Molecule binding to GPRC5d and use thereof

Abstract

Provided in the present application are a single-domain antibody specifically binding to GPRC5D, a bispecific T cell engager or chimeric antigen receptor, etc. containing the single-domain antibody, and the use thereof in the treatment of multiple myeloma.

Description

GPRC5D-bound molecules and their applications

[0001] Cross-citation of related applications

[0002] This application claims priority to Chinese Patent Application No. CN202410822419.9, filed on June 24, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to a single-domain antibody that specifically binds to GPRC5D, and a GPRC5D binding molecule comprising the single-domain antibody. This application also relates to the use of the single-domain antibody or GPRC5D molecule of this application in the treatment of, for example, multiple myeloma. Background Technology

[0004] Plasma cells are a type of white blood cell that produces antibodies and participates in the immune response in a normal body. When these cells grow abnormally and thus suppress the production of healthy cells in the bone marrow, it can lead to multiple myeloma (MM). In MM patients, plasma cells produce abnormal immunoglobulins that invade and destroy nearby bone tissue, causing symptoms such as pain, fractures, anemia, and hypercalcemia.

[0005] As the second most common malignant tumor, multiple myeloma is generally considered incurable by the medical community. One characteristic of the clinical course of MM is relapse over time, and with each relapse, progression-free survival gradually shortens. As of 2018, the 5-year survival rate for MM patients was approximately 58%. In 2021, the FDA approved the first CAR-T cell therapy targeting BCMA for the treatment of adult patients with relapsed MM. In 2022, the FDA approved TECVAYLI, a bispecific antibody targeting BCMA and CD3. TMTeritumumab. These BCMA-targeted drugs have achieved unexpected efficacy in relapsed MM patients, especially in triple-drug-exposed MM patients with a very poor prognosis. However, the number of patients who relapse after receiving BCMA treatment is increasing year by year, possibly due to the downregulation of cell antigen expression caused by BCMA therapy and the cleavage of the extracellular domain of BCMA on the cell surface by γ-secretase (Del Giudice, ML, 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).

[0006] G protein-coupled receptors (GPCRs) play a series of important functions in the human body. Changes in their activity can lead to abnormalities in cell signaling pathways, affecting the occurrence and development of diseases such as inflammation, cardiovascular disease, mental disorders, hormonal imbalances, and cancer. GPCR C5 family subtype D (GPRC5D), a member of the GPCR family, is one of the promising candidate targets for treating multidisciplinary patients (MM), especially those with relapsed MM.

[0007] GPRC5D is an orphan receptor, and no ligand for it has been found to date. Studies have shown that GPRC5D is expressed only in a small number of normal tissues such as hair follicles and testes, as well as in the bone marrow of patients with multiple myeloma (MM). The expression levels in hair follicles and testes are very low, and high GPRC5D expression is associated with a poorer prognosis in MM (Atamaniuk J, Gleiss A, Porpaczy E et al (2012) Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma. Eur J Clin Invest 42(9):953-960). In 65% of MM patients, the RNA expression level of GPRC5D in the bone marrow is thousands of times higher than that in serum. Most importantly, GPRC5D expression is independent of BCMA and is not affected by BCMA therapy (Smith EL, Harrington K, Staehr M 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). Therefore, GPRC5D-targeted drugs are expected to become a new treatment option for both relapsed and refractory end-line patients and relapsed cases that have previously received BCMA-targeted therapy, and have great clinical value. Currently, there are several CAR-T products targeting GPRC5D under investigation, all of which have shown high overall response rates (ORR) in relapsed MM patients. However, in addition to common cytokine release syndrome and neurotoxicity, new toxicities have been observed in these CAR-T therapies, including cerebellar toxicity, skin toxicity, and taste disturbances. In addition, there are bispecific antibodies targeting GPRC5D and CD3 under investigation (Del Giudice, ML, Galimberti, S. & Buda, G. (2023) ibid.). Janssen’s GPRC5D×CD3 bispecific antibody TALVEY™ taquituzumab-tgvs received accelerated approval from the FDA on August 10, 2023, for patients with relapsed / refractory multiple myeloma who have received at least four prior lines of therapy (including immunomodulatory agents, anti-CD38 antibodies, and proteasome inhibitors).

[0008] Given the significant role of GPRC5D targeted therapy in the treatment of multiple myeloma, there is a need to develop more GPRC5D antibodies with superior properties. Summary of the Invention

[0009] The inventors of this application have screened several targeted GPRC5D single-domain antibodies. Single-domain antibodies possess characteristics such as high binding specificity, strong tissue penetration, ease of modification and optimization, low immunogenicity, good hydrophilicity, and good developability. They can be used to construct heavy chain antibodies, antibody-drug conjugates (ADCs), bispecific T-cell connectors (TCEs), chimeric antigen receptors (CARs), or recombinant T-cell receptors. In particular, single-domain antibodies exhibit special advantages when used in CAR-T and bispecific T-cell connector TCE therapies.

[0010] Specifically, the GPRC5D single-domain antibody of this application exhibits comparable or superior binding affinity to GPRC5D-expressing cells (e.g., human myeloma cells) compared to existing GPRC5D antibodies, such as the GPRC5D antibody portion in Talquetamab and the GPRC5D antibody portion in LM305.

[0011] The single-domain antibody of this application can act on multiple myeloma cells by constructing heavy chain antibodies and performing antibody-dependent cytotoxicity (ADCC) and / or antibody-dependent phagocytosis (ADCP), or by constructing antibody-drug conjugates (ADCs), bispecific T-cell connectors (TCEs), chimeric antigen receptors (CARs), recombinant T-cell receptors, etc.

[0012] Therefore, in one aspect, this application provides an isolated single-domain antibody capable of binding to GPRC5D (e.g., human GPRC5D), which may contain a variable region, the variable region may contain a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1 region, the CDR2 region and the CDR3 region may respectively contain as follows: (1) SEQ ID NOs: 1, 2 and 3; (2) SEQ ID NOs: 5, 6 and 7; (3) SEQ ID NOs: 9, 10 and 11; (4) SEQ ID NOs: 13, 14 and 15; (5) SEQ ID NOs: 17, 18 and 19; (6) SEQ ID NOs: 21, 22 and 23; (7) SEQ ID NOs: 25, 26 and 27; (8) SEQ ID NOs: 29, 30 and 31; (9) SEQ ID NOs: 33, 34 and 35; (10) SEQ ID NOs: 37, 38 and 39; (11) SEQ ID NOs:41, 42 and 43; or (12) amino acid sequences of SEQ ID NOs:45, 46 and 47, or amino acid sequences containing 1-3 amino acid substitutions (e.g., 1, 2 or 3) in each CDR compared to the above amino acid sequences.

[0013] The variable region of the isolated single-domain antibody of this application may contain an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NOs:4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, or 48.

[0014] In some embodiments, the single-domain antibody of this application may include a variable region, which may contain CDR1, CDR2, and CDR3 regions, wherein the CDR1, CDR2, and CDR3 regions may each contain amino acid sequences as shown in SEQ ID NOs:1, 2, and 3, respectively. The variable region may contain an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:4.

[0015] In some embodiments, the single-domain antibody of this application may include a variable region, which may contain CDR1, CDR2, and CDR3 regions, wherein the CDR1, CDR2, and CDR3 regions may each contain amino acid sequences as shown in SEQ ID NOs:5, 6, and 7, respectively. The variable region may contain an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8.

[0016] In some embodiments, the single-domain antibody of this application may include a variable region, which may contain CDR1, CDR2, and CDR3 regions, wherein the CDR1, CDR2, and CDR3 regions may each contain amino acid sequences as shown in SEQ ID NOs:9, 10, and 11, respectively. The variable region may contain an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:12.

[0017] In some embodiments, the single-domain antibody of this application may include a variable region comprising CDR1, CDR2, and CDR3 regions, wherein the CDR1, CDR2, and CDR3 regions may each contain amino acid sequences as shown in SEQ ID NOs:13, 14, and 15, respectively. The variable region may contain an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:16.

[0018] In some embodiments, the single-domain antibody of this application may include a variable region, which may contain CDR1, CDR2, and CDR3 regions, wherein the CDR1, CDR2, and CDR3 regions may each contain amino acid sequences as shown in SEQ ID NOs:17, 18, and 19, respectively. The variable region may contain an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:20.

[0019] The single-domain antibody of this application may include a variable region, which may contain a CDR1 region, a CDR2 region, and a CDR3 region, wherein the CDR1 region, CDR2 region, and CDR3 region may have at least 95% sequence identity with the CDR1 region, CDR2 region, and CDR3 region of the variable region shown in SEQ ID NOs:4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, or 48, respectively.

[0020] The single-domain antibody can be derived from alpacas or humanized.

[0021] In another aspect, this application provides a GPRC5D binding molecule that may contain the single-domain antibody of this application.

[0022] The GPRC5D binding molecule can be a single-domain antibody of this application.

[0023] The GPRC5D binding molecule can be a fusion protein of the single-domain antibody and the immunoglobulin heavy chain constant region of this application. The heavy chain constant region can be a variable region of the IgG, IgD, IgA, IgM, or IgE heavy chain, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or a functional fragment thereof (e.g., the Fc region). In some embodiments, the heavy chain constant region may include a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the heavy chain constant region may include both a CH2 domain and a CH3 domain. In some embodiments, the heavy chain constant region may have Fc receptor and / or complement system protein binding capacity, particularly strong Fc receptor and / or complement system protein binding capacity, such as the IgG1 heavy chain constant region, or a modified IgG4 heavy chain constant region with Fc receptor and / or complement system protein binding capacity, or a functional fragment thereof, such as the Fc region. In some embodiments, the heavy chain constant region can be the human IgG1 heavy chain constant region or a functional fragment thereof, such as the Fc region. In some embodiments, the human IgG1 heavy chain constant region may contain the amino acid sequence shown in SEQ ID NO:50. In some embodiments, the Fc region of human IgG1 may contain the amino acid sequence shown in SEQ ID NO:49. The C-terminus of the single-domain antibody may be linked to the N-terminus of the heavy chain constant region or a functional fragment thereof.

[0024] The GPRC5D binding molecule can be a dimer containing two of the above-mentioned single-domain antibody-immunoglobulin heavy chain constant region fusion proteins (such as single-domain antibody-Fc fusion proteins), the two fusion proteins being linked by, for example, one or more disulfide bonds.

[0025] The GPRC5D binding molecule can be a heavy chain antibody (HCAb) or its antigen-binding fragment, which may contain the single-domain antibody of this application. In some embodiments, the heavy chain antibody or its antigen-binding fragment may contain the single-domain antibody of this application linked to the aforementioned immunoglobulin heavy chain constant region. The heavy chain antibody may contain two heavy chains, or be composed of two heavy chains, wherein at least one of the heavy chains may contain the single-domain antibody of this application linked to the aforementioned immunoglobulin heavy chain constant region. The heavy chain antibody or its antigen-binding fragment may be alpaca-derived, chimeric, or humanized. In some embodiments, the heavy chain antibody or its antigen-binding fragment may be a dimer containing the aforementioned single-domain antibody, or a single-domain antibody-immunoglobulin heavy chain constant region fusion protein (such as a single-domain antibody-Fc fusion protein).

[0026] The GPRC5D binding molecules of this application, including the single-domain antibodies of this application, can specifically bind to GPRC5D (e.g., human GPRC5D), particularly to cells expressing GPRC5D such as myeloma cells, and have in vivo antitumor activity.

[0027] This application also provides immunoconjugates containing the single-domain antibody or GPRC5D binding molecule of this application, which can be linked to therapeutic agents such as cytotoxic molecules or anticancer agents. This application also provides bispecific molecules containing the single-domain antibody or GPRC5D binding molecule of this application, which can be linked to a second functional group, such as a second antibody, having a binding specificity different from that of the single-domain antibody or GPRC5D binding molecule of this application. In some embodiments, the second functional group can specifically bind to T cells, particularly specifically to CD3 on T cells. In another aspect, this application provides a chimeric antigen receptor (CAR) or genetically modified T cell receptor (TCR) containing the single-domain antibody or GPRC5D binding molecule of this application. This application also provides immune cells containing the above-mentioned CAR and / or TCR, including T cells, NK cells, etc. This application also provides an oncolytic virus encoding or carrying the single-domain antibody or GPRC5D binding molecule of this application.

[0028] This application also includes a nucleic acid molecule encoding a single-domain antibody, a GPRC5D binding molecule (including a heavy chain antibody or its antigen-binding fragment), an immunoconjugate, a bispecific molecule, or a CAR / TCR, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector or having the nucleic acid molecule integrated into its genome.

[0029] This application also provides a method for preparing the single-domain antibody, GPRC5D binding molecule (including heavy chain antibody or its antigen-binding fragment), immunoconjugate, bispecific molecule, or CAR / TCR nucleic acid molecule using the aforementioned host cells, comprising: (i) expressing the single-domain antibody, GPRC5D binding molecule (including heavy chain antibody or its antigen-binding fragment), immunoconjugate, bispecific molecule, or CAR / TCR nucleic acid molecule in the host cells, and (ii) isolating the single-domain antibody, GPRC5D binding molecule (including heavy chain antibody or its antigen-binding fragment), immunoconjugate, bispecific molecule, or CAR / TCR nucleic acid molecule from the host cells or their cultures.

[0030] This application also provides a composition that may comprise the single-domain antibody of this application, a GPRC5D binding molecule (including a heavy chain antibody or its antigen-binding fragment), an immunoconjugate, a bispecific molecule, a CAR / TCR, immune cells carrying a CAR / TCR, an oncolytic virus, a nucleic acid molecule, an expression vector, or a host cell. The composition of this application may be a pharmaceutical composition comprising a therapeutically effective amount of the single-domain antibody of this application, a GPRC5D binding molecule (including a heavy chain antibody or its antigen-binding fragment), an immunoconjugate, a bispecific molecule, a CAR / TCR, immune cells carrying a CAR / TCR, an oncolytic virus, a nucleic acid molecule, an expression vector, or a host cell, and may also comprise a pharmaceutically acceptable vector.

[0031] In another aspect, this application provides a method for treating or alleviating GPRC5D-related disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of this application to the subject.

[0032] GPRC5D-related diseases can be GPRC5D-related cancers, including, but not limited to, multiple myeloma, plasma cell leukemia, and plasmacytoma. In some embodiments, GPRC5D-related diseases can be multiple myeloma. In some embodiments, the pharmaceutical composition of this application can be administered in combination with at least one anticancer agent, such as a PD-L1 antibody. In another embodiment, the pharmaceutical composition of this application can be administered in combination with cytokines (e.g., IL-2 and / or IL-21) or co-stimulatory antibodies (e.g., CD137 antibody and / or GITR antibody). In another embodiment, the pharmaceutical composition of this application can be administered in combination with a chemotherapeutic agent, which can be a cytotoxic agent. The subject can be a mammal, particularly a human.

[0033] All documents referenced or mentioned in this application (including, but not limited to, all documents, patents, and published patent applications cited herein) (“References”), all documents referenced or mentioned in the References, and manufacturer’s manuals, instructions, product specifications, and product pages of any product mentioned in this application or any of the References are incorporated herein by reference and may be used in the practice of this invention. More specifically, all referenced documents are incorporated herein by reference as if they were individually incorporated. Any Genbank sequences mentioned herein are incorporated by reference.

[0034] It should be noted that in this application, particularly in the claims, terms such as “comprising” and “including” may have the meanings conferred by the Chinese Patent Law; while terms such as “consistent with…” have the meanings conferred by the Chinese Patent Law, such as allowing the existence of elements not explicitly stated, but excluding elements existing in the prior art or elements that affect the basic or new characteristics of the present invention.

[0035] Other features and advantages of the present disclosure will become clearer based on the following specific descriptions and embodiments, which should not be construed as limiting. All documents, Genbank records, patents, and published patent applications cited in this application are expressly included herein by reference. Attached Figure Description

[0036] The following detailed description, given by way of example but not intended to limit the invention to the specific embodiments described, can be better understood in conjunction with the accompanying drawings.

[0037] Figure 1 shows the binding affinity of the anti-GPRC5D chimeric antibody of this application to 3T3 / GPRC5D cells as determined by FACS.

[0038] Figure 2 shows the binding affinity of the anti-GPRC5D chimeric antibody of this application to human myeloma cells MM.1S as determined by FACS.

[0039] Figure 3 illustrates the epitope competition between the anti-GPRC5D chimeric antibody of this application and the GPRC5D antibody moiety in taquitocilizumab.

[0040] Figure 4 illustrates the epitope competition between the anti-GPRC5D chimeric antibody of this application and the GPRC5D antibody moiety in LM305. Detailed Implementation

[0041] Unless otherwise specified, the terms used herein have their common meanings as found in dictionaries, textbooks, and technical reference books, or as commonly understood by those skilled in the art. The following descriptions of some terms are for the purpose of understanding this application only and are not intended to impose any particular limitations on these terms, unless otherwise specified.

[0042] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural form of the object referred to, unless the context clearly specifies otherwise.

[0043] The term "or" refers to a single element among the listed selectable elements, unless the context explicitly indicates otherwise.

[0044] The terms “comprising” or “including” mean that the stated elements, integers, or steps are included, but do not exclude the inclusion of any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps.

[0045] The term "GPRC5D" refers to subtype D of the C5 family of G protein-coupled receptors. This term includes variants, homologs, orthologs, and parallel homologs. For example, antibodies specific to human GPRC5D can cross-react with GPRC5D proteins from another species, such as monkeys, under certain circumstances.

[0046] The term "human GPRC5D" refers to a GPRC5D protein with a human amino acid sequence, such as a GPRC5D protein with the amino acid sequence of NCBI index number NP_061124.1 (Mi X, Penson A, Abdel-Wahab O and Mailankody S. (2023) Genetic Basis of Relapse after GPRC5D-Targeted CAR TCells N Engl J Med 389(15):1435-1437).

[0047] In this article, the term "antibody" refers to an immunoglobulin molecule that specifically recognizes and binds to a target via an antigen-binding site, which is typically located within the variable region of the immunoglobulin molecule.

[0048] In some contexts, the term "antibody" as used herein specifically refers to a heavy chain antibody or its antigen-binding portion. The terms "heavy chain antibody" or "HCAb" refer to a functional antibody that contains a heavy chain but lacks a light chain. Naturally occurring heavy chain antibodies are found in camel-dwelling animals (camels, llamas, or alpacas). Each camel-derived heavy chain antibody contains a variable region and a constant region of the heavy chain; the variable region is called the V region. H H domain, VH H fragments or single-domain / nanobody (sdAb). V H H interacts with the antigen. V H The heavy chain constant region (H) comprises three complementation-determining regions (CDRs) and four framework regions (FRs), arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The heavy chain constant region may contain a hinge region, a CH2 domain, and a CH3 domain. A missing CH1 domain may be replaced by an extended hinge region. In chimeric or humanized heavy chain antibodies, the heavy chain constant region may contain human IgG, such as the constant regions of IgG1, IgG2, or IgG4. The constant region can mediate the binding of the heavy chain antibody to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component of the complement system (C1q). A "functional fragment" of the heavy chain constant region refers to the portion of the constant region that retains the activity that enables the antibody to bind to host tissues or factors, thereby guiding activities such as ADCC, CDC, ADCP, etc. The antibody of this application contains a heavy chain variable region or a functional fragment thereof that possesses or has strong Fc receptor and / or complement system protein binding activity.

[0049] An "antigen-binding fragment" or "antigen-binding moiety" of a heavy chain antibody refers to one or more fragments of the heavy chain antibody that retain a specific binding affinity for an antigen (such as GPRC5D). The antigen-binding function of heavy chain antibodies is known to be achieved through fragments of the heavy chain antibody. Examples of antigen-binding fragments or antigen-binding moieties of heavy chain antibodies include, but are not limited to, (i) a separated complementarity-determining region (CDR); (ii) a monovalent V H H segment; (iii) contains two unit valences V H (iv) A divalent segment of H; containing V H A monovalent segment connected to the H segment and a functional segment of the heavy chain constant region, such as a V segment connecting the CH2 domain of the heavy chain constant region, or the CH2 and CH3 domains. H H segment; (v) contains two V segments respectively connected to the heavy chain constant region or a segment thereof. H A divalent segment of H; (vi) multiple monovalent V segments connected by a connector or directly. H H-structure domain.

[0050] In some contexts, "antibody" as used herein refers to a single-domain antibody or nanobody. The term "sdAb" refers to a single antigen-binding polypeptide containing a single monomeric variable region capable of binding to an antigen without pairing with other corresponding CDR-containing polypeptides. This variable region contains three complementarity-determining regions (CDRs). In some cases, single-domain antibodies, also known as V antibodies, are derived from camel HCAb through modification. HH domain or HCAb fragment. Single-domain antibodies are the antigen-binding parts of heavy chain antibodies. Camel sdAb is the smallest known antigen-binding fragment (Hamers-Casterman et al., (1993) Nature 363:446-8; Greenberg et al., (1995) Nature 374:168-73; Hassanzadeh-Ghassabeh et al., (2013) Nanomedicine (Lond), 8:1013-26).

[0051] In this document, "GPRC5D binding molecule" refers to a molecule containing the single-domain antibody of this application, which has the affinity / activity to bind to GPRC5D, including the single-domain antibody itself, a monovalent fusion protein of the single-domain antibody and a heavy chain variable region such as the Fc region, and a dimer of the fusion protein (such as a heavy chain antibody).

[0052] As used herein, the term "isolated" single-domain antibody or heavy-chain antibody refers to an antibody that is substantially free of other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to the GPRC5D protein is substantially free of antibodies that specifically bind to antigens other than the GPRC5D protein. However, an isolated antibody that specifically binds to the human GPRC5D protein may have cross-binding to other antigens, such as GPRC5D proteins from other species. Furthermore, isolated antibodies are substantially free of other cellular material and / or chemicals.

[0053] The term "alpaca-derived antibody" refers to an antibody whose variable region backbone and CDR region are derived from alpaca lineage immunoglobulin sequences. The alpaca-derived antibody of this application may contain amino acid residues not encoded by alpaca lineage immunoglobulin sequences, such as mutations introduced through in vitro random mutations or point mutations, or through in vivo somatic mutations. However, the term "alpaca-derived antibody" does not include antibodies in which CDR sequences derived from other mammalian species are inserted into the alpaca backbone sequence.

[0054] The term "chimeric antibody" refers to an antibody obtained by combining non-human (e.g., alpaca) genetic material with human genetic material. Or, more broadly, a chimeric antibody is an antibody that combines genetic material from one species with genetic material from another species.

[0055] The term "humanized antibody" refers to an antibody derived from a non-human species (e.g., alpaca) whose protein sequence has been modified to increase its similarity to antibodies naturally generated in the human body.

[0056] In this document, an antibody that "specifically binds to human GPRC5D" refers to an antibody that binds to human GPRC5D (and GPRC5D from other non-human species) but substantially does not bind to non-GPRC5D proteins. Preferably, the antibody binds to human GPRC5D protein with "high affinity".

[0057] The term "EC" 50 "Half-maximal effect concentration" (SMP) refers to the antibody concentration that produces a 50% maximum effect.

[0058] The term "IC" 50 "Half-inhibition concentration" refers to the concentration of a drug or inhibitor required to inhibit a specified biological process by half.

[0059] The terms “antibody-dependent cytotoxicity,” “antibody-dependent cell-mediated cytotoxicity,” or “ADCC” refer to cell-mediated immune defense in which immune system effector cells actively bind to cell membrane surface antigens and antibodies, such as the GPRC5D single-domain antibody or heavy chain antibody of this application, to lyse target cells.

[0060] The term “antibody-dependent phagocytosis” or “ADCP” refers to an immune elimination mechanism in which an antibody, such as the GPRC5D heavy chain antibody of this application, binds to a target cell and recruits immune effector cells, such as phagocytes, via a constant region, such as Fc, thereby facilitating the phagocytosis of the target cell by the immune effector cells.

[0061] The term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammalians, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cattle, and horses, are preferred.

[0062] The term "therapeutic effective amount" refers to the amount of the antibody or binding molecule of this application sufficient to prevent or alleviate symptoms associated with a disease or condition (e.g., cancer). Therapeutic effective amount is related to the disease being treated, and the actual effective amount can be readily determined by those skilled in the art.

[0063] In this article, "sequence identity" refers to the percentage of nucleotides / amino acids in a sequence that are identical to those in a reference sequence after sequence alignment. If necessary, spaces are introduced in the sequence alignment to achieve the maximum percentage of sequence similarity between the two sequences. Those skilled in the art can use various methods, such as computer software, to perform pairwise or multiple sequence alignments to determine the percentage of sequence similarity between two or more nucleic acid or amino acid sequences. Such computer software includes, for example, ClustalOmega, T-coffee, Kalign, and MAFFT.

[0064] Several aspects of this application are described in more detail below.

[0065] The single-domain antibody and the GPRC5D binding molecule containing the single-domain antibody of this application can specifically bind to human GPRC5D, particularly to cells expressing GPRC5D, such as myeloma cells, and the binding affinity is comparable to or better than that of existing antibodies such as the GPRC5D antibody portion in taquituzumab and the GPRC5D antibody portion in LM305. It is foreseeable that the single-domain antibody or GPRC5D binding molecule of this application can be used to treat multiple myeloma or other GPRC5D-related diseases, and the efficacy is comparable to or better than existing products.

[0066] Preferred single-domain antibodies and / or heavy-chain antibodies of this application are monoclonal antibodies. Furthermore, the single-domain antibodies may be alpaca-derived or humanized, and the heavy-chain antibodies may be, for example, alpaca-derived, chimeric, or humanized.

[0067] The variable region CDR of the single-domain antibodies and heavy chain antibodies or their antigen-binding portions in this application is determined using the IMGT numbering system. As is well known in the art, the variable region CDR can be determined using, for example, the Kabat, Chothia, AbM, or Contact numbering systems / methods. The variable region sequences and their SEQ ID NOs of exemplary single-domain antibodies or binding molecules (including heavy chain antibodies or their antigen-binding portions) in this application are listed in Table 2.

[0068] The single-domain antibody of this application can be linked to a heavy chain constant region, such as the IgG1 constant region or a functional fragment thereof. The constant region linked to the single-domain antibody of this application can be natural or genetically modified, and has FcR binding force and / or complement system protein binding force, particularly strong FcR binding force and / or complement system protein binding force.

[0069] It is well known in the field that the CDR3 domain, independent of CDR1 and / or CDR2, can independently determine the binding specificity of an antibody to the same antigen, and can predict the generation of multiple antibodies with the same binding specificity based on the CDR3 sequence.

[0070] The single-domain antibody or GPRC5D binding molecule of this application (such as a heavy chain antibody and its antigen-binding portion) may include the heavy chain variable region CDR2 of the single-domain antibody of this application and the CDRs of other single-domain antibodies that bind human GPRC5D, such as the heavy chain variable regions CDR1 and / or CDR3. Preferably, these antibodies (a) competitively bind GPRC5D; (b) retain functional properties; (c) bind the same epitope; and / or (d) have similar binding affinity to the GPRC5D single-domain antibody and / or binding molecule of this application.

[0071] In another embodiment, the single-domain antibody or binding molecule of this application (such as a heavy chain antibody and its antigen-binding portion) may include a heavy chain variable region sequence or CDR1, CDR2, and CDR3 sequences that have one or more conserved modifications to the GPRC5D single-domain antibody or binding molecule of this application (such as a heavy chain antibody and its antigen-binding portion). It is known in the art that some conserved sequence modifications do not result in the loss of antigen binding. See, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al. al., (1992) J. Immunol. 149: 1605-12; Kelley and O'Connell (1993) Biochem. 32: 6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10: 341-6 and Beers et al. al., (2000) Clin. Can. Res. 6:2835-43.

[0072] Therefore, in one embodiment, the single-domain antibody or GPRC5D binding molecule of this application (such as a heavy chain antibody and its antigen-binding moiety) includes a heavy chain variable region, which includes CDR1, CDR2, and CDR3, wherein:

[0073] (a) The heavy chain variable region CDR1 contains the sequences listed in Table 2, and / or their conservative modifications; and / or

[0074] (b) The heavy chain variable region CDR2 contains the sequences listed in Table 2, and / or their conservative modifications; and / or

[0075] (c) The heavy chain variable region CDR3 contains the sequences listed in Table 2, and / or their conservative modifications; and

[0076] (d) This single-domain antibody specifically binds to human GPRC5D.

[0077] As used herein, the term "conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter the binding properties of the antibody or binding molecule. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or binding molecule of this application using standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conserved amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues with similar side chains are known in the art. These groups of amino acid residues include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the antibody of this application may be replaced by other amino acid residues in the same side chain group, and the resulting antibody or binding molecule may be tested for retaining function (i.e., the function described above) using the functional assays described herein.

[0078] The single-domain antibody or GPRC5D binding molecule of this application (such as heavy chain antibody and its antigen-binding moiety) has one or more of the following functional characteristics, such as high specificity binding to human GPRC5D, especially to cells expressing human GPRC5D such as human myeloma cells, and in vivo antitumor activity.

[0079] In various embodiments, the single-domain antibody, heavy chain antibody, or its antigen-binding portion may be, for example, alpaca-derived, chimeric, or humanized.

[0080] The single-domain antibody or GPRC5D binding molecule (such as a heavy chain antibody or its antigen-binding portion) of this application can be used as a starting material to prepare a genetically modified single-domain antibody or binding molecule. The single-domain antibody or binding molecule can be modified by V... H Genetic modification can be performed on one or more residues within the H region (e.g., in one or more CDR regions and / or one or more backbone regions) to improve binding affinity and / or increase similarity to naturally occurring antibodies in certain species. Alternatively, antibodies can be genetically modified by modifying residues in constant regions, for example, to alter the effector function of the antibody.

[0081] In some implementations, CDR region implantation can be used to genetically modify the variable regions of antibodies. Single-domain antibodies primarily interact with target antigens through amino acid residues located in the three heavy chain complementarity-determining regions (CDRs). For this reason, the amino acid residues within the CDRs are more diverse among individual antibodies than the sequences outside the CDRs. Because the CDR sequence is responsible for the main antibody-antigen interactions, recombinant antibodies that mimic the characteristics of specific natural antibodies can be expressed by constructing expression vectors containing the CDR sequence of a specific natural antibody and inserting it into the backbone sequences of different antibodies with different properties.

[0082] Therefore, another embodiment of this application relates to isolated monoclonal single-domain antibodies or GPRC5D binding molecules containing such single-domain antibodies, such as heavy chain antibodies or their antigen-binding moieties, which include a heavy chain variable region V. H H, V H H includes CDR1, CDR2, and CDR3 having the sequences described above in this application. Although these single-domain antibodies or binding molecules contain V of this application... H HCDR sequences can contain different backbone sequences.

[0083] Such backbone sequences can be obtained from publicly available DNA databases or references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy chain variable region genes can be obtained from the Vbase Human Germline Sequence Database (www.mrc-cpe.cam.ac.uk / vbase) and from Kabat et al., (1991), ibid.; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol. 24:827-836. As another embodiment, germline DNA sequences for human heavy chain variable region genes can be obtained from the Genbank database. For example, the Genbank accession numbers for the heavy chain germline sequences in the following HCo7 HuMAb mice are 1-69 (NG--0010109,NT--024637&BC070333), 3-33 (NG--0010109&NT--024637), and 3-7 (NG--0010109&NT--024637). As another example, the Genbank accession numbers for the heavy chain germline sequences from Hco12 HuMAb mice are 1-69 (NG--0010109,NT--024637&BC070333), 5-51 (NG--0010109&NT--024637), 4-34 (NG--0010109&NT--024637), 3-30.3 (CAJ556644), and 3-23 (AJ406678).

[0084] Single-domain antibody protein sequences were compared with protein sequence databases using one of the sequence similarity search methods known in the art, called gap BLAST (Altschul et al., (1997)).

[0085] The preferred backbone sequences used for the single-domain antibodies, heavy chain antibodies, or their antigen-binding sites in this application are those that are structurally similar to the backbone sequences used for the single-domain antibodies, heavy chain antibodies, or their antigen-binding sites in this application. H The CDR1, CDR2, and CDR3 sequences can be inserted into a backbone region that has the same sequence as the germline immunoglobulin gene from which the backbone sequence is derived, or the CDR sequence can be inserted into a backbone region containing one or more mutations compared to the germline sequence. For example, in some cases, it is beneficial to mutate residues in the backbone region to maintain or enhance the antigen-binding properties of the antibody (see, for example, US Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0086] Another type of variable region modification is to modify V H Amino acid residues within the CDR1, CDR2, and / or CDR3 regions are mutated to improve one or more binding properties (e.g., affinity) of the target antibody. Mutations can be introduced through point mutations or PCR-mediated mutations, and their effects on antibody binding or other functional properties can be evaluated using in vitro or in vivo assays known in the art. Preferably, conserved modifications known in the art are introduced. Mutations can be amino acid substitutions, additions, or deletions, but substitution is preferred. Furthermore, typically no more than one, two, three, four, or five residues within the CDR regions are altered.

[0087] Furthermore, in another embodiment, this application provides an isolated GPRC5D single-domain antibody, a heavy chain antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region, which includes: (a) V H H CDR1 region, containing the sequence of this application, or an amino acid sequence with one, two, three, four, or five amino acid substitutions, deletions, or additions; (b) V H The H CDR2 region contains the sequence of this application, or an amino acid sequence with one, two, three, four, or five amino acid substitutions, deletions, or additions; and (c)V H The HCDR3 region contains the sequence of this application, or an amino acid sequence with one, two, three, four, or five amino acid substitutions, deletions, or additions.

[0088] The genetically modified antibody in this application includes V HGenetic modifications are made to the backbone residues of H to alter, for example, antibody properties. Backbone modifications include mutations in one or more residues in the backbone region, or even one or more CDR regions, to remove T-cell epitopes, thereby reducing the antibody's potential immunogenicity. This method is also known as "deimmunization," and is described in more detail in U.S. Patent Publication 20030153043.

[0089] In addition to modifications within the backbone or CDR region, the heavy chain antibody of this application can be genetically modified to include genetic modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antibody-dependent cytotoxicity. Furthermore, the antibody of this application can be chemically modified (e.g., by attaching one or more chemical functional groups to the antibody), or modified to alter its glycosylation, to change one or more functional properties of the antibody.

[0090] In one implementation, C H1 The hinge region is modified, altered, for example, by increasing or decreasing the number of cysteine ​​residues in the hinge region. This method is further described in U.S. Patent 5,677,425. Modification of C... H1 Cysteine ​​residues in the hinge region can, for example, promote the assembly of heavy and light chains or increase / decrease antibody stability.

[0091] In another embodiment, the Fc hinge region of the heavy chain antibody is mutated to increase or decrease the antibody's biological half-life. More specifically, one or more amino acid mutations are introduced into the C10 region of the Fc hinge segment. H2 -C H3 The linker region thus weakens the SpA binding affinity of the antibody relative to the natural Fc-hinge domain. This method is described in more detail in U.S. Patent 6,165,745.

[0092] In another embodiment, the glycosylation of the antibody is modified. For example, deglycosylated antibodies (i.e., antibodies lacking glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the antibody's affinity for the antigen. Such glycosylation modification can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region backbone glycosylation sites, thereby eliminating glycosylation at that location. Such deglycosylation can increase the antibody's affinity for the antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0093] Furthermore, antibodies with altered glycosylation types can be prepared, such as low-fucosylated antibodies with reduced fucose residues, or antibodies with increased bifurcated GlcNac structures. The altered glycosylation forms have been shown to increase the ADCC activity of the antibodies. Such glycosylation modifications can be performed, for example, by expressing the antibody in host cells with altered glycosylation systems. Cells with altered glycosylation systems are known in the art, including, but not limited to, FUT8 knockout cell lines, the mutant CHO cell line Lec13, the rat fusion tumor cell line YB2 / 0, cell lines containing small interfering RNA specifically targeting the FUT8 gene, and cell lines co-expressing β-1,4-N-acetylglucosamine transferase III and Golgi α-mannosidase II. These can be used as host cells for expressing the recombinant antibody of this application to prepare antibodies with altered glycosylation.

[0094] Another modification to the antibody described herein is polyethylene glycol (PEGylation). Antibodies can be PEGylated, for example, to increase the antibody's biological (e.g., serum) half-life. To PEGylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that attach one or more PEG groups to the antibody or antibody fragment. Preferably, PEGylation is carried out by an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). The term "polyethylene glycol" as used herein includes any form of PEG used to derive other proteins, such as mono(C1-C1) PEG. 10 Alkyl- or aryl-oxy polyethylene glycol or polyethylene glycol maleimide. In some embodiments, the antibody to be PEGylated is a deglycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of this application. See, for example, EPO 154 316 and EP 0 401 384.

[0095] The antibodies of this application can be characterized by a variety of physical properties to detect and / or differentiate their classification.

[0096] For example, antibodies may contain one or more glycosylation sites in the variable region of the heavy chain. These glycosylation sites may cause increased antibody immunogenicity or altered antibody pK values ​​due to changed antigen binding (Marshall et al. (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al. (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al. (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation is known to occur in motifs containing NXS / T sequences. In some cases, GPRC5D antibodies are preferred as not containing variable region glycosylation. This can be achieved by selecting antibodies that do not contain glycosylation motifs in the variable region or by mutating residues in the glycosylation region.

[0097] In a preferred embodiment, the antibody does not contain an asparagine isomer site. Deamidation of asparagine may occur in the NG or DG sequence, creating isoaspartic residues that introduce kinks into the polypeptide chain and reduce its stability (isoaspartic effect).

[0098] On the other hand, this application provides nucleic acid molecules encoding the heavy chain variable region, CDR, and / or other fragments of the single-domain antibody or GPRC5D binding molecule of this application (including the heavy chain antibody or its antigen-binding portion). The nucleic acid molecules may be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. When purified from other cellular components or other contaminants such as other cellular nucleic acids or proteins using standard techniques, the nucleic acid is "isolated" or "substantially pure." The nucleic acid of this application may be, for example, DNA or RNA, and may or may not contain intron sequences.

[0099] The nucleic acid molecules described in this application can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas, the cDNA encoding the heavy chain of the hybridoma-prepared antibody can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding such antibodies can be collected from the gene library.

[0100] Preferred nucleic acid molecules of this application include V encoding a single-domain antibody of GPRC5D. H H sequences and / or CDRs. Once the encoded V is obtained HDNA fragments of H can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain (heavy chain antibody) genes. The term "operably ligated" refers to the joining of two DNA fragments so that the amino acid sequences encoded by both fragments are within the reading frame.

[0101] Encoding V H The isolated DNA from the H region can be operatively linked to the V region. H H encodes DNA and encodes the heavy chain constant region (C H1 C H2 and C H3 Another DNA molecule is transformed into a full-length heavy chain gene. The sequences of human heavy chain constant regions are known in the field, and DNA fragments including these regions can be obtained by standard PCR amplification. Heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but are preferably IgG1 constant regions.

[0102] The single-domain antibodies, heavy-chain antibodies, or their antigen-binding portions described in this application can be generated in host cells transfected with tumors using, for example, recombinant DNA technology combined with gene transfection methods (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, a coding portion or full-length heavy-chain DNA obtained by standard molecular biotechnology is inserted into one or more expression vectors, thereby operatively linking the gene with transcriptional and translational regulatory sequences. In this case, the term "operatively linked" means that the antibody gene is linked to the vector so that the transcriptional and translational control sequences within the vector perform their intended functions of regulating antibody gene transcription and translation.

[0103] The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody genes. Such regulatory sequences have been described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that guide high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses such as the adenovirus major late promoter (AdMLP), and polyomaviruses. Alternatively, non-viral regulatory sequences, such as ubiquitin promoters or β-globin promoters, may be used. In addition, the regulatory elements are composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and long terminal repeats of human T-cell leukemia virus type I (Takebe et al., (1988) Mol. Cell. Biol. 8: 466-472). The expression vector and expression control sequence are selected to be compatible with the expression host cells used.

[0104] The variable region is used to construct a heavy chain antibody gene by inserting it into an expression vector that already encodes the heavy chain constant region of the desired isotype, thereby enabling V... H H and C in the carrier H Operable linking. Alternatively, the recombinant expression vector can encode a signal peptide that promotes the secretion of antibody chains from host cells. The antibody chain gene can be cloned into the vector, allowing the signal peptide to link to the amino terminus of the antibody chain gene within the reading frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin).

[0105] In addition to the antibody chain gene and regulatory sequence, the recombinant expression vector of this application may carry other sequences, such as sequences regulating vector replication in host cells (e.g., replication origin) and selectable marker genes. Selectable marker genes can be used to select host cells into which the vector has been introduced (see, for example, U.S. Patents 4,399,216; 4,634,665 and 5,179,017). For example, selectable marker genes typically confer drug resistance, such as resistance to G418, hygromycin, or methotrexate, to host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for methotrexate selection / amplification in DHFR host cells) and the neo gene (for G418 selection).

[0106] For heavy chain expression, the expression vector encoding the heavy chain is transfected into host cells using standard techniques. The term "transfection" encompasses various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-dextrose transfection. Although expression of the antibody described herein is theoretically feasible in prokaryotic or eukaryotic host cells, expression in eukaryotic cells is preferred, and most preferably in mammalian host cells, because eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete appropriately folded and immunologically active antibodies.

[0107] Preferred mammalian host cells for expressing the recombinant antibodies of this application include FUT8 knockout cell lines, variant CHO cell line Lec13, rat fusion tumor cell line YB2 / 0, cell lines containing small interfering RNA specifically targeting the FUT8 gene, cell lines co-expressing β-1,4-N-acetylglucosidase III and Golgi α-mannosidase II, Chinese hamster ovary (CHO) cells (including dhfr-CHO cells administered with DHFR selectable markers, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, DHFR selectable markers described, for example, in RJ Kaufman and PASharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, antibodies are prepared by culturing the host cells for a period of time sufficient for antibody expression in the host cells, or preferably sufficient for antibody secretion into the culture medium in which the host cells grow. The antibodies can be recovered from the culture medium using protein purification methods.

[0108] On the other hand, this application relates to bispecific molecules comprising a single-domain antibody or GPRC5D binding molecule (including a heavy chain antibody or its antigen-binding moiety) of this 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 having three or more specificities.

[0109] The single-domain antibody or GPRC5D binding molecule (including heavy chain antibodies or their antigen-binding moieties) of this 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 intercalators, 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 via a cleavable linker, such as a peptide linker, disulfide linker, or hydrazone linker. For example, the linker can be a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, GS linker, etc. ADCs can be prepared as described in U.S. Patents 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02 / 096910; WO 07 / 038,658; WO 07 / 051,081; WO 07 / 059,404; WO 08 / 083,312; and WO 08 / 103,693; and U.S. Patent Publications 20060024317; 20060004081; and 20060247295. In particular, the cytotoxic molecule can be any small molecule compound or protein molecule that damages target cells, such as a tubulin polymerization inhibitor, DNA damaging agent, etc.

[0110] Bispecific molecules can appear in a variety of forms and sizes. At one end of the size spectrum, bispecific molecules retain the traditional antibody form, except that they have two binding arms, each with different specificities, instead of two binding arms with the same specificity. At the other extreme are bispecific molecules consisting of two single-domain antibodies linked by peptide chains. Mid-sized bispecific molecules consist of an F(ab) fragment linked by a peptide linker and a single-domain antibody. These and other forms of bispecific molecules can be prepared by genetic engineering, somatic cell hybridization, or chemical methods. See, for example, Cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644 (1998); and van Spriel et al., Immunology Today, 21(8), 391-397 (2000).

[0111] The single-domain antibody or GPRC5D binding molecule (including heavy chain antibody or its antigen-binding portion) of this application can be linked to peptides or proteins that specifically bind to T cells, such as antibodies that specifically bind to CD3 molecules on T cells. Such a bispecific molecule can be called a "T cell adaptor (TCE)," meaning a bispecific molecule that can simultaneously bind to both T cells and target cells and guide the T cell to produce cytotoxicity against the target cell.

[0112] This application also provides a chimeric antigen receptor comprising a GPRC5D single-domain antibody. The chimeric antigen receptor may comprise (a) an extracellular antigen-binding domain containing a GPRC5D single-domain antibody; (b) a transmembrane domain; and (c) an intracellular signal transduction domain. This application also provides an immune cell, such as a T cell or NK cell, comprising the chimeric antigen receptor of this application.

[0113] The T cell receptor can also be modified to include the single-domain antibody of this application, and to activate T cells and induce cytotoxicity against target cells carrying the GPRC5D molecule when the single-domain antibody binds to GPRC5D. This application also provides a T cell comprising the recombinant T cell receptor of this application.

[0114] Oncolytic viruses preferentially infect and kill cancer cells. The single-domain antibody or binding molecule (including heavy chain antibody or its antigen-binding portion) of this application can be used in conjunction with oncolytic viruses. Furthermore, oncolytic viruses encoding the single-domain antibody or binding molecule (including heavy chain antibody or its antigen-binding portion) of this application can be introduced into the human body.

[0115] On the other hand, this application provides a pharmaceutical composition comprising the single-domain antibody, binding molecule (including heavy chain antibody or its antigen-binding portion), immunoconjugate, bispecific molecule, CAR / TCR, CAR / TCR-carrying immune cells, oncolytic virus, nucleic acid molecule, expression vector, or host cell, formulated together with a pharmaceutically acceptable carrier. The composition may optionally contain one or more other pharmaceutically effective ingredients, such as another antitumor antibody, or an immune-enhancing antibody, or a non-antibody antitumor agent, or an immune enhancer. The pharmaceutical composition of this application may be used in combination with, for example, another anticancer agent.

[0116] Pharmaceutical compositions may contain any number of excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of appropriate excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).

[0117] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or bolus). Depending on the route of administration, the active ingredient may be encapsulated in a material to protect it from acids and other natural conditions that may inactivate it. “Parenteral administration” refers to methods other than intestinal and topical application, typically administered by injection, including but not limited to intravenous, intramuscular, intra-arterial, intramembranous, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcystic, subarachnoid, spinal, supradural, and intrasternal injections and boluses. Alternatively, the antibody of this application may be administered via non-parenteral routes, such as topical, epidermal, or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical application.

[0118] Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high concentrations of drugs.

[0119] The amount of active ingredient prepared together with the carrier material into a single dosage form will vary depending on the therapeutic subject and specific administration mode, and is essentially the amount of the composition that produces the therapeutic effect. In percentage terms, this amount is approximately 0.01% to approximately 99% of the active ingredient bound to a pharmaceutically acceptable carrier.

[0120] The dosing regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a rapid infusion can be administered, multiple fractions can be administered over time, or the dose can be proportionally reduced or increased depending on the severity of the treatment condition. Particularly advantageous are parenteral compositions formulated in convenient and uniformly dose-unit formats. A dose-unit format refers to physically separate units suitable for a single dose to the therapeutic subject; each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect when used with the pharmaceutical carrier. Alternatively, antibodies can be administered as sustained-release formulations, in which case the required dosing frequency is reduced.

[0121] For the administration of single-domain antibodies or binding molecules (including heavy chain antibodies or their antigen-binding portions), the dosage may be approximately 0.001-100 mg / kg of host body weight.

[0122] The therapeutically effective amount of the pharmaceutical composition of this application causes a reduction in the severity of disease symptoms and an increase in the frequency and duration of asymptomatic periods. For example, in the treatment of a subject suffering from multiple myeloma, the therapeutically effective amount preferably inhibits the growth of multiple myeloma cells by at least about 20%, particularly at least about 40%, even more particularly at least about 60%, and even more particularly at least about 80% compared to an untreated subject. The therapeutically effective amount of the pharmaceutical composition of this application can stop the progression of the disease, reduce the number of multiple myeloma cells, alleviate symptoms in the subject, who may be a human or another mammal.

[0123] The pharmaceutical composition may be a sustained-release agent, including implants and microcapsule delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0124] Pharmaceutical compositions can be administered via medical devices, such as (1) needle-free subcutaneous injection devices (e.g., 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) microinfusion pumps (U.S. Patent 4,487,603); (3) transdermal drug delivery devices (U.S. Patent 4,486,194); (4) bolus injection devices (U.S. Patents 4,447,233 and 4,447,224); and (5) permeation devices (U.S. Patents 4,439,196 and 4,475,196).

[0125] In some embodiments, the single-domain antibodies or binding molecules of this application may be formulated to ensure suitable in vivo distribution. For example, to ensure that the therapeutic antibodies or binding molecules of this application cross the blood-brain barrier, the antibodies may be formulated in liposomes, which may also additionally contain targeting functional groups to enhance selective delivery to specific cells or organs. See, for example, U.S. Patents 4,522,811; 5,374,548, etc.

[0126] The pharmaceutical compositions of this application have various in vitro and in vitro-intravenous applications, relating to the treatment of, for example, multiple myeloma, plasma cell leukemia, or plasmacytoma. The pharmaceutical compositions can be administered to human subjects to, for example, inhibit the progression of multiple myeloma in vivo.

[0127] In view of the ability of the pharmaceutical composition of this application to inhibit the proliferation and survival of multiple myeloma cells, this application provides a method for treating or alleviating multiple myeloma in a subject, including administering the pharmaceutical composition of this application to the subject. Multiple myeloma that can be treated with the antibody of this application may be newly diagnosed, refractory, or relapsed.

[0128] This application provides a combination therapy in which the pharmaceutical composition of this application is administered in combination with one or more other antibody or non-antibody therapeutic agents, which effectively inhibits the proliferation and survival of multiple myeloma cells in a subject. In one embodiment, this application provides a method for inhibiting myeloma cell growth in a subject, comprising administering the pharmaceutical composition of this application and one or more other antibodies, such as a PD-L1 antibody, to the subject. In some embodiments, the subject is a human. In another aspect, this application provides a cancer treatment method in which the pharmaceutical composition of this application is administered in combination with a chemotherapeutic agent, which may be a cytotoxic agent. Other therapies that can be combined with the pharmaceutical composition of this application include, but are not limited to, administration of immunogenic agents, administration of interleukin-2 (IL-2), radiotherapy, surgery, or hormone removal.

[0129] The combination of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions, wherein each agent is contained in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

[0130] Furthermore, if multiple combination therapies are administered and the drugs are administered sequentially, the order of administration at each time point can be reversed or kept the same, and sequential administration can be combined with simultaneous administration or any combination thereof.

[0131] This application is further described through the following embodiments, which should not be construed as limiting. All figures, all references, Genebank sequences, patents, and published patent applications cited throughout this application are incorporated herein by reference in their entirety.

[0132] Various aspects and implementations of this application will be discussed with reference to the accompanying drawings and the following embodiments. Other aspects and implementations will be apparent to those skilled in the art. All documents described herein are incorporated herein by reference in their entirety. Although this application has been described in conjunction with exemplary embodiments, many equivalent modifications and variations were apparent to those skilled in the art at the time of this application. Therefore, the exemplary embodiments of this application are exemplary and not limiting. Various changes can be made to the described embodiments without departing from the spirit and scope of this application.

[0133] Example 1. Preparation of anti-GPRC5D single-domain antibody

[0134] 1) Immunization

[0135] In accordance with current animal welfare regulations, one alpaca was immunized with mRNA lipid nanoparticles (LNPs) encoding human GPRC5D (manufacturer: Chengshi Biosciences, catalog number: PB-GPRC5D-110). Immunization was administered via multiple subcutaneous injections. The initial immunization used 500 μg of mRNA-LNP, and subsequent immunizations used 300 μg of mRNA-LNP. Seven days after each immunization, 2 ml of serum was collected, and antibody titers were detected using flow cytometry (FACS). Starting with the third immunization, 50 ml of serum was collected seven days after each immunization, and PBMCs were extracted. Phage libraries were constructed based on the serum titer results.

[0136] 2) Immune library construction

[0137] Based on serum titer results, PBMCs obtained after the third and fourth immunizations were mixed for phage library construction of single-domain antibodies. In short, total RNA was extracted from PBMCs, reverse transcribed into cDNA, and primer combinations were used to construct a phage library for single-domain antibodies. H Amplification of H. The purified amplified fragment was inserted into the vector to construct a phage library.

[0138] 3) Single-domain antibody library screening and selection

[0139] The single-domain antibody phage display library stock solution was expressed and precipitated with PEG / NaCl, resuspended in PBS, and panned. In the first round, 3T3 cells expressing GPRC5D, referred to as 3T3 / GPRC5D (constructed using pcDNA3.1 vector, 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. NEngl J Med 389(15):1435-1437)) and 3T3 parent cells were panned. In the second round, MM.1S human myeloma cells (manufacturer: Cell Bank of Chinese Academy of Sciences, catalog number: SCSP-5017) were used for cell panning. Phage eluent was infected with Escherichia coli TG1 at 37°C. M13K07 helper phage or IPTG was used for amplification and transfection to prepare phages for subsequent rounds of panning. Infected TG1 cells were plated on LB-Amp+ plates, and single clones were picked. The binding activity of the single-clonal phage to the two cell lines was verified by FACS using 3T3 / GPRC5D (Built-up Construction) and MM.1S human myeloma cells (manufacturer: Cell Bank of Chinese Academy of Sciences, catalog number: SCSP-5017). Eighteen clones were selected based on a FACS mean fluorescence intensity ratio greater than 5-fold, and their fluorescence intensity ratios are shown in Table 1.

[0140] Table 1. Binding of phage supernatants to FACS in 3T3 / GPRC5D and MM.1S cells from 18 bacteriophage supernatants

[0141] 4) Sequencing of single-domain antibodies and construction of single-domain antibody-Fc recombinant proteins.

[0142] The 18 positive monoclonal antibodies were sequenced. Based on sequence diversity analysis, 12 monoclonal antibodies were selected and fused with human IgG1 Fc (SEQ ID NO:49). These antibodies were then recombinantly expressed in ExpiCHO-STM cells (manufacturer: Thermo Fisher Scientific, catalog number: A29127) using the pcDNA3.4 vector, with the C-terminus of the antibody variable region linked to the N-terminus of the Fc region. The recombinantly expressed protein is structurally similar to the heavy chain antibody and will be referred to as a chimeric antibody below. The variable region and CDR sequences of the 12 selected antibodies are shown in Table 2.

[0143] Table 2. Sequences and SEQ ID NO of antibody variable region and CDR.

[0144] Example 2. Binding of anti-GPRC5D chimeric antibody to 3T3 / GPRC5D cells

[0145] The binding affinity of the 12 recombinant chimeric antibodies of this application to 3T3 / GPRC5D cells was tested using FACS. Each antibody was serially diluted 3-fold in PBS containing 0.1% BSA, starting at a concentration of 100 nM. 50 μl of antibody was then mixed with approximately 1 × 10⁻⁶ BSA in 50 μl of PBS. 5 3T3 / GPRC5D cells were incubated at 4°C for 40 minutes. Afterwards, the cells were washed three times with PBS, and then 100 μl of 1 μg / ml goat anti-human IgG (Fcγ specific) fluorescent secondary antibody (manufacturer: Jackson Immuno Research, catalog number: 109-605-098) was added. The cells were incubated at 4°C for 30 minutes, and fluorescence signal was measured in a BD Canto analyzer.

[0146] Based on the variable region sequence of the GPRC5D-targeting antibody in Janssen's taquitumab and the variable region sequence of the antibody portion in Lixin Pharmaceuticals' LM305, along with the heavy chain constant region (SEQ ID NO:50) and light chain κ constant region (SEQ ID NO:51), IgG antibodies were recombinantly expressed in ExpiCHO-STM cells as positive controls, and will be referred to as taquitumab and LM305 in the following text. Human IgG was used as a negative control.

[0147] The average fluorescence intensity of each antibody is shown in Figure 1, and the half-maximal effect concentration (EC50) is also shown in Figure 1. 50 As shown in Table 3, the results indicate that all recombinant chimeric antibodies in this application exhibited good binding affinity to 3T3 / GPRC5D cells overexpressing GPRC5D. Except for AHP44377 and Yangshen LM305 ECMO cells... 50 In addition, the EC of the other antibodies in this application 50 Both are superior to the two Yang Shen (a type of ginseng).

[0148] Table 3. ECG binding between anti-GPRC5D antibody and 3T3 / GPRC5D cells 50

[0149] Example 3. Binding of anti-GPRC5D chimeric antibody to MM.1S cells

[0150] The binding affinity of the 12 recombinant chimeric antibodies in this application to MM.1S human myeloma cells (manufacturer: Cell Bank of Chinese Academy of Sciences, catalog number: SCSP-5017) was tested using FACS.

[0151] Each antibody was serially diluted 3-fold in PBS containing 0.1% BSA, starting at a concentration of 100 nM. Take 50 μl of antibody and mix it with approximately 1 × 10⁻⁶ BSA in 50 μl of PBS. 5MM.1S cells were incubated at 4°C for 40 minutes. Afterwards, the cells were washed three times with PBS, and then 100 μl of 1 μg / ml goat anti-human IgG (Fcγ specific) fluorescent secondary antibody (manufacturer: Jackson ImmunoResearch, catalog number: 109-605-098) was added and incubated at 4°C for 30 minutes. Fluorescence signal was then measured in BD CantoII.

[0152] The average fluorescence intensity of each antibody is shown in Figure 2, and the half-maximal effect concentration (EC50) is also shown in Figure 2. 50 As shown in Table 4, the results indicate that all recombinant chimeric antibodies in this application exhibited binding curves superior to the two positive control antibodies. Among them, AHP44402, AHP44358, AHP44369, AHP44338, AHP44411, AHP44377, AHP44383, AHP44378, and AHP44368 showed better half-maximal effective concentrations (EC50) than the two positive control antibodies. 50 .

[0153] Table 4. ECG binding between anti-GPRC5D antibody and MM.1S cells 50

[0154] Example 4. Epitope competition between anti-GPRC5D chimeric antibody and taquituzumab

[0155] To test whether the recombinant chimeric antibody of this application binds to the same epitope as the yang-sensitive antibody taquitolimab, 3T3 / GPRC5D cells were used, and FACS was performed to test whether each antibody of this application competes with the epitope of the yang-sensitive antibody.

[0156] Taquitumab was biotinylated using N-[6-(Biotinamino)hexanoyl]-6-aminohexanoic acid N-succinimide ester (manufacturer: Aladdin, catalog number: B122220-25mg). Briefly, taquitumab and the biotinylated agent were mixed at a molar ratio of 10:1 and incubated at room temperature for 2 hours. The biotinylated antibody was then dialyzed against PBS at pH 7.4 at 4°C. The biotinylated taquitumab was then tested for binding to 3T3 / GPRC5D cells using FACS, following the method described in Example 2. 50 0.3 μg / ml was selected for the following epitope competition test.

[0157] 50 μl of biotin-conjugated taquitumab at a concentration of 0.3 μg / ml was mixed with approximately 1 × 10⁻⁶ ppm of the solution. 53T3 / GPRC5D cells were incubated at 4°C for 15 minutes. Then, each antibody from this application was serially diluted 3-fold in PBS containing 0.1% BSA, starting at 300 nM, resulting in 12 concentrations. 100 μl of each antibody from this application was added to a suspension of biotin-conjugated taquituzumab and cells, and incubated at 4°C for 40 minutes. After washing the cells three times with PBS, 100 μl of 0.5 μg / ml BSA fluorescent secondary antibody (manufacturer: Jackson ImmunoResearch, catalog number: 016-600-084) was added, and the cells were incubated at 4°C for 30 minutes. Fluorescence signal was then measured in a BD Cantoll.

[0158] Figure 3 shows the average fluorescence intensity of taquiltuzumab on cells after co-incubation with the antibodies of this application, and the IC50 of each antibody. 50 As shown in Table 5, the results show that talutuzumab and the 12 candidate antibodies of this application exhibit dose-dependent competition, indicating that these 12 candidate antibodies are likely to bind to the same or similar epitopes of GPRC5D as talutuzumab.

[0159] Table 5. Epitope competition IC50 between the antibody and taquituzumab in this application 50

[0160] Example 5. Epitope competition between anti-GPRC5D chimeric antibody and LM305

[0161] To test whether the recombinant chimeric antibody of this application binds to the same epitope as the LM305 comparison product of the yang ginseng, 3T3 / GPRC5D cells were used, and FACS was used to test the competition between each antibody of this application and the epitope of the yang ginseng.

[0162] LM305 monoclonal antibody was biotinylated using N-[6-(biotinamino)hexanoyl]-6-aminohexanoic acid N-succinimide ester (manufacturer: Aladdin, catalog number: B122220-25mg). Briefly, LM305 monoclonal antibody and biotinylated agent were mixed at a molar ratio of 10:1, incubated at room temperature for 2 hours, and then the biotinylated antibody was dialyzed against PBS at pH 7.4 at 4°C. The biotinylated LM305 was then tested for binding to 3T3 / GPRC5D cells using FACS, following the method described in Example 2. 50 0.1 μg / ml was selected for the following epitope competition test.

[0163] 50 μl of biotin-conjugated LM305 monoclonal antibody at a concentration of 0.1 μg / ml was mixed with approximately 1 × 10⁻⁶ ppm of the antibody in 50 μl of water. 53T3 / GPRC5D cells were incubated at 4°C for 15 minutes. Then, each antibody from this application was serially diluted 3-fold in PBS containing 0.1% BSA, starting at 300 nM, resulting in 12 concentrations. 100 μl of each antibody from this application was added to a suspension of biotin-conjugated LM305 and cells, and incubated at 4°C for 40 minutes. After washing the cells three times with PBS, 100 μl of 0.5 μg / ml SA fluorescent secondary antibody (manufacturer: Jackson ImmunoResearch, catalog number: 016-600-084) was added, and incubated at 4°C for 30 minutes. Fluorescence signal was then measured in a BD Cantoll.

[0164] Figure 4 shows the average fluorescence intensity of LM305 on cells after co-incubation with the antibodies of this application, and the IC50 of each antibody. 50 As shown in Table 6, the results show that LM305 and the 12 candidate antibodies of this application exhibit dose-dependent competition, indicating that these 12 candidate antibodies are likely to bind to the same or similar epitopes of GPRC5D as LM305.

[0165] Table 6. IC50 of epitope competition between the antibody of this application and LM305

[0166] Some of the sequences involved in the application are listed below.

[0167] Human IgG1 Fc

[0168] Human IgG1 constant region

[0169] Human κ constant region

[0170] Although this application has been described in conjunction with one or more embodiments, it should be understood that this application is not limited to these embodiments. The description in this application is intended to cover all variations and equivalents, all of which are included within the spirit and scope of the appended claims. All references cited herein are incorporated herein by reference in their entirety.

Claims

1. An isolated monoclonal single-domain antibody capable of specifically binding to GPRC5D, comprising a CDR1 region, a CDR2 region, and a CDR3 region, wherein, CDR1, CDR2 and CDR3 contain amino acid sequences as shown in (1) SEQ ID NOs:5, 6 and 7; (2) SEQ ID NOs:9, 10 and 11; (3) SEQ ID NOs:1, 2 and 3; (4) SEQ ID NOs:13, 14 and 15; (5) SEQ ID NOs:17, 18 and 19; (6) SEQ ID NOs:21, 22 and 23; (7) SEQ ID NOs:25, 26 and 27; (8) SEQ ID NOs:29, 30 and 31; (9) SEQ ID NOs:33, 34 and 35; (10) SEQ ID NOs:37, 38 and 39; (11) SEQ ID NOs:41, 42 and 43; or (12) SEQ ID NOs:45, 46 and 47, or amino acid sequences with 1-3 amino acid substitutions in each CDR compared to the above amino acid sequences.

2. The single-domain antibody according to claim 1, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NOs:8, 12, 4, 16, 20, 24, 28, 32, 36, 40, 44, or 48.

3. A GPRC5D binding molecule comprising i) a single-domain antibody as described in claim 1 or 2, and ii) a constant region of an immunoglobulin heavy chain, wherein i) and ii) form a fusion protein.

4. The GPRC5D binding molecule according to claim 3, wherein the immunoglobulin heavy chain constant region has Fc receptor binding force and / or complement system binding force.

5. The GPRC5D binding molecule according to claim 4, wherein the immunoglobulin heavy chain constant region is the IgG1 heavy chain constant region or its Fc region.

6. The GPRC5D binding molecule according to claim 3, wherein it is a dimer of the fusion protein.

7. An immunoconjugate comprising i) a single-domain antibody as claimed in claim 1 or 2, or a GPRC5D binding molecule as claimed in any one of claims 3-6, and ii) a cytotoxic molecule.

8. A bispecific binding molecule comprising i) a single-domain antibody as described in claim 1 or 2, or a GPRC5D binding molecule as described in any one of claims 3-6, and ii) an antibody or its antigen-binding portion capable of specifically binding to a non-GPRC5D molecule.

9. A chimeric antigen receptor or recombinant T-cell receptor comprising, in its extracellular domain, the single-domain antibody of claim 1 or 2.

10. A nucleic acid molecule encoding the single-domain antibody of claim 1 or 2, or the GPRC5D binding molecule of any one of claims 3-6.

11. An expression vector comprising the nucleic acid molecule of claim 10.

12. A host cell comprising the expression vector of claim 11, or having the nucleic acid molecule of claim 10 integrated into its genome.

13. A composition comprising the single-domain antibody of claim 1 or 2, the GPRC5D binding molecule of any one of claims 3-6, the antibody-drug conjugate of claim 7, the bispecific binding molecule of claim 8, the chimeric antigen receptor or recombinant T-cell receptor of claim 9, the nucleic acid molecule of claim 10, the expression vector of claim 11, or the host cell of claim 12.

14. The composition of claim 13, which is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

15. Use of the pharmaceutical composition of claim 14 in the preparation of a medicament for treating tumors associated with GPRC5D.

16. The use according to claim 15, wherein the tumor is multiple myeloma, plasma cell leukemia, or plasma cell tumor.

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