Antibody binding to GPRC5d and use thereof

Abstract

Provided in the present application is an antibody specifically binding to GPRC5D or an antigen-binding portion thereof. The present application further relates to a bispecific T cell engager or chimeric antigen receptor, etc., that comprises the antibody or the antigen-binding portion thereof, and the use thereof in the treatment of multiple myeloma.

Description

Antibodies binding to GPRC5D and uses thereof

[0001] Cross reference to related applications

[0002] This application claims priority to Chinese Patent Application No. CN202410824863.4, filed on June 24, 2024, the entirety of which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to an antibody or antigen-binding portion thereof that binds to GPRC5D, and uses thereof in the treatment of, for example, multiple myeloma. BACKGROUND

[0004] Multiple myeloma (MM) is the second most common malignancy, accounting for 2% of all cancer deaths, and is characterized by the abnormal proliferation of plasma cells in the bone marrow. Healthy plasma cells produce antibodies that recognize and attack, for example, microorganisms, while cancerous plasma cells in multiple myeloma accumulate within the bone marrow, crowd out healthy blood cells, and produce abnormal antibody proteins that trigger complications.

[0005] Multiple myeloma is almost incurable, with a very high relapse rate, and the 5-year survival rate of patients is about 58%. Recently approved BCMA therapies, such as CAR-T and bispecific T-cell engagers, have achieved unprecedented remission rates in relapsed MM patients. However, most patients are doomed to disease progression, and the presence of residual multiple myeloma cells with low expression of BCMA in the body may be one of the causes of relapse.

[0006] Since the expression of GPRC5D was found on malignant tumor plasma cells, CAR-T cells and bispecific T cell engagers targeting this molecule have been gradually developed and have achieved good results in patients who relapsed after BCMA therapy. Bispecific tafurid, which targets GPRC5D and CD3, has been selected by the US FDA as the Breakthrough Therapy Designation of 2022 based on its phase 1 clinical trial, and has been approved by the FDA for accelerated approval on August 10, 2023, for the treatment of patients with relapsed / refractory multiple myeloma who have received at least 4 treatments, including immunomodulators, anti-CD38 antibodies and proteasome inhibitors (Smith EL et al., (2019) GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells. Sci Transl Med. 11(485): eaau7746; Del Giudice, M. L., Galimberti, S. & Buda, G. (2023) Beyond BCMA, why GPRC5D could be the right way: treatment strategies with immunotherapy at relapse after anti-BCMA agents. Cancer Immunol Immunother (2023). https: / / doi.org / 10.1007 / s00262-023-03559-4).

[0007] The full name of GPRC5D is G protein-coupled receptor C5 family subtype D, which belongs to an orphan receptor and is a member of the G protein-coupled receptor (GPCR) family. GPCR is the largest and most diverse membrane receptor protein family in eukaryotes, which plays a series of important functions in the human body, and about 30-40% of the drugs on the market target GPCR. GPRC5D, as a therapeutic target for MM, has two advantages. First, GPRC5D is highly expressed on the surface of multiple myeloma cells, while its expression in normal tissues is limited to areas such as hair follicles and testes, and the expression amount is low. Second, the expression of GPRC5D is independent of BCMA and is not affected by BCMA therapy, so it can be used for the treatment of patients who have received BCMA therapy and show certain resistance.

[0008] Currently under investigation for GPRC5D therapeutics include CAR-T products and bispecific T cell engagers. BMS-986393, a CAR-T therapy, achieved 100% overall response rate in patients who had not received prior BCMA therapy and 78% in patients who had received prior BCMA therapy in Phase 1 clinical; tafuridomab is being tested in multiple clinical trials as monotherapy and in combination (Manisha Bhutani, & Saad Z. Usmani (2023) GPRC5D: The Next Frontier for Immunotherapy in Multiple Myeloma, The Hematologist 20(1) https: / / doi.org / 10.1182 / hem.V20.1.202314).

[0009] In view of the significant role of GPRC5D-targeted therapy in the treatment of multiple myeloma, there is a need to develop more GPRC5D antibodies with good therapeutic effect and high safety. SUMMARY

[0010] The inventors of the present application have prepared several GPRC5D antibodies, which have comparable or superior binding ability to GPRC5D-expressing cells (e.g., human myeloma cells) and / or comparable or superior internalization properties compared to prior art GPRC5D antibodies, such as the GPRC5D antibody portion in tafuridomab and the GPRC5D antibody portion in LM305.

[0011] These antibodies or antigen-binding portions thereof of the present application can act on multiple myeloma cells via antibody-dependent cellular cytotoxicity (ADCC), and / or antibody-dependent cellular phagocytosis (ADCP), etc., or by constructing antibody drug conjugates (ADCs), bispecific T cell engagers (TCEs), chimeric antigen receptors (CARs), recombinant T cell receptors, etc.

[0012] Accordingly, in a first aspect, the present application relates to an isolated monoclonal antibody (e.g., mouse-derived, chimeric, or humanized antibody), or antigen binding portion thereof, capable of specifically binding to GPRC5D (e.g., human, monkey GPRC5D), which can comprise i) a heavy chain variable region which can comprise a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 can comprise the amino acid sequences set forth in (1) SEQ ID NOs: 1, 2, and 3, respectively; (2) SEQ ID NOs: 9, 10, and 11, respectively; (3) SEQ ID NOs: 17, 18, and 19, respectively; (4) SEQ ID NOs: 25, 26, and 27, respectively; (5) SEQ ID NOs: 33, 34, and 35, respectively; (6) SEQ ID NOs: 41, 42, and 43, respectively; (7) SEQ ID NOs: 49, 50, and 51, respectively; (8) SEQ ID NOs: 57, 58, and 59, respectively; (9) SEQ ID NOs: 65, 66, and 67, respectively; (10) SEQ ID NOs: 73, 74, and 75, respectively; (11) SEQ ID NOs: 81, 82, and 83, respectively; or (12) SEQ ID NOs: 89, 90, and 91, respectively; and / or ii) a light chain variable region which can comprise a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 can comprise the amino acid sequences set forth in (1) SEQ ID NOs: 4, 5, and 6, respectively; (2) SEQ ID NOs: 12, 13, and 14, respectively; (3) SEQ ID NOs: 20, 21, and 22, respectively; (4) SEQ ID NOs: 28, 29, and 30, respectively; (5) SEQ ID NOs: 36, 37, and 38, respectively; (6) SEQ ID NOs: 44, 45, and 46, respectively; (7) SEQ ID NOs: 52, 53, and 54, respectively; (8) SEQ ID NOs: 60, 61, and 62, respectively; (9) SEQ ID NOs: 68, 69, and 70, respectively; (10) SEQ ID NOs: 76, 77, and 78, respectively; (11) SEQ ID NOs: 84, 85, and 86, respectively; or (12) SEQ ID NOs: 92, 93, and 94, respectively. Also provided are variants of the above antibodies or antigen binding portions, which comprise up to about 3 amino acid residue substitutions, e.g., 1, 2, or 3 amino acid residue substitutions, in each CDR as compared to the above antibodies or antigen binding portions thereof.

[0013] The isolated monoclonal antibody or antigen-binding portion thereof of the present application can comprise a heavy chain variable region and a light chain variable region, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 can comprise the amino acid sequences set forth in (1) SEQ ID NOs: 1, 2, 3, 4, 5, and 6; (2) SEQ ID NOs: 9, 10, 11, 12, 13, and 14; (3) SEQ ID NOs: 17, 18, 19, 20, 21, and 22; (4) SEQ ID NOs: 25, 26, 27, 28, 29, and 30; (5) SEQ ID NOs: 33, 34, 35, 36, 37, and 38; (6) SEQ ID NOs: 41, 42, 43, 44, 45, and 46; (7) SEQ ID NOs: 49, 50, 51, 52, 53, and 54; (8) SEQ ID NOs: 57, 58, 59, 60, 61, and 62; (9) SEQ ID NOs: 65, 66, 67, 68, 69, and 70; (10) SEQ ID NOs: 73, 74, 75, 76, 77, and 78; (11) SEQ ID NOs: 81, 82, 83, 84, 85, and 86; or (12) SEQ ID NOs: 89, 90, 91, 92, 93, and 94, respectively. Variants of the above antibodies or antigen-binding portions are also provided that comprise up to about 3 amino acid residue substitutions in each CDR compared to the above antibodies or antigen-binding portions thereof, e.g., 1, 2, or 3 amino acid residue substitutions.

[0014] The heavy chain variable region of the antibody or antigen-binding portion thereof of the present application can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, or 95.

[0015] The light chain variable region of the antibody or antigen-binding portion thereof of the present application can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, or 96.

[0016] The heavy chain variable region and the light chain variable region of the antibody or antigen-binding portion thereof of the present application can each comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to (1) SEQ ID NOs: 7 and 8; (2) SEQ ID NOs: 15 and 16; (3) SEQ ID NOs: 23 and 24; (4) SEQ ID NOs: 31 and 32; (5) SEQ ID NOs: 39 and 40; (6) SEQ ID NOs: 47 and 48; (7) SEQ ID NOs: 55 and 56; (8) SEQ ID NOs: 63 and 64; (9) SEQ ID NOs: 71 and 72; (10) SEQ ID NOs: 79 and 80; (11) SEQ ID NOs: 87 and 88; or (12) SEQ ID NOs: 95 and 96.

[0017] The isolated monoclonal antibody or antigen-binding portion thereof of the present application can comprise a heavy chain constant region and / or a light chain constant region, wherein the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region. The heavy chain constant region can be an IgG, IgD, IgA, IgM, or IgE heavy chain constant region, preferably a heavy chain constant region that naturally or after modification has Fc receptor and / or complement system protein binding ability, or a functional fragment thereof, such as a fragment comprising the hinge region, CH2, and CH3 of the heavy chain constant region. In one embodiment, the heavy chain constant region can be an IgG1 heavy chain constant region, such as a human IgG1 heavy chain constant region. In some embodiments, the heavy chain constant region can comprise the amino acid sequence set forth in SEQ ID NO: 97. The light chain constant region can be a kappa or lambda light chain constant region, such as a human kappa light chain constant region. In some embodiments, the light chain constant region can comprise the amino acid sequence set forth in SEQ ID NO: 98.

[0018] The antibody of the present application can in some embodiments comprise or consist of two heavy chains and two light chains, wherein each heavy chain comprises the heavy chain constant region sequence, the heavy chain variable region sequence, and / or the CDR sequence described above, and each light chain comprises the light chain constant region sequence, the light chain variable region sequence, and / or the CDR sequence described above. In some embodiments, the antibody or antigen-binding portion thereof of the present application can be a Fab, F(ab')2 fragment, Fv, scFv, or (scFv)2, etc.

[0019] The antibody or antigen-binding portion thereof of the present application can be, for example, mouse-derived, chimeric, or humanized.

[0020] The present application also provides immunoconjugates comprising an antibody or antigen-binding portion thereof of the present application, which can be linked to a therapeutic agent, such as a cytotoxic molecule or an anti-cancer agent. The present application also provides bispecific molecules comprising an antibody or antigen-binding portion thereof of the present application, which can be linked to a second functional group, such as a second antibody, which has a different binding specificity than the antibody or binding portion thereof of the present application. In some embodiments, the second functional group can specifically bind to a T cell, in particular, the second functional group can specifically bind to CD3 on a T cell. In another aspect, the present application provides a chimeric antigen receptor (CAR) or a genetically engineered T cell receptor (TCR) comprising an antibody or antigen-binding portion thereof of the present application. The present application also provides an immune cell, including a T cell, a NK cell, etc., comprising the above-mentioned CAR and / or TCR. The present application also provides an oncolytic virus encoding or carrying an antibody or antigen-binding portion thereof of the present application.

[0021] The present application also includes nucleic acid molecules encoding an antibody or antigen-binding portion thereof, an immunoconjugate, a bispecific molecule, a CAR, or a TCR of the present application. The present application can also provide an expression vector and a host cell. The expression vector can comprise a nucleic acid molecule of the present application. The host cell can comprise an expression vector of the present application, or a nucleic acid molecule of the present application integrated in its genome.

[0022] The present application also provides a method of using a host cell of the present application to produce an antibody or antigen-binding portion thereof, an immunoconjugate, a bispecific molecule, a CAR, or a TCR of the present application, comprising: (i) expressing the antibody or antigen-binding portion thereof, the immunoconjugate, the bispecific molecule, the CAR, or the TCR in the host cell, and (ii) isolating the antibody or antigen-binding portion thereof, the immunoconjugate, the bispecific molecule, the CAR, or the TCR from the host cell or its culture.

[0023] The present application also provides a composition comprising an antibody or antigen-binding portion thereof, an immunoconjugate, a bispecific molecule, a CAR / TCR, an immune cell carrying a CAR / TCR, an oncolytic virus, a nucleic acid molecule, an expression vector, or a host cell of the present application. In some embodiments, the composition can be a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding portion thereof, an immunoconjugate, a bispecific molecule, a CAR / TCR, an immune cell carrying a CAR / TCR, an oncolytic virus, a nucleic acid molecule, an expression vector, or a host cell, and can further comprise a pharmaceutically acceptable carrier.

[0024] In another aspect, the present application provides a method of treating or slowing a GPRC5D-associated disease in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present application.

[0025] The GPRC5D-related disease can be a GPRC5D-related cancer, such as multiple myeloma, plasma cell leukemia, or plasmacytoma. In some embodiments, the pharmaceutical composition of the present application can be administered with at least one anti-cancer agent, such as a PD-L1 antibody, etc. In another embodiment, the pharmaceutical composition of the present application is administered with a cytokine (such as IL-2 and / or IL-21) or a costimulatory antibody (such as a CD137 antibody and / or a GITR antibody). In another embodiment, the pharmaceutical composition of the present application can be administered with a chemotherapeutic agent, which can be a cytotoxic agent. The subject can be a mammal, particularly a human.

[0026] The present application also provides a method of inhibiting the growth of a GPRC5D + cell, comprising contacting the GPRC5D + cell with the composition of the present application. The GPRC5D + cell can be a myeloma cell.

[0027] The present application also protects the use of the composition of the present application in the manufacture of a medicament for treating a GPRC5D-related disease, and in the manufacture of a medicament for inhibiting the growth of a GPRC5D + cell.

[0028] It should be noted that in the present application, particularly in the claims, the terms such as "comprising", "including" or the like can have the meaning ascribed to them under the Chinese Patent Law; and the terms such as "consisting essentially of' have the meaning ascribed to them under the Chinese Patent Law, e.g., allowing for the presence of elements that do not materially affect the basic or novel characteristics of the application.

[0029] Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in conjunction with the accompanying drawings of which: BRIEF DESCRIPTION OF DRAWINGS

[0030] The following detailed description is presented in connection with the appended drawings. It is not intended to limit the application to the precise form described. In the drawings:

[0031] Figure 1 shows the binding of the anti-GPRC5D chimeric antibodies of the present application to 3T3 / GPRC5D cells tested by FACS.

[0032] Figure 2 shows the binding of the anti-GPRC5D chimeric antibodies of the present application to human myeloma cells MM.1S cells tested by FACS.

[0033] Figure 3 shows the binding of the anti-GPRC5D chimeric antibodies of the present application to HEK293T / monkey GPRC5D cells as tested by FACS.

[0034] Figure 4 shows the internalization of the anti-GPRC5D chimeric antibodies of the present application by 3T3 / GPRC5D cells.

[0035] Figure 5 shows that the anti-GPRC5D chimeric antibodies of the present application compete for the epitope of the GPRC5D antibody portion in talacotumab.

[0036] Figure 6 shows that the anti-GPRC5D chimeric antibodies of the present application compete for the epitope of the GPRC5D antibody portion in LM305. DETAILED DESCRIPTION

[0037] The terms used in the specification, unless otherwise specifically defined, have their ordinary meaning as understood by one of ordinary skill in the art. The following description of some terms is provided for the purpose of understanding the present application, and is not intended to be specially limiting, unless otherwise specifically indicated.

[0038] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0039] The term "or" means a single element of the list, unless the context clearly dictates otherwise.

[0040] The term "comprising" or "including" means including but not limited to, such that other elements, integers or steps are not precluded. In the present document, when the term "comprising" or "including" is used, it also covers the case where the elements, integers or steps are combined, unless otherwise indicated.

[0041] The term "GPRC5D" refers to G protein-coupled receptor C5 family, subtype D. The term includes variants, homologs, orthologs, and paralogs. For example, an antibody specific for human GPRC5D can in some cases cross-react with GPRC5D protein of another species, such as monkey.

[0042] The term “human GPRC5D” refers to a GPRC5D protein having a human amino acid sequence, for example a GPRC5D protein having the amino acid sequence of NCBI Accession Number NP_061124.1 (Mi X, Penson A, Abdel-Wahab O and Mailankody S. (2023) Genetic Basis of Relapse after GPRC5D-Targeted CAR T Cells N Engl J Med 389(15): 1435-1437). “Monkey GPRC5D” refers to a GPRC5D protein having a monkey amino acid sequence, for example a GPRC5D protein having the amino acid sequence of NCBI Accession Number XP_005570250.2.

[0043] The term “antibody” herein is intended to include IgG, IgA, IgD, IgE and IgM full length antibodies and any antigen binding fragment (i.e., antigen binding portion) thereof. Full length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, i.e., CH H1 , CH H2 , and CH H3 . Each light chain is comprised of a light chain variable region (abbreviated herein as V L or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL L . The V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors (FcRs) of various immune system cells (e.g., effector cells) and the first component (Clq) of the classical complement system. A “functional fragment” of an antibody constant region refers to a fragment of the constant region that retains certain desired functions, for example a fragment of the heavy chain constant region that retains FcR / complement system component binding activity, such as an Fc fragment.

[0044] The term "antigen-binding portion" of an antibody (or simply "antibody portion"), used herein, refers to one or more fragments of an antibody that retain its ability to specifically bind to an antigen (e.g., a GPRC5D protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L , and C H1 domains of a single arm of V H , V H1 domains of a single arm of V L , V H domains of a single arm of V H ; (vi) an isolated complementarity determining region (CDR); and (vii) a dAb- V L , a fragment comprising a single variable domain and a heavy chain constant domain. Furthermore, although the two domains of the Fv fragment, V L , and V H , are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L , and V H regions pair to form monovalent molecules. Such single chain antibodies are also intended to be encompassed within the term. These antibody fragments are obtained using conventional techniques known to those with ordinary skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0045] The term "isolated antibody" as used herein refers to an antibody that is substantially free of other antibodies having different antigenic specificities. For example, an isolated antibody that specifically binds to a GPRC5D protein is substantially free of antibodies that specifically bind antigens other than the GPRC5D protein. However, an isolated antibody that specifically binds to a human GPRC5D protein can have cross-reactivity to other antigens, such as GPRC5D proteins of other species. Moreover, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0046] The term "monoclonal antibody" or "monoclonal" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0047] The term "mouse-derived antibody" refers to an antibody whose variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. Mouse-derived antibodies of the application can contain amino acid residues that are not encoded by mouse germline immunoglobulin sequences, e.g., mutations introduced by in vitro random or site-directed mutagenesis or by in vivo somatic mutation. However, the term "mouse-derived antibody" does not encompass antibodies in which CDR sequences derived from another mammalian species have been grafted into a mouse framework sequence.

[0048] The term "chimeric antibody" refers to an antibody that has been derived by combining genetic material of non-human origin, such as mouse origin, with genetic material of human origin. Alternatively, more generally, a chimeric antibody refers to an antibody that has combined genetic material of one species with genetic material of another species.

[0049] The term "humanized antibody" refers to an antibody that has been derived from a non-human species, such as mouse, whose protein sequence has been altered to increase similarity to antibodies that are naturally produced in the human body.

[0050] The terms "specifically recognizes" or "specifically binds" a target, such as human GPRC5D, as used herein, means that an antibody or antigen-binding fragment is able to distinguish the target biological molecule from one or more reference molecules, and has a binding affinity or binding activity for the target biological molecule that is greater than, e.g., 1-fold, 5-fold, 10-fold, etc., than for the other reference molecules. Methods for determining specificity include, but are not limited to, SPR, Western blot, ELISA, RIA, ECL, IRMA assays, and peptide scanning.

[0051] The term "EC 50 " or "half maximal effective concentration" refers to the concentration of an antibody that elicits 50% of the maximum effect.

[0052] The term "IC 50 " or "half maximal effective concentration" refers to the concentration of an antibody that elicits 50% of the maximum effect.

[0053] The term "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refers to a form of cell-mediated immune defense in which immune system effector cells actively lyse target cells that have bound an antibody, such as a GPRC5D single-domain antibody or heavy chain antibody of the application, to their cell membrane surface.

[0054] The term "antibody-dependent cellular phagocytosis" or "ADCP" refers to an immune elimination mechanism in which an antibody, such as a GPRC5D heavy chain antibody of the application, binds to a target cell and recruits an immune effector cell, such as a phagocyte, via a constant region, such as an Fc, thereby facilitating phagocytosis of the target cell by the immune effector cell.

[0055] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, e.g., non-human primates, sheep, dogs, cats, cows, and horses.

[0056] The term "therapeutically effective amount" refers to an amount of an antibody of the present application sufficient to prevent or slow the progression of symptoms associated with a disease or disorder, e.g., cancer. A therapeutically effective amount is related to the disease being treated, where one of skill in the art can readily determine the actual effective amount.

[0057] "Sequence identity" herein refers to the percentage of nucleotides / amino acid residues in a sequence that are identical with those of a reference sequence, after aligning the sequences for optimal comparison. Spaces are introduced into the sequence alignment to achieve the maximum percent sequence identity, if necessary. One of skill in the art can determine the percent sequence identity between two or more nucleic acid or amino acid sequences by a variety of methods, e.g., using computer software, such as Clustal Omega, T-coffee, Kalign, and MAFFT.

[0058] GPRC5D antibodies of the present application have comparable or superior GPRC5D-expressing cell (e.g., human myeloma cell) binding and / or comparable or superior internalization properties compared to prior art GPRC5D antibodies, e.g., the GPRC5D antibody portion in talquetamab, and the GPRC5D antibody portion in LM305.

[0059] Preferred antibodies of the present application are monoclonal antibodies. In addition, the antibodies or antigen-binding portions thereof can be, e.g., mouse-derived, chimeric, or humanized.

[0060] The heavy chain variable region and light chain variable region sequences or sequence identifiers of the antibodies of the present application or antigen-binding portions thereof are set forth in Table 2. The heavy chain variable region CDRs and light chain variable region CDRs are determined by the Contact numbering system, and the CDR sequences or sequence identifiers determined thereby are set forth in Table 2. The heavy chain variable region CDRs and light chain variable region CDRs of the antibodies of the present application or antigen-binding portions thereof can also be determined based on the full-length sequences of the variable regions by the IMGT, Chothia, Kabat, or AbM numbering systems.

[0061] An antibody of the application can comprise a heavy chain constant region, e.g., a native or engineered one having FcR and / or complement system protein binding capacity, particularly high FcR and / or complement system protein binding capacity. In some embodiments, the heavy chain constant region can be an IgGl constant region, comprising, e.g., the amino acid sequence set forth in SEQ ID NO: 97. The light chain constant region can be a kappa constant region, e.g., a human kappa constant region, comprising, e.g., the amino acid sequence set forth in SEQ ID NO: 98.

[0062] VH and / or VL sequences of other GPRC5D antibodies that bind to human GPRC5D H and / or VH L sequences (or CDR sequences) can be "mixed and matched" with VH H and / or VL L sequences of an antibody of the application. Preferably, when VH H and VL L sequences (or CDRs therein) are mixed and matched, the particular VH H / VH L pair is one that specifically binds to human GPRC5D. H sequences can be substituted for structurally similar VH H sequences. Similarly, it is preferred that the VH H / VH L pair is one in which the VH L sequences are substituted for structurally similar VH L sequences.

[0063] Thus, in one embodiment, an antibody of the application, or antigen-binding portion thereof, comprises:

[0064] (a) a heavy chain variable region comprising an amino acid sequence set forth in Table 2; and

[0065] (b) a light chain variable region comprising an amino acid sequence set forth in Table 2, or CDRs of another GPRC5D antibody, wherein the antibody specifically binds to human GPRC5D. L

[0066] Thus, in one embodiment, an antibody of the application, or antigen-binding portion thereof, comprises:

[0067] (a) CDR1, CDR2, and CDR3 of a heavy chain variable region set forth in Table 2; and

[0068] (b) CDR1, CDR2, and CDR3 of a light chain variable region set forth in Table 2, or CDRs of another GPRC5D antibody, wherein the antibody specifically binds to human GPRC5D.

[0069] ​In another embodiment, an antibody or antigen-binding portion thereof of the application comprises the CDR2 of the heavy chain variable region of a GPRC5D antibody and the CDRs of another antibody that binds human GPRC5D, e.g., the CDR1 and / or CDR3 of the heavy chain variable region, and / or the CDR1, CDR2, and / or CDR3 of the light chain variable region of another GPRC5D antibody.

[0070] Furthermore, it is well known in the art that the CDR3 domain, independent of the CDR1 and / or CDR2, alone determines the binding specificity of an antibody to a cognate antigen, and that multiple antibodies with the same binding specificity can be predicted to be generated based on the CDR3 sequence.

[0071] In another embodiment, an antibody or antigen-binding portion thereof of the application comprises the CDR2 of the heavy chain variable region of a GPRC5D antibody and the CDR3 of the heavy chain and / or light chain variable region of at least one GPRC5D antibody, or the CDR3 of the heavy chain and / or light chain variable region of another GPRC5D antibody, wherein the antibody or antigen-binding portion thereof is capable of specifically binding human GPRC5D. Preferably, these antibodies or antigen-binding portions thereof (a) compete for binding to GPRC5D; (b) retain a functional property; (c) bind to the same epitope; and / or (d) have a similar binding affinity as a GPRC5D antibody or antigen-binding portion thereof of the application. In another embodiment, the antibody or antigen-binding portion thereof can further comprise the CDR2 of the light chain variable region of a GPRC5D antibody or antigen-binding portion thereof of the application, or the CDR2 of the light chain variable region of another GPRC5D antibody, wherein the antibody or antigen-binding portion thereof specifically binds human GPRC5D. In another embodiment, an antibody of the application can comprise the CDR1 of the heavy chain / light chain variable region of a GPRC5D antibody or antigen-binding portion thereof of the application, or the CDR1 of the heavy chain and / or light chain variable region of another GPRC5D antibody, wherein the antibody or antigen-binding portion thereof specifically binds human GPRC5D.

[0072] In another embodiment, an antibody or antigen-binding portion thereof of the application comprises a heavy chain and / or light chain variable region sequence or CDR1, CDR2, and CDR3 sequence of a GPRC5D antibody or antigen-binding portion thereof of the application with one or more conservative modifications. It is known in the art that some conservative sequence modifications do not remove antigen binding. See, e.g., Brummell et al., (1993) Biochem 32:1180-8.

[0073] Thus, in one embodiment, an antibody or antigen-binding portion thereof comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region and the light chain variable region each comprising a CDR1, a CDR2, and a CDR3, wherein:

[0074] (a) the heavy chain variable region CDR1 comprises a sequence as listed in Table 2, and / or conservative modifications thereof; and / or

[0075] (b) the heavy chain variable region CDR2 comprises a sequence as listed in Table 2, and / or conservative modifications thereof; and / or

[0076] (c) the heavy chain variable region CDR3 comprises a sequence as listed in Table 2, and / or conservative modifications thereof; and / or

[0077] (d) the light chain variable region CDR1, and / or CDR2, and / or CDR3 comprises a sequence as listed in Table 2, and / or conservative modifications thereof; and

[0078] (e) the antibody or antigen binding portion thereof specifically binds to human GPRC5D.

[0079] The term "conservative sequence modification" as used herein refers to amino acid modifications that do not significantly affect or alter the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antigen binding portion thereof of the application by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Groups of amino acid residues with similar side chains are well known in the art. These groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, any one or more amino acid residues in a CDR region of an antibody or antigen binding portion thereof of the application can be replaced with other amino acid residues of the same side chain group and the resulting antibody can be tested for retention of function (i.e., the functions described above) using the functional assays described herein.

[0080] An antibody or antigen binding portion thereof of the application can be prepared as a genetically modified antibody starting with an antibody having one or more V H / V L sequences of a GPRC5D antibody or antigen binding portion thereof of the application. The antibody can be genetically modified by modifying one or both variable regions (i.e., V H and / or V Lone or more residues within (e.g., in one or more CDR regions and / or one or more framework regions) to improve binding affinity and / or increase similarity to naturally occurring antibodies of certain species. For example, an antibody can be genetically modified by modifying residues in the constant regions, e.g., to alter the effector function of the antibody.

[0081] The variable region modification can be mutating one or more of the V H and / or V L amino acid residues within the CDR1, CDR2, and / or CDR3 regions to improve one or more binding properties (e.g., affinity) of the antibody of interest. Point mutations or PCR-mediated mutations can be made to introduce the mutations, and their effects on antibody binding or other functional properties can be evaluated in in vitro or in vivo assays known in the art. Preferably, conservative modifications known in the art are introduced. The mutations can be amino acid substitutions, additions, or deletions, but are preferably substitutions. Moreover, typically no more than one, two, three, four, or five residues within the CDR regions are altered.

[0082] In another embodiment, the application provides an isolated GPRC5D monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region and a light chain variable region, which comprises: (a) a V H CDR1 region comprising a sequence of the application, or an amino acid sequence that has one, two, three, four, or five amino acid substitutions, deletions, or additions; (b) a V H CDR2 region comprising a sequence of the application, or an amino acid sequence that has one, two, three, four, or five amino acid substitutions, deletions, or additions; (c) a V H CDR3 region comprising a sequence of the application, or an amino acid sequence that has one, two, three, four, or five amino acid substitutions, deletions, or additions; (d) a V L CDR1 region comprising a sequence of the application, or an amino acid sequence that has one, two, three, four, or five amino acid substitutions, deletions, or additions; (e) a V L CDR2 region comprising a sequence of the application, or an amino acid sequence that has one, two, three, four, or five amino acid substitutions, deletions, or additions; and (f) a V L CDR3 region comprising a sequence of the application, or an amino acid sequence that has one, two, three, four, or five amino acid substitutions, deletions, or additions.

[0083] Genetically engineered antibodies of the application include those in which one or more of the V H and / or V Lthose in which modifications are made to the framework residues, for example, to alter antibody properties. Framework modifications include mutating one or more residues in the framework region, or even one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also known as "deimmunization."

[0084] In addition, as an alternative to modifications within the framework or CDR regions, antibodies of the application can be genetically engineered to include modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antibody-dependent cellular cytotoxicity. In addition, antibodies of the application can be chemically modified (e.g., one or more chemical functional groups can be added to the antibody), or modified to alter its glycosylation, to alter one or more functional properties of the antibody.

[0085] In one embodiment, the C H1 In one embodiment, the C H1 In one embodiment, the C

[0086] In another embodiment, the Fc hinge region of the antibody is mutated to increase or decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the C H2 -C H3 In one embodiment, the C

[0087] In another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be prepared (i.e., an antibody lacking glycosylation). Glycosylation can be altered, for example, to increase the binding affinity of the antibody for antigen. Such glycosylation modifications can be accomplished by, for example, altering one or more of the glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made that eliminate one or more of the variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the binding affinity of the antibody for antigen. See, e.g., U.S. Patents 5,714,350 and 6,350,861.

[0088] In addition, antibodies with altered types of glycosylation, e.g., hypofucosylated antibodies with reduced amounts of fucosyl residues, or antibodies with increased bisecting GlcNac structures can be made. Altered glycosylation has been demonstrated to increase the ADCC activity of antibodies. Such glycosylation modifications can be made, e.g., by expressing the antibody in a host cell with an altered glycosylation system. Cells with altered glycosylation systems are known in the art, including, but not limited to, Slc35cl knockout cell lines, FUT8 knockout cell lines, variant CHO cell lines Lec13, rat hybridoma cell line YB2 / 0, cell lines comprising small interfering RNA specific for the FUT8 gene, cell lines co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. They can be used as host cells for expression of the recombinant antibodies of the application to make antibodies with altered glycosylation

[0089] Another modification of the antibodies herein is PEGylation. Antibodies can be PEGylated, e.g., to increase the bio(serum) half-life of the antibody. To PEGylate an antibody, the antibody or fragment thereof is typically reacted with a polyethylene glycol (PEG), e.g., a reactive ester or aldehyde derivative of PEG, under conditions that allow one or more PEG groups to become attached to the antibody or antibody fragment. Preferably, the PEGylation is carried out by acylation or alkylation with a reactive PEG molecule (or a similarly reactive water-soluble polymer). As used herein, the term "polyethylene glycol" includes any of the forms of PEG that are used to derivatize other proteins, such as mono-(C1-C6)alkoxy- or aryloxy polyethylene glycol or polyethylene glycol maleimide. In certain embodiments, the antibody that is desired to be PEGylated is a deglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the application. See, e.g., EPO 154 316 and EP 0 401 384. 10 )alkoxy- or aryloxy polyethylene glycol or polyethylene glycol maleimide. In certain embodiments, the antibody that is desired to be PEGylated is a deglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the application. See, e.g., EPO 154 316 and EP 0 401 384.

[0090] The antibodies or antigen-binding portions thereof of the application can be characterized by their various physical properties to detect and / or distinguish among the classes.

[0091] For example, the antibodies or antigen-binding portions thereof can comprise one or more glycosylation sites in the light chain or heavy chain variable region. These glycosylation sites can lead to increased immunogenicity of the antibody, or altered pK of the antibody due to altered antigen binding. Glycosylation is known to occur in motifs containing an N-X-S / T sequence. In some cases, it is preferred that the GPRC5D antibodies or antigen-binding portions thereof do not comprise variable region glycosylation. This can be achieved by selecting antibodies that do not comprise glycosylation motifs in the variable region, or by mutating residues in the glycosylation region.

[0092] In preferred embodiments, the antibody or antigen-binding portion thereof does not comprise an asparagine isomerization site. Deamidation of asparagine can occur at N-G or D-G sequences, creating an isoaspartate residue that introduces a kink into the polypeptide chain and reduces its stability (isoaspartate effect).

[0093] Monoclonal antibodies of the application can be produced using somatic cell hybridization (hybridoma) methods. Alternatively, they can be obtained by phage display techniques, viral or oncogenic transformation of B lymphocytes, and the like. Methods for making chimeric antibodies are also well known in the art. Antibodies of the application or antigen-binding portions thereof can also be produced in host cell transfectomas using, for example, recombinant DNA techniques in conjunction with gene transfection methods (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding a partial or full-length light chain and heavy chain is obtained by standard molecular biology techniques and inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" means that an antibody gene is ligated into a vector such that transcriptional and translational regulatory sequences within the vector show their established regulatory effects on the antibody gene.

[0094] The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control transcription or translation of the antibody gene. Preferred regulatory sequences for mammalian host cell expression include viral elements that are obtained from, for example, cytomegaloviruses (CMV), Simian Virus 40 (SV40), adenoviruses, and / or enhancers and promoters obtained from retroviruses such as Rous Sarcoma Virus, Rhabdoviruses, and Beta-phytohemagglutinin, and / or enhancers and promoters obtained from animal viruses, for example, polyoma virus and adenovirus, most preferably the adenovirus major late promoter (AdMLP) and the multi- plasty virus. Alternatively, non-viral regulatory elements can be used, for example, the ubiquitin promoter or the beta-globin promoter. In addition, the regulatory elements are composed of sequences from different origins, for example, the SRa promoter system, which comprises sequences from the early promoter of SV40 and the long terminal repeat of the human T-cell leukemia virus type I. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used.

[0095] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors. In preferred embodiments, the variable region is constructed into a full-length antibody gene by insertion into an expression vector that already encodes the heavy chain constant region and the light chain constant region of the desired isotype, such that the V H with the C H are operably linked, the V L with the C LOperably linked. Alternatively, a recombinant expression vector can encode a signal peptide that facilitates secretion of antibody chains from a host cell. Antibody chain genes can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of an antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0096] In addition to antibody chain genes and regulatory sequences, a recombinant expression vector of the application can carry additional sequences, such as a sequence enabling the vector to replicate in the host cell in question (e.g., an origin of replication) and a selectable marker gene. The selectable marker gene facilitates selection of host cells into which the vector has been introduced. For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include dihydrofolate reductase (DHFR) gene (for methotrexate selection / amplification) and neo gene (for G418 selection).

[0097] For expression of the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The term "transfection" is intended to encompass a wide variety of techniques for introducing foreign DNA into a prokaryotic or eukaryotic host cell, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although expression of antibodies or antigen-binding portions thereof of the application in prokaryotic or eukaryotic host cells is theoretically possible, it is preferred that the antibodies be expressed in eukaryotic cells, most preferably mammalian host cells, as eukaryotic cells, particularly mammalian cells, are more likely to assemble and secrete properly folded and immunologically active antibodies than are prokaryotic cells.

[0098] Preferred mammalian host cells for expressing the recombinant antibodies of the application include Slc35C1 knockout cell lines, FUT8 knockout cell lines, variant CHO cell line Lec13, rat myeloma cell line YB2 / 0, cell lines comprising small interfering RNA specific for the FUT8 gene, co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II, Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells in which the dhfr gene has been inactivated, together with the dhfr selectable marker), NSO myeloma cells, COS cells, and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibody is produced by the host cells in a culture medium using a process known in the art. Antibodies or antigen-binding portions thereof can be recovered from the culture medium using protein purification methods.

[0099] In another aspect, the application provides nucleic acid molecules encoding the heavy chain / light chain variable regions or CDRs of the antibodies or antigen binding portions thereof of the application. The nucleic acid can be in whole cells, in a cell lysate, or in a partially purified or substantially pure form. The nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques. The nucleic acids of the application can be, for example, DNA or RNA, and can or can not contain intronic sequences.

[0100] The nucleic acids of the application can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes), cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acids encoding such antibodies can be collected from the library.

[0101] Preferred nucleic acid molecules of the application include those encoding the V H and V L sequences or CDRs of the GPRC5D monoclonal antibodies of the application. Once a DNA fragment encoding a V H or V L is obtained, the DNA fragment can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region-encoding DNA to a full-length antibody chain-encoding DNA, a Fab fragment-encoding DNA, or a scFv-encoding DNA. In these manipulations, the DNA fragment encoding the V H or V L is operatively linked to another DNA segment, such as that encoding the constant region of an antibody or a flexible linker. The term "operatively linked" means that the two DNA fragments are connected in such a way that both DNA fragments are expressed in reading frame.

[0102] The isolated DNA encoding the V H region can be converted to a full-length heavy chain-encoding gene by operatively linking the V H encoding DNA to another DNA molecule, such as that encoding the heavy chain constant region (C H1、 C H2 and C H3 ). The sequences of human heavy chain constant regions are known, and DNA fragments H encoding these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is preferably an IgGl constant region. For a Fab fragment heavy chain gene, the DNA H1Another DNA molecule encoding the constant region is ligated.

[0103] DNA encoding the V L region can be converted to a full-length light chain gene by operatively linking the DNA encoding the V L region to another DNA molecule encoding the light chain constant region, C L The sequences of human light chain constant region genes are known in the art, and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa and lambda constant region.

[0104] To create a scFv gene, the DNA fragments encoding the V H and V L regions can be operatively linked to another fragment encoding a flexible linker, such that the V H and V L sequences can be expressed as a contiguous single-chain protein, in which the V H and V L regions are connected by the flexible linker.

[0105] The antibody or antigen-binding portion thereof of the present application can be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxic molecules, alkylating agents, DNA minor groove binding molecules, DNA intercalating agents, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In an ADC, the antibody and the therapeutic agent can be cross-linked by a linker, which can be cleavable, such as a peptide linker, a disulfide linker, or a hydrazone linker. More preferably, the linker is a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. Methods for preparing ADCs are known in the art. An ADC has utility in the context of antibody internalization. An internalizing antibody can be conjugated to a cytotoxic molecule, such that the cytotoxic molecule specifically damages the cell to which the internalizing antibody is directed. In particular, the cytotoxic molecule can be internalized by the antibody into the target cell. The cytotoxic molecule can be any small molecule compound or protein molecule that causes damage to the target cell, such as a microtubulin polymerization inhibitor, a DNA damaging agent, and the like.

[0106] In another aspect, the application relates to bispecific molecules comprising an antibody or antigen-binding portion thereof of the application linked to at least one other functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The term "bispecific molecule" includes molecules with three or more specificities.

[0107] Bispecific molecules can come in a variety of formats and sizes. At one end of the size spectrum, bispecific molecules retain the traditional antibody format, except that they have two binding arms, each with a different specificity, instead of two binding arms with the same specificity. At the other extreme are bispecific molecules composed of two single-chain antibody fragments (scFv) connected by a peptide linker, known as Bs(scFv)2 constructs. Bispecific molecules of intermediate size include two different F(ab) fragments connected by a peptide linker. These and other formats of bispecific molecules can be prepared by genetic engineering, somatic hybridization, or chemically.

[0108] An antibody or antigen-binding portion thereof of the application can be linked to a peptide or protein that specifically binds to a T cell, such as an antibody that specifically binds to a CD3 molecule on a T cell. A bispecific molecule so constructed can be referred to as a "T cell engager (TCE)", i.e., a bispecific molecule that can simultaneously bind to a T cell and a target cell and direct the T cell to produce cytotoxicity against the target cell.

[0109] The application also provides a chimeric antigen receptor comprising a GPRC5D single-chain antibody (scFv) comprising the heavy and light chain CDRs, or the heavy and light chain variable regions, described herein. The GPRC5D chimeric antigen receptor can comprise (a) an extracellular antigen binding domain comprising a GPRC5D scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain. The application also provides an immune cell, such as a T cell or NK cell, comprising the chimeric antigen receptor of the application.

[0110] A T cell receptor can also be engineered to comprise an antibody or antigen-binding portion thereof of the application, such as a scFv, and to activate the T cell and elicit cytotoxicity against a target cell bearing a GPRC5D molecule upon binding of the antibody or antigen-binding portion thereof to GPRC5D. The application also provides a T cell comprising a recombinant T cell receptor of the application.

[0111] Oncolytic viruses preferentially infect and kill cancer cells. An antibody or antigen-binding portion thereof of the application can be used with an oncolytic virus. In addition, an oncolytic virus encoding an antibody or antigen-binding portion thereof of the application can be introduced into a human.

[0112] In another aspect, the application provides a composition comprising an antibody or antigen binding portion thereof, immunoconjugate, bispecific molecule, CAR / TCR, immune cell carrying a CAR / TCR, oncolytic virus, nucleic acid molecule, expression vector, or host cell of the application. In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. The composition can optionally comprise one or more other pharmaceutically effective ingredients, such as another anti-tumor antibody, or an immune-enhancing antibody, or a non-antibody anti-tumor agent, or an immune-enhancing agent. The compositions of the application can be used in combination with, for example, another anti-cancer agent.

[0113] Pharmaceutical compositions can include any number of excipients. Excipients that can be used include carriers, surface active agents, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents and combinations thereof. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).

[0114] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or topical (e.g., by injection or infusion) administration. Depending on the route of administration, the active ingredient can be coated with a material to protect it from the acid and other natural

[0115] Pharmaceutical compositions can be in the form of a sterile solution or dispersion for intravenous administration. They can also be formulated in a microemulsion, liposome, or other ordered structure that is suitable for high drug concentration.

[0116] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host and the particular mode of administration. In general, a range of from about 0.01 to about 99% of the compositions are used for such purposes. In percentage terms, this would amount to from about 0.01 to about 99% of the effective ingredient combined with a pharmaceutically acceptable carrier.

[0117] The dosage regimen is adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a rapid onset of effect can be obtained by administration of a bolus dose, multiple doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier. Alternatively, the antibody can be administered as a sustained release formulation, where desired frequency of administration is reduced.

[0118] For administration of the antibody, the dosage can be about 0.001-100 mg / kg of host body weight. An exemplary treatment regime involves administration once per week.

[0119] A "therapeutically effective amount" of a pharmaceutical composition of the application causes a decrease in the severity of a disease symptom, an increase in the frequency and duration of symptom-free periods. For example, for the treatment of a subject afflicted with multiple myeloma, a "therapeutically effective amount" preferably inhibits the growth of multiple myeloma cells by at least about 20%, particularly by at least about 40%, even more particularly by at least about 60%, and more particularly by at least about 80%, as compared to untreated subjects. A therapeutically effective amount of a pharmaceutical composition of the application can halt progression of the disease, reduce the number of multiple myeloma cells, alleviate symptoms in the subject, etc. The subject can be a human or another mammal.

[0120] The pharmaceutical compositions can be in a sustained release formulation, including implants, and microencapsule delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0121] The pharmaceutical compositions can be administered via a medical device, such as (1) needleless subcutaneous injection devices (U.S. Patents 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) miniosmotic pumps (U.S. Patent 4,487,603); (3) transdermal patch devices (U.S. Patent 4,486,194); (4) bolus devices (U.S. Patents 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Patents 4,439,196 and 4,475,196).

[0122] In certain embodiments, the components in the compositions of the application can be formulated to ensure proper in vivo distribution. For example, to ensure that the therapeutic antibodies or antigen-binding portions thereof of the application cross the blood-brain barrier, the antibodies can be formulated in a liposome, which can additionally comprise a targeting functional group to enhance selective delivery to a particular cell or organ.

[0123] The present application also relates to in vivo gene therapy, in which a nucleic acid molecule encoding an antibody or antigen-binding portion thereof, immunoconjugate, or bispecific molecule, etc. of the present application is introduced directly into a subject. For example, a nucleic acid sequence encoding an antibody or antigen-binding portion of the present application is introduced into a target cell via local injection of a nucleic acid construct with or without a suitable delivery vehicle, such as an adeno-associated viral vector. Other alternative viral vectors include, but are not limited to, retroviral, adenoviral, herpes simplex viral, and papilloma viral vectors. In vivo physical transfer of the viral vector can be achieved by local injection of the desired nucleic acid construct or other suitable delivery vehicle comprising the desired nucleic acid sequence, liposome-mediated transfer, direct injection (naked DNA), or microparticle bombardment (gene gun).

[0124] The pharmaceutical compositions of the present application have a variety of in vitro and in vivo utilities, relating to, for example, the treatment of multiple myeloma. The pharmaceutical compositions can be administered to a human subject to, for example, inhibit the progression of multiple myeloma in vivo.

[0125] In view of the ability of the pharmaceutical compositions of the present application to inhibit the proliferation and survival of multiple myeloma cells, the present application provides methods of treating or ameliorating multiple myeloma in a subject comprising administering to the subject a pharmaceutical composition of the present application. The multiple myeloma that can be treated by the antibodies of the present application can be newly diagnosed, or refractory or relapsed.

[0126] The present application provides combination therapies in which a pharmaceutical composition of the present application is administered with one or more other antibody or non-antibody based therapeutic agents effective to inhibit the proliferation and survival of multiple myeloma cells in a subject. In one embodiment, the present application provides a method of inhibiting the growth of myeloma cells in a subject comprising administering to the subject a pharmaceutical composition of the present application and one or more other antibodies, such as a PD-L1 antibody. In certain embodiments, the subject is a human. In another aspect, the present application provides a method of treating cancer in which a pharmaceutical composition of the present application is administered with a chemotherapeutic agent, which can be a cytotoxic agent. Other therapies that can be combined with the pharmaceutical compositions of the present application include, but are not limited to, administration of an immunogenic agent, administration of interleukin 2 (IL-2), radiation therapy, surgery, or hormone removal.

[0127] The combinations of therapeutic agents discussed herein can be administered simultaneously, as a single composition in a pharmaceutically acceptable carrier, or as separate compositions, where each agent is in a pharmaceutically acceptable carrier, simultaneously. In another embodiment, the combinations of therapeutic agents can be administered sequentially.

[0128] Furthermore, if multiple combination therapy administrations are performed, and the agents are administered sequentially, the order of sequential administration at each time point can be reversed or remain the same, and the sequential administration can be combined with simultaneous administration or any combination thereof.

[0129] Aspects and embodiments of the present application will be discussed with reference to the accompanying drawings and the following examples. Other aspects and embodiments will be apparent to those skilled in the art. All documents described herein are incorporated by reference herein in their entirety. Although the present application has been described in connection with the exemplary embodiments, numerous modifications and variations are possible in light of the above teachings. Thus, the exemplary embodiments are meant to be illustrative only and not limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the application and its spirit and scope.

[0130] Example 1. Generation of anti-GPRC5D monoclonal antibodies

[0131] 1) Immunization

[0132] Twelve Balb / c mice were immunized with mRNA-lipid nanoparticles (LNP) of human GPRC5D (manufacturer: CureVac, lot number: PB-GPRC5D-110) according to current animal welfare regulations. For immunization, the antigen was diluted with PBS and injected subcutaneously via the back. Each animal received four doses, with 40 pg of mRNA-LNP per immunization. Seven days after each immunization, 10 pl serum was taken and tested for antibody titers in a flow cytometry (FACS)-based method.

[0133] 2) Screening of hybridomas secreting anti-GPRC5D antibodies

[0134] Four days after the last immunization, the spleen cells of selected mice were extracted in a sterile environment and fused with Sp2 / 0 cells according to the standard hybridoma production protocol. The fused cells were cultured in DMEM medium containing 1xHAT (hypoxanthine, aminopterin, thymidine) and 10% fetal bovine serum for 6-7 days. A total of 90 96-well plate hybridomas were screened, and the binding ability of all hybridoma supernatants to the GPRC5D overexpression cell line (constructed by Nanjing Pobu) was analyzed by FACS, and positive clones against GPRC5D were screened. A total of 1793 parental hybridoma mother clones bound to the GPRC5D overexpression cell line 3T3 / GPRC5D (FACS mean fluorescence intensity ratio > 14). According to the standard of FACS mean fluorescence intensity ratio greater than 50 times, 51 hybridoma mother clones were selected, subcloned by limiting dilution, and cultured in DMEM medium containing 1xHAT (hypoxanthine, aminopterin, thymidine) and 10% fetal bovine serum. The cells were cultured until positive monoclonal cells were obtained before a new round of screening was performed using FACS. A total of 37 single clones were selected, and the supernatants of hybridoma subclones were positively detected by FACS, and the results are shown in Table 1. The 37 subclones were sequenced to obtain their antibody sequences. According to the variable region sequences of the GPRC5D antibody part in Janssen talquetumab, plus the heavy chain constant region (SEQ ID NO: 97) and the light chain constant region (SEQ ID NO: 98), IgG antibodies were recombinantly expressed in CHO cells as a positive control, hereinafter referred to as talquetumab.

[0135] Table 1. FACS binding of 37 hybridoma subclone supernatants to 3T3 / GPRC5D cells

[0136] 3) Sequencing of antibodies and construction of chimeric antibodies

[0137] The above positive single clones were sequenced, and 12 clones were selected and fused with human IgG1 heavy chain constant region (SEQ ID NO: 97) and kappa light chain (SEQ ID NO: 98), and an expression plasmid was constructed using a mammalian system expression vector pcDNA3.4, and the plasmid was transiently transfected into CHO cells for recombinant expression. The chimeric antibodies obtained by recombinant expression were purified by Protein A.

[0138] The variable region and CDR sequences of the 12 recombinantly expressed antibodies are shown in Table 2.

[0139] Table 2. Sequences of antibody variable regions and CDRs and SEQ ID NO.

[0140] Example 2. Binding of anti-GPRC5D chimeric antibodies to 3T3 / GPRC5D cells

[0141] To test the binding of the chimeric antibodies obtained in Example 1 to GPRC5D, FACS was performed using 3T3 / GPRC5D cells overexpressing human GPRC5D (purchased from Promega, the nucleotide sequence of GPRC5D has NCBI index number NM_018654.2 (Mi X et al., (2023) Genetic Basis of Relapse after GPRC5D-Targeted CAR T Cells. N Engl J Med 389(15): 1435-1437)).

[0142] Each antibody was diluted in a gradient of 3-fold in PBS containing 0.1% BSA, with a starting concentration of 300 nM. 50 μl of antibody was taken, and about 1 x 1053T3 / GPRC5D cells were added in 50 μl. After incubation at 4°C for 40 minutes, the cells were washed 3 times with PBS, and 100 μl of 1 μg / ml goat anti-human IgG (H+L) fluorescent secondary antibody (manufacturer: Jackson ImmunoResearch, product number: 109-605-088) was added, incubated at 4°C for 30 minutes, and then the fluorescent signal was measured on a BD Canto. 5 3T3 / GPRC5D cells, incubated at 4°C for 40 minutes. Then, the cells were washed 3 times with PBS, and 100 μl of 1 μg / ml goat anti-human IgG (H+L) fluorescent secondary antibody (manufacturer: Jackson ImmunoResearch, product number: 109-605-088) was added, incubated at 4°C for 30 minutes, and then the fluorescent signal was measured on a BD Canto.

[0143] According to the variable region sequences of the GPRC5D antibody part in Janssen talquetumab and the variable region sequences of the antibody part in Lianximed LM305, plus the heavy chain constant region (SEQ ID NO: 97) and the light chain constant region (SEQ ID NO: 98), IgG antibodies were recombinantly expressed in ExpiCHO-STM cells, as positive control, hereinafter referred to as talquetumab and LM305, respectively. Human IgG was used as negative control.

[0144] The average fluorescence intensity of each antibody is shown in Figure 1, and the half-effective concentration EC50is shown in Table 3. 50 As shown in Table 3. The results show that the 12 chimeric antibodies of the present application all show better binding to GPRC5D overexpressing cells than talquetumab.

[0145] Table 3. EC50of anti-GPRC5D antibodies binding to 3T3 / GPRC5D cells 50

[0146] Example 3. Binding of anti-GPRC5D chimeric antibodies to MM.1S cells

[0147] The chimeric antibodies of the present application were tested for their binding to human myeloma cells MM.1S (Vendor: Cell Bank of Chinese Academy of Sciences, Catalog No: SCSP-5017) by FACS.

[0148] Each antibody was 3-fold serially diluted in PBS containing 0.1% BSA, with an initial concentration of 300 nM. 50 μl of antibody was taken and mixed with about 1 x 10 5 MM.1S cells were incubated at 4 degrees Celsius for 40 minutes. After that, the cells were washed with PBS for 3 times, and then 100 μl of 1 μg / ml goat anti-human IgG (H+L) fluorescent secondary antibody (Vendor: Jackson ImmunoResearch, Catalog No: 109-605-088) was added, incubated at 4 degrees Celsius for 30 minutes, and then fluorescence signal detection was performed in a BD Canto II.

[0149] Table 4. EC50 of anti-GPRC5D antibodies binding to MM.1S cells 50

[0150] The mean fluorescence intensity of each antibody is shown in Figure 2, and the half maximal effective concentration EC50 of each antibody is shown in Table 4. 50 As shown in Table 4, the results showed that most of the chimeric antibodies of the present application exhibited good binding ability to tumor cells expressing GPRC5D, among which 24C4B8, 90C7B11, 51H10D3, and 52C6A6 showed better binding than the two positive control antibodies.

[0151] Example 4. Binding of anti-GPRC5D chimeric antibodies to cynomolgus monkey GPRC5D

[0152] Based on the above experiments, 7 antibodies were taken and tested for their species cross-binding to cells expressing cynomolgus monkey GPRC5D by FACS.

[0153] Specifically, 1 μg of pcDNA3.4 plasmid containing expression of cynomolgus monkey GPRC5D (NCBI: XP_005570250.2) was transiently transfected into about 1 x 10 6 HEK293T cells. After 24 hours, 50 μl of cell culture fluid containing about 1 x 10 5 HEK293T cells was taken, and the final concentration of the antibodies of the present application was 10.0 nM, 3.3 nM, or 1.1 nM, respectively, and incubated at 4 degrees Celsius for 40 minutes. After that, the cells were washed with PBS for 3 times, and then 100 μl of 1 μg / ml goat anti-human IgG (H+L) fluorescent secondary antibody was added, incubated at 4 degrees Celsius for 30 minutes, and then fluorescence signal detection was performed in a BD Canto II.

[0154] The mean fluorescence intensity of each antibody is shown in Figure 3, and the mean fluorescence intensity ratio (293T / GPRC5D: 293T) is listed in Table 5. The results show that the 7 candidate antibodies of the present application all exhibit better cynomolgus monkey GPRC5D binding ability than that of talimogene laherparapvec. Except for 53A6H9, the antibodies of the present application exhibit better cynomolgus monkey GPRC5D binding ability than that of LM305.

[0155] Table 5. FACS binding values of anti-GPRC5D antibodies to cynomolgus monkey GPRC5D

[0156] Example 5. Internalization of anti-GPRC5D chimeric antibodies in 3T3 / GPRC5D cells

[0157] Using 3T3 / GPRC5D cells, the internalization efficiency of the antibodies of the present application was tested.

[0158] The antibodies of the present application were diluted 2-fold in PBS, with an initial concentration of 15.0 nM. Then, the diluted antibodies were mixed with a labeling reagent working solution (manufacturer: Sartorius, item number: 4649) at a volume ratio of 1:1, and incubated at 37 degrees Celsius for 15 minutes to allow full coupling. 50 μl of the coupling mixture was mixed with 50 μl of a volume of about 1 x 10 4 3T3 / GPRC5D cells, and placed in a cell incubator at 37 degrees Celsius for 24 hours, while real-time imaging detection was performed using a instrument (Germany). The red total integral fluorescence intensity in the cells was calculated using the Live-Cell Analysis System software to represent the internalization rate of the antibodies.

[0159] The results of the antibody internalization after 24 hours of incubation are shown in Figure 4 and Table 6. The results show that 53A6H9, 52C6A6, and 58A9D9 exhibit better internalization ability than that of the positive control talimogene laherparapvec and LM305. 24C4B8 and 90C7B11 exhibit internalization ability comparable to that of the positive control LM305.

[0160] Table 6. Internalization values of anti-GPRC5D antibodies to 3T3 / GPRC5D cells

[0161] Example 6. Epitope competition of anti-GPRC5D chimeric antibodies with talimogene laherparapvec

[0162] To test whether the chimeric antibodies of the present application bind to the same epitope as the positive control talimogene laherparapvec, each antibody of the present application was tested for epitope competition with the positive control using the 3T3 / GPRC5D cells described above by FACS.

[0163] Biotin conjugation of talimogene laherparapovec was performed using the biotinylation reagent N-[6-(Biotinamido)hexanoyl]-6-aminohexanoic acid N- succinimidyl ester (Vendor: Avidity, Cat# B122220-25mg). Briefly, talimogene laherparapovec and the biotinylation reagent were mixed at a molar ratio of 10:1 and incubated at room temperature for 2 hours. The biotinylated antibody was then dialyzed into PBS, pH 7.4 at 4 degrees Celsius. The biotin conjugated talimogene laherparapovec was tested for its binding EC 50 and 0.3 pg / ml was chosen for the following epitope competition test.

[0164] The antibodies of the present application were 3-fold serially diluted in PBS with a starting concentration of 300 nM to give a total of 12 concentrations. 50 mI of antibody was mixed with about 1 x 105 5 3T3 / GPRC5D cells and incubated at 4 degrees Celsius for 40 minutes. After that, the cells were washed with PBS for 3 times, 100 mI of biotin conjugated talimogene laherparapovec at a final concentration of 0.3 pg / ml was added and incubated at 4 degrees Celsius for 40 minutes. SA fluorescent secondary antibody (Vendor: Jackson ImmunoResearch, Cat# 016-600-084) was added and incubated at 4 degrees Celsius for 30 minutes. Fluorescent signal was measured in a BD Canto.

[0165] The mean fluorescent intensity of talimogene laherparapovec after co-incubation with the antibodies of the present application is shown in Figure 5, and the half-inhibitory concentration IC 50 as shown in Table 7. The results showed that talimogene laherparapovec and the 7 candidate antibodies of the present application presented dose-dependent competition, indicating that the 7 candidate antibodies of the present application most likely bind to the same or similar epitope on GPRC5D as talimogene laherparapovec.

[0166] Table 7. Epitope competition IC50 of anti-GPRC5D antibodies with talimogene laherparapovec 50

[0167] Example 7. Epitope competition of anti-GPRC5D chimeric antibodies with LM305

[0168] To test whether the chimeric antibodies of the present application bind to the same epitope as the positive control LM305, the antibodies of the present application were tested for their epitope competition with the positive control using 3T3 / GPRC5D cells by FACS.

[0169] LM305 mAb was biotin conjugated using the biotinylation reagent N-[6-(Biotinamido)hexanoyl]-6-aminohexanoic acid N-succinimidyl ester (Vendor: Avidity, Cat# B122220-25mg). Briefly, LM305 mAb and biotinylation reagent were mixed at a molar ratio of 10:1, incubated at room temperature for 2 hours, and then the biotinylated antibody was dialyzed into PBS, pH 7.4 at 4 degrees Celsius. The biotin conjugated LM305 was tested for its binding EC 50 to 3T3 / GPRC5D cells by the method of Example 2, and 0.1 pg / ml was chosen for the following epitope competition test.

[0170] The antibodies of the present application were 3-fold serially diluted in PBS, starting at a concentration of 300 nM, to give a total of 12 concentrations. 50 mI of antibody was added to about 1 x 105 5 3T3 / GPRC5D cells, and incubated at 4 degrees Celsius for 40 minutes. After that, the cells were washed with PBS for 3 times, and then 100 mI of biotin conjugated LM305 at a final concentration of 0.1 pg / ml was added, and incubated at 4 degrees Celsius for 40 minutes. SA fluorescent secondary antibody (Vendor: Jackson ImmunoResearch, Cat# 016-600-084) was added, and incubated at 4 degrees Celsius for 30 minutes, and then fluorescent signal was measured in a BD Canto.

[0171] Table 8. Epitope competition IC of anti-GPRC5D antibodies with LM305 50

[0172] The mean fluorescence intensity of LM305 after co-incubation with the antibodies of the present application is shown in Figure 6, and the half-inhibitory concentration IC 50 of the antibodies is shown in Table 8. The results show that LM305 and the 7 candidate antibodies of the present application present dose-dependent competition, indicating that the 7 candidate antibodies likely bind to the same or similar epitope on GPRC5D as LM305.

[0173] Some sequences referred to in the application are listed below.

[0174] Human IgGl constant region

[0175] Human kappa constant region

[0176] While the application has been described in connection with one or more embodiments, it will be understood that the application is not limited to such embodiments. The description in this application is intended to cover all variants and equivalents and is intended to include all the subject matter within the scope of the claims. All documents cited herein are incorporated by reference in their entirety.

Claims

1. An isolated monoclonal antibody or its antigen-binding moiety thereof, capable of specifically binding to GPRC5D, comprising i) a heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3, and ii) a light chain variable region comprising VL CDR1, VL CDR2, and VL CDR3. VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively contain (1) SEQ ID NO: 17, 18, 19, 20, 21 and 22; (2) SEQ ID NO: 33, 34, 35, 36, 37 and 38; (3) SEQ ID NO: 9, 10, 11, 12, 13 and 14; (4) SEQ ID NO: 49, 50, 51, 52, 53 and 54; (5) SEQ ID NO: 57, 58, 59, 60, 61 and 62; (6) SEQ ID NO: 81, 82, 83, 84, 85 and 86; (7) SEQ ID NO: 41, 42, 43, 44, 45 and 46; (8) SEQ ID NO: 1, 2, 3, 4, 5 and 6; (9) SEQ ID NO:25, 26, 27, 28, 29 and 30; (10) SEQ ID NO:65, 66, 67, 68, 69 and 70; (11) SEQ ID NO:73, 74, 75, 76, 77 and 78; or (12) the amino acid sequences shown in SEQ ID NO:89, 90, 91, 92, 93 and 94, or amino acid sequences containing 1-3 amino acid substitutions in each CDR compared to the above amino acid sequences.

2. The isolated monoclonal antibody or its antigen-binding portion according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs:23, 39, 15, 55, 63, 87, 47, 7, 31, 71, 79, or 95.

3. The isolated monoclonal antibody or its antigen-binding portion according to claim 1, wherein the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs:24, 40, 16, 56, 64, 88, 48, 8, 32, 72, 80, or 96.

4. The isolated monoclonal antibody or its antigen-binding portion according to claim 1, wherein the heavy chain variable region and the light chain variable region respectively comprise amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with (1) SEQ ID NO: 23 and 24; (2) SEQ ID NO: 39 and 40; (3) SEQ ID NO: 15 and 16; (4) SEQ ID NO: 55 and 56; (5) SEQ ID NO: 63 and 64; (6) SEQ ID NO: 87 and 88; (7) SEQ ID NO: 47 and 48; (8) SEQ ID NO: 7 and 8; (9) SEQ ID NO: 31 and 32; (10) SEQ ID NO: 71 and 72; (11) SEQ ID NO: 79 and 80; or (12) SEQ ID NO: 95 and 96.

5. The isolated monoclonal antibody or its antigen-binding portion according to claim 1 further comprises a heavy chain constant region and / or a light chain constant region.

6. The isolated monoclonal antibody or its antigen-binding moiety according to claim 5, wherein the heavy chain constant region has Fc receptor binding capacity and / or complement system protein binding capacity.

7. The isolated monoclonal antibody or its antigen-binding portion according to claim 1, wherein it is mouse-derived, chimeric, or humanized.

8. An immunoconjugate comprising i) an antibody or its antigen-binding portion as described in any one of claims 1-7, and ii) a cytotoxic molecule, wherein i) and ii) are linked via a adapter or directly linked.

9. A bispecific molecule comprising i) an antibody or an antigen-binding portion thereof as described in any one of claims 1-7, and ii) a second functional group, wherein i) and ii) bind to different antigens or different epitopes of the same antigen, and i) and ii) are connected together.

10. A chimeric antigen receptor comprising i) an extracellular antigen-binding domain, ii) a transmembrane domain, and iii) an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises an antibody or an antigen-binding portion thereof as described in any one of claims 1-7.

11. An immune cell comprising the chimeric antigen receptor of claim 10.

12. An oncolytic virus expressing the antibody or antigen-binding portion thereof as described in any one of claims 1-7.

13. A nucleic acid molecule encoding an antibody or antigen-binding portion thereof as described in any one of claims 1-7, an immunoconjugate as described in claim 8, a bispecific molecule as described in claim 9, or a chimeric antigen receptor as described in claim 10.

14. An expression vector comprising the nucleic acid molecule of claim 13.

15. A host cell comprising the expression vector of claim 14, or having the nucleic acid molecule of claim 13 integrated into its genome.

16. A composition comprising an antibody or antigen-binding portion thereof as described in any one of claims 1-7, an immunoconjugate as described in claim 8, a bispecific molecule as described in claim 9, a chimeric antigen receptor as described in claim 10, an immune cell as described in claim 11, an oncolytic virus as described in claim 12, a nucleic acid molecule as described in claim 13, an expression vector as described in claim 14, or a host cell as described in claim 15.

17. The composition of claim 16, which is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

18. Use of the composition of claim 16 or 17 in the preparation of a medicament for treating or alleviating tumors associated with GPRC5D.

19. The use according to claim 18, wherein the tumor is multiple myeloma, plasma cell leukemia, or plasma cell tumor.

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