and CHOS-hGPRC5D cells expressing hGPRC5D were centrifuged and resuspended in DPBS solution containing 2% BSA (FACS buffer, 4°C), and 5 × 10 5 Cells were added to a U-bottom 96-well plate at 100 μl per well, and gradient-diluted antibodies were added. The cells were incubated at 4°C for 1 hour, centrifuged, and the supernatant was discarded. 100 μl of anti-human IgG Fc-APC secondary antibody was added per well and incubated at 4°C for 1 hour. The cells were then washed once with FACS buffer, resuspended in 200 μl of FACS buffer, and the fluorescent signal was read using a BD Canto II. Results showed that the ch-72C7 antibody bound to HEK293-hGPRC5D (Figure 1) and CHOS-hGPRC5D (Figure 2).

[0201] MM.1R cells, NCI-H929 cells, and RPMI-8226 cells (GPRC5D high expression, ATCC, CL-188) were cultured in RPMI1640 medium containing 10% FBS. The cells were digested with TrypLE trypsin, centrifuged, and resuspended in DPBS solution containing 2% BSA (FACS buffer, 4°C). 5 × 10 5 Cells were added to a U-bottom 96-well plate at 100 μl per well, and gradient-diluted antibodies were added. The mixture was incubated at 4°C for 1 hour, centrifuged, and the supernatant was discarded. 100 μl of anti-human IgG Fc-APC secondary antibody was added per well and incubated at 4°C for 1 hour. The cells were then washed once with FACS buffer, resuspended in 200 μl of FACS buffer, and the fluorescent signal was read using a BD Canto II. Results showed that the ch-72C7 antibody bound to MM.1R cells (Figure 3), NCI-H929 cells (Figure 4), and RPMI-8226 cells (Figure 5). [Table 7]

[0202] Example 3 Evaluation of ADCC effect of antibodies Target cells were tumor cells naturally expressing GPRC5D (NCI-H929, Nanjing Kebai Biosciences; MM.1R, Nanjing Kebai Biosciences), and effector cells were stably transfected with the in-house constructed Jurkat-NFAT-Luc-CD16 cell line, expressing the CD16 receptor and NFAT response element. Experiments were performed in 96-well flat-bottom cell plates (Corning 3903). Gradient-diluted antibodies were added to the target cells and incubated at 37°C for 30 minutes. 60,000 effector cells were added per 10,000 target cells and incubated at 37°C for 6 hours. After the incubation, One-Glo™ reagent (Promega, E6110) was added for fluorescence development, and the cell plates were read using a Tecan Spark10 microplate reader. Data analysis was performed using GraphPad, with the horizontal axis representing the logarithm of antibody concentration and the vertical axis representing the luminescence readings from the corresponding wells. The EC50 values ​​for antibody-dependent cellular cytotoxicity of anti-GPRC5D antibodies were fitted to the curve. The results showed that the blank control isotype (ISO) had no cytotoxic effect on NCI-H929 cells and MM.1R cells. Both the ch-72C7 and CG5B596 antibodies had cytotoxic effects on tumor cells NCI-H929 (Figure 6) and RPMI-8226 (Figure 7), which naturally express GPRC5D. Compared to the CG5B596 antibody, the ch-72C7 antibody exhibited stronger ADCC activity.

[0203] Example 4 Humanized antibody design and expression The ch-72C7 antibody was compared with the IMGT database, and the human Framework sequence with the highest homology to its VH / VL was selected. CDR grafting was performed, and computational chemistry simulations were performed to maintain antigen binding. The amino acid sequence of the humanized antibody is shown in Table 8. [Table 8-1] [Table 8-2]

[0204] The VH and VL regions of these antibodies were linked to the human IgG1 Fc region and kappa light chain constant region, and the antibody heavy and light chain sequences were inserted into the pcDNA3.4 vector. They were transiently expressed in HEK293 cells, and the antibodies were purified using protein A or G.

[0205] At the same time, the VH and VL of the ch-72C7 antibody were replaced with the VH and VL of a human IgG1 antibody, respectively, to form a chimeric antibody, 22mono, which was used as a control to evaluate the humanization results of each antibody. [Table 9]

[0206] Example 5 Affinity testing of humanized GPRC5D antibodies A binding affinity assay for human GPRC5D was performed using the recombinant humanized antibody expressed by the method of Example 4. The recombinant GPRC5D antigen (Human GPRC5D protein-Flag-His tag (51.8 KD) ACRO Cat: GPD-H52D3) was immobilized on a chip and detected using the Octet® R8 Biomolecular Interaction Analysis System. The results are shown in Table 10. [Table 10]

[0207] The results showed that all four humanized GPRC5D antibodies had excellent binding affinity to the human GPRC5D antigen, with 22Mono5JO4 having the highest binding affinity to the antigen. Because the binding affinity to the human GPRC5D antigen exceeds that of the original mouse antibody ch-72C7, it is expected that the binding to the human GPRC5D antigen and cytotoxicity against disease cells expressing the human GPRC5D antigen will be at least comparable to, and even superior to, the mouse antibody ch-72C7.

[0208] While the present application has been particularly shown and described with reference to specific embodiments, some of which are preferred embodiments, it should be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the present application as disclosed herein.

Claims

1. An anti-GPRC5D antibody or an antigen-binding fragment thereof, comprising three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the heavy chain complementarity determining regions are the heavy chain variable region (V) shown in SEQ ID NO:

13. Ｈ ) and the light chain complementarity determining region is the same as the light chain variable region (V Ｌ ) an antibody or antigen-binding fragment thereof, wherein the three light chain complementarity-determining regions are the same as those contained within

2. An anti-GPRC5D antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, a) said HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, or SEQ ID NO: 9 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions; b) said HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 or a variant thereof having 3, 2 or no more than 1 amino acid substitutions, or SEQ ID NO: 10 or a variant thereof having 3, 2 or no more than 1 amino acid substitutions; c) the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 or a variant thereof having 3, 2 or no more than 1 amino acid substitutions, or SEQ ID NO: 11 or a variant thereof having 3, 2 or no more than 1 amino acid substitutions; d) said LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, or SEQ ID NO: 7 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions; e) the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions, or SEQ ID NO: 8 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions; and f) An antibody or antigen-binding fragment thereof, wherein the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3 or a variant thereof having no more than 3, 2 or 1 amino acid substitutions.

3. a) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, or b) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 8 or a variant thereof having 3, 2 or 1 or less amino acid substitutions, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof having 3, 2 or 1 or less amino acid substitutions; An antibody or antigen-binding fragment thereof according to any one of the preceding claims.

4. 10. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13 or a variant thereof having 3, 2, or no more than 1 amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12 or a variant thereof having 3, 2, or no more than 1 amino acid substitutions.

5. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is humanized.

6. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V Ｈ ) and / or light chain variable region (V Ｌ ), and a) the heavy chain variable region comprises an amino acid sequence selected from the group of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, or a variant thereof having no more than 3, 2, or 1 amino acid substitution; b) The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the light chain variable region is selected from the amino acid sequence of the group of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or variants thereof having no more than 3, 2, or 1 amino acid substitutions.

7. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V Ｈ ) and / or light chain variable region (V Ｌ ), and a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15, or a variant thereof having three, two, or one or fewer amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, or a variant thereof having three, two, or one or fewer amino acid substitutions; b) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17 or a variant thereof having three, two, or one or fewer amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof having three, two, or one or fewer amino acid substitutions; c) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof with no more than 3, 2, or 1 amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 or a variant thereof with no more than 3, 2, or 1 amino acid substitutions; or d) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof with no more than 3, 2, or 1 amino acid substitutions, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20 or a variant thereof with no more than 3, 2, or 1 amino acid substitutions; 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the variant still retains specific binding affinity to GPRC5D.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the amino acid substitution is not within a CDR region.

9. 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims, which is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide bond stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide bond stabilized diabody (ds diabody), single chain antibody molecule (scFv), or scFv dimer (bivalent diabody).

10. 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims, further comprising an immunoglobulin constant region, optionally comprising the constant region of a human immunoglobulin, or optionally comprising the constant region of a human IgG.

11. The antibody or antigen-binding fragment thereof of claim 9, wherein the human IgG constant region is derived from IgG1, IgG2, IgG3, or IgG4.

12. 11. The antibody or antigen-binding fragment thereof of claim 9 or 10, wherein the constant region comprises one or more amino acid residue substitutions or modifications, which confers increased CDC or ADCC compared to the wild-type constant region.

13. 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims, which is bispecific or multispecific.

14. A nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 13.

15. A vector comprising the nucleic acid of claim 14.

16. A host cell comprising the nucleic acid of claim 14 or the vector of claim 15.

17. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 and a payload conjugated thereto, wherein the payload is selected from the group consisting of a radioactive label, a fluorescent label, an enzyme substrate label, an affinity purification tag, a tracking molecule, an anticancer drug and a cytotoxic molecule.

18. A chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an immune cell signaling domain, wherein the antigen-binding domain specifically binds to GPRC5D, and the CAR comprises the antigen-binding fragment of any one of claims 1 to 13.

19. A nucleic acid encoding the chimeric antigen receptor (CAR) of claim 18.

20. A cell comprising the nucleic acid sequence of claim 19.

21. A genetically modified cell that expresses the CAR of claim 19.

22. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the nucleic acid according to claim 14, the vector according to claim 15, the conjugate according to claim 18, the nucleic acid according to claim 19, or the cell according to claim 20 or 21, and a pharmaceutically acceptable carrier.

23. 21. A method for treating or preventing a disease, condition, or symptom, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 13, or the nucleic acid of claim 14, the vector of claim 15, the conjugate of claim 18, the nucleic acid of claim 19, the cell of claim 20 or 21, or the composition of claim 22.

24. 24. The method of claim 23, wherein the disease, condition or symptom is selected from the group of cancer, immune disease, inflammation.

25. A method for stimulating an immune cell (such as a T cell or NK cell)-mediated immune response against a cell or tissue expressing GPRC5D in a mammal, the method comprising administering to the mammal an effective amount of genetically modified cells to express the CAR of claim 18.

26. 21. A method of treating a mammal having a GPRC5D-related disease or condition, comprising administering to said mammal an effective amount of the cells of claim 20, thereby treating said mammal.

27. 27. The method of claim 26, wherein the cells are autologous T cells or autologous NK cells.

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