Fully human antibodies and chimeric antigen receptors (CARs) targeting GPRC5D and uses thereof

Abstract

The present specification provides a fully human antibody targeting GPRC5D and a chimeric antigen receptor (CAR) comprising the fully human antibody. The present specification further provides a host cell, such as a CAR-T cell, expressing the CAR. The present specification further provides the use of these fully human antibodies, CARs, and CAR-T cells in the field of treating tumors (e.g., multiple myeloma).

Description

[Technical field]

[0001] This application claims priority to a PCT patent application filed with the State Intellectual Property Office of China on December 21, 2021, application number PCT / CN2021 / 140153, and to a PCT patent application filed with the State Intellectual Property Office of China on March 15, 2022, application number PCT / CN2022 / 080836, both of which are incorporated by reference in their entireties.

[0002] The present application relates to the field of biopharmaceuticals, in particular to fully human antibodies and CARs targeting GPRC5D, as well as methods for their preparation and use. [Background technology]

[0003] Multiple myeloma is a plasma cell tumor that occurs in the bone marrow. The tumor causes diseases such as hypercalcemia, anemia, renal dysfunction, osteonecrosis, and bone marrow failure. Currently, multiple myeloma is the second most common blood tumor. [1] According to the multiple myeloma incidence statistics published by the World Health Organization (WHO) in 2020, the incidence and mortality rates of Asians are 36% and 42%, respectively, ranking first among all continents. The incidence and mortality ratio of multiple myeloma worldwide is 1.8:1.1, and in Asia it is 1.1:0.76, indicating that the survival rate of multiple myeloma patients is low. One of the main reasons is that the median age of patients is high at about 66 years, and the incidence rate of people under 40 years old is around 2%. In addition, multiple myeloma has almost no chance of being cured with conventional chemotherapy, immunomodulatory drugs, and monoclonal antibody therapy, and even forced remission is accompanied by a very high relapse rate, i.e., relapsed / refractory multiple myeloma (RRMM), with a 5-year survival rate of only 51%. [1],[2] .

[0004] In recent years, bispecific T cell engagers (BiTEs) and adoptive T cell therapy (ACT) have made great strides in the treatment of multiple myeloma. Targeted therapy products for multiple myeloma mainly target BCMA, CD38, CD138, GPRC5D, etc. [2] CD38 and CD138 are expressed on normal tissue cells and hematopoietic stem cells, and after removal using targeted therapy, they often have serious side effects, damage normal organs, or damage the autoimmune system, while BCMA and GPRC5D are mainly expressed on plasma cells or plasma cells in myeloma, and plasma cells can be replenished by relying on the continuous renewal of the body's own B cells. Taking bispecific T cell-inducing antibody products as an example, BCMA-targeted products mainly include REGN5458 developed by Regeneron Pharmaceuticals, Inc. (REGN) and Teclistamab developed by Johnson & Johnson. [2][3] REGN5458 is currently in Phase I clinical trials. According to a report at the ASH conference in 2022, 54 patients received intravenous drug injections with a response rate of 71.4% and a maximum remission rate of 57.1%, while 51 patients received subcutaneous drug injections with a response rate of 60.4% and a maximum remission rate of 39.6%. [4] Teclistamab was launched in October 2022 and had an overall response rate of 61.8%, with 28.2% of patients achieving complete or stringent remission. [5] Talqulemab, a representative bispecific antibody targeting GPRC5D, submitted a BLA in December 2022 and applied for marketing. A completed Phase I / II clinical trial showed that the overall response rate at a dose of 0.4 mg / kg was 73%, the maximum remission rate was 58%, and the complete remission rate was over 29%. [6] It is already in Phase III clinical trials.

[0005] bb2121, a CAR-T product developed by Bristol Myers Squibb (BMS) targeting BCMA for multiple myeloma, was approved by the FDA in May 2021 as a marketed CAR-T product for multiple myeloma. The product had an overall response rate (ORR) of 73%, with 33% of patients achieving complete remission (CR). [7] Nanjing Legend's BCMACAR-T, launched in 2022, has improved efficacy compared to BMS, with an overall response rate (ORR) of 98%, 80% of patients achieving complete remission (CR), and a median progression-free survival of 22.8 months. [8] The two-year survival rate of both commercially available products is less than 50%. It is known that existing BCMACAR-T treatment products have a high relapse rate in the late stage. In addition, since the therapy removes all plasma cells, side effects such as a decrease in blood cells and a decrease in immunoglobulin remain. GPRC5D expression is more specific than BCMA, and is only expressed in plasma cells of myeloma patients, is hardly expressed in normal tissues, and significant RNA and protein expression can be detected only in hair follicle tissue. [9],

[10] Furthermore, we found that there was no correlation between BCMA expression and GPRC5D, and that although both are co-expressed in plasma cells, their expression profiles are relatively independent.

[11] Targeting GPRC5D as a therapeutic target may be useful in patients with low BCMA expression who have failed BCMA CAR-T therapy.

[0006] The GPRC5D-targeted CAR-T product currently under investigation is mainly MCARH109 developed by Eureka. In a Phase I clinical trial, according to data from the 2022 ASH Annual Report, the overall response rate was 71% (12 / 17) and the maximum remission rate was 59% (10 / 17) in 17 patients who received GPRC5D CAR-T injections.

[12] Another GPRC5D CAR-T therapy has been developed by Oricell and has already been IND approved, therefore, the medical market is still in need of novel GPRC5D CAR-T products with high efficacy and low immunogenicity. Summary of the Invention

[0007] According to one aspect, the present disclosure provides a GPRC5D-binding peptide comprising a heavy chain variable region of an antibody molecule, wherein HCDR1, HCDR2, and HCDR3 in the heavy chain variable region are HCDR1 is GGSFSGYY (SEQ ID NO:1), HCDR2 is INHSGST (SEQ ID NO:2), Combination 1) in which the HCDR3 sequence is ARARRYGGRTRFDP (SEQ ID NO:3); HCDR1 is GFIFSSYG (SEQ ID NO:4), the sequence of HCDR2 is ISSSGDYT (SEQ ID NO:5); Combination 2) in which the HCDR3 sequence is ARMSFRRYDH (SEQ ID NO:6); and HCDR1 is GFSFSGYI (SEQ ID NO:7), the sequence of HCDR2 is TSSSGTET (SEQ ID NO:8); 3) in which the sequence of HCDR3 is ARYYSKYGRSYHVDS (SEQ ID NO:9).

[0008] In some embodiments, the GPRC5D-binding peptide is an antibody molecule or an antigen-binding fragment thereof. In some embodiments, the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:10, 11 or 12, or the heavy chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:10, 11 or 12, and is capable of specifically binding to GPRC5D.

[0009] In some embodiments, the GPRC5D binding peptide is a single domain antibody molecule. In some embodiments, the GPRC5D-binding peptide is a fully human antibody molecule. In some embodiments, the binding ability of the GPRC5D-binding peptide to cells expressing GPRC5D on their surface is such that the EC50 value, as detected by FACS, is 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, or 5 nM or less.

[0010] In another aspect, the present disclosure provides a fusion protein comprising the above-described GPRC5D-binding peptide. In some embodiments, the fusion protein comprises at least two GPRC5D-binding peptides. In some embodiments, the HCDR1, HCDR2 and HCDR3 of the two GPRC5D binding peptides are each selected from different combinations above.

[0011] In some embodiments, in the fusion protein, one of the two GPRC5D-binding peptides comprises an amino acid sequence as set forth in SEQ ID NO:10 or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence as set forth in SEQ ID NO:10 and is capable of specifically binding to GPRC5D, and the other comprises an amino acid sequence as set forth in SEQ ID NO:11 or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence as set forth in SEQ ID NO:11 and is capable of specifically binding to GPRC5D. One of the two GPRC5D-binding peptides comprises an amino acid sequence as shown in SEQ ID NO:10 or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence as shown in SEQ ID NO:10 and is capable of specifically binding to GPRC5D, and the other comprises an amino acid sequence as shown in SEQ ID NO:12 or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence as shown in SEQ ID NO:12 and is capable of specifically binding to GPRC5D. Alternatively, one of the two GPRC5D-binding peptides comprises the amino acid sequence shown in SEQ ID NO:11 or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:11 and is capable of specifically binding to GPRC5D, and the other comprises the amino acid sequence shown in SEQ ID NO:12 or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:12 and is capable of specifically binding to GPRC5D.

[0012] In some embodiments, the fusion protein comprises at least three of the GPRC5D-binding peptides, and the HCDR1, HCDR2 and HCDR3 of the three GPRC5D-binding peptides are each a combination of the above three. In some embodiments, the first of the three GPRC5D-binding peptides of the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence set forth in SEQ ID NO:10, and is capable of specifically binding to GPRC5D. The second comprises an amino acid sequence set forth in SEQ ID NO:11 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence set forth in SEQ ID NO:11, and is capable of specifically binding to GPRC5D. and the third comprises an amino acid sequence set forth in SEQ ID NO:12 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence set forth in SEQ ID NO:12, and is capable of specifically binding to GPRC5D.

[0013] In some embodiments, the fusion protein further comprises an Fc fragment. In some embodiments, the fusion protein further comprises another binding peptide capable of specifically binding to an antigen different from GPRC5D.

[0014] In some embodiments, the alternative binding peptide is a single domain antibody or a single chain antibody. In some embodiments, the fusion protein further comprises a detectable or purification tag. In some embodiments, the detectable tag has enzymatic activity.

[0015] In another aspect, the present disclosure provides a nucleic acid molecule encoding the above-mentioned GPRC5D-binding peptide or the above-mentioned fusion protein. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:13, 14 or 15.

[0016] In another aspect, the present disclosure includes an expression vector comprising the above-described nucleic acid molecule. In another aspect, the present disclosure provides a host cell comprising the above-described nucleic acid molecule or expression vector. According to another aspect, the present specification provides a multispecific antibody molecule comprising at least a first functional portion and a second functional portion, wherein the first functional portion comprises the above-mentioned GPRC5D binding peptide and the second functional portion has a binding specificity different from that of the first functional portion.

[0017] In some embodiments, the second functional moiety has binding specificity for an immune cell. In some embodiments, the second functional moiety has binding specificity for a T cell. In some embodiments, said second functional moiety has binding specificity for CD3.

[0018] In another aspect, the present disclosure provides an immunoconjugate comprising the above-described GPRC5D-binding peptide linked to a therapeutic agent. In some embodiments, the therapeutic agent is a drug. In some embodiments, the therapeutic agent is cytotoxic. In some embodiments, the therapeutic agent is a radioisotope.

[0019] According to another aspect, the present specification provides a pharmaceutical composition comprising 1) the above-mentioned GPRC5D-binding peptide, the above-mentioned fusion protein, the above-mentioned multispecific antibody molecule, or the above-mentioned immunoconjugate, and 2) a pharma- ceutically acceptable carrier. The above GPRC5D-binding peptides, fusion proteins, multispecific antibody molecules, immunoconjugates or pharmaceutical compositions are useful for the treatment of GPRC5D-related diseases.

[0020] In another aspect, the present disclosure provides the use of the above-mentioned GPRC5D-binding peptide, fusion protein, multispecific antibody molecule or immunoconjugate in the preparation of a medicament for treating a GPRC5D-related disease. In some embodiments, the GPRC5D-related disease is cancer or an autoimmune disease, and the cancer is preferably a plasma cell malignancy disease, such as multiple myeloma, or a B-cell malignancy, such as Hodgkin's lymphoma or non-Hodgkin's lymphoma.

[0021] According to another aspect, the present specification provides a method for treating a GPRC5D-related disease, comprising administering to a subject in need thereof an effective amount of the above-mentioned GPRC5D-binding peptide, fusion protein, multispecific antibody molecule, immunoconjugate or pharmaceutical composition. In some embodiments, the GPRC5D-related disease is cancer or an autoimmune disease, and the cancer is preferably a plasma cell malignancy disease, such as multiple myeloma, or a B-cell malignancy, such as Hodgkin's lymphoma or non-Hodgkin's lymphoma.

[0022] According to another aspect, the present disclosure provides a method for producing a method for manufacturing a method for a semiconductor device comprising: 1) contacting the biological sample with the above-mentioned GPRC5D-binding peptide, fusion protein, or multispecific antibody molecule to form a binding complex with the GPRC5D; and step 2) determining the amount of GPRC5D in the biological sample based on the amount of the binding complex produced in step 1).

[0023] According to another aspect, the present disclosure provides a method for producing a method for manufacturing a method for a semiconductor device comprising: Step 1) linking the GPRC5D-binding peptide to a detectable tag; 2) contacting the GPRC5D-binding peptide having the detectable tag with the biological sample; and 3) determining the amount of GPRC5D in the biological sample by detecting the amount of the detectable tag.

[0024] In another aspect, the present disclosure provides a detection kit comprising the above-described GPRC5D-binding peptide, fusion protein or multispecific antibody molecule. In another aspect, the present disclosure provides a pharmaceutical kit comprising the above-described GPRC5D-binding peptide, fusion protein or multispecific antibody molecule.

[0025] According to another aspect, the present disclosure provides a method for producing a method for manufacturing a method for a semiconductor device comprising: Step 1) determining the amount of GPRC5D in a biological sample derived from the subject using the above detection kit; and 2) comparing the amount of GPRC5D present in the sample with a normal amount of GPRC5D present in a population to determine whether or not the GPRC5D-related disease is present or its status.

[0026] In some embodiments, the GPRC5D-related disease is cancer or an autoimmune disease, preferably a plasma cell malignancy disease such as multiple myeloma, or a B-cell malignancy disease such as Hodgkin's lymphoma or non-Hodgkin's lymphoma.

[0027] According to another aspect, the present disclosure provides a method for producing a method for manufacturing a method for a semiconductor device comprising: 1) identifying a GPRC5D-related disease status of said subject using the method described above; 2) treating the subject with the drug; The present invention provides a method for determining the therapeutic effect of a drug for treating a GPRC5D-related disease in a subject, comprising: repeating step 1) to identify changes in the state of the GPRC5D-related disease in the subject, and step 3) to determine the therapeutic effect of the drug based on the changes.

[0028] In some embodiments, the GPRC5D-related disease is cancer or an autoimmune disease, and the cancer is preferably a plasma cell malignancy disease, such as multiple myeloma, or a B-cell malignancy, such as Hodgkin's lymphoma or non-Hodgkin's lymphoma.

[0029] According to another aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising one or more antibody molecules or antigen-binding fragments thereof, the extracellular antigen-binding domain of which targets GPRC5D, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of said antibody molecule are the sequence of HCDR1 is GGSFSGYY (SEQ ID NO:1); the sequence of HCDR2 is INHSGST (SEQ ID NO:2); Combination 1) in which the HCDR3 sequence is ARARRYGGRTRFDP (SEQ ID NO:3); the sequence of HCDR1 is GFIFSSYG (SEQ ID NO:4); the sequence of HCDR2 is ISSSGDYT (SEQ ID NO:5); Combination 2) in which the HCDR3 sequence is ARMSFRRYDH (SEQ ID NO:6); and the sequence of HCDR1 is GFSFSGYI (SEQ ID NO:7); the sequence of HCDR2 is TSSSGTET (SEQ ID NO:8); 3) in which the sequence of HCDR3 is ARYYSKYGRSYHVDS (SEQ ID NO:9).

[0030] In some embodiments, the extracellular antigen-binding domain comprises a plurality (e.g., two) of said antibody molecules or antigen-binding fragments thereof linked in tandem, preferably wherein said antibody molecules or antigen-binding fragments thereof are linked via a linker peptide. In some embodiments, the two antibody molecules or antigen-binding fragments thereof are the same or different.

[0031] In some embodiments, the antibody molecule is a single domain antibody. In some embodiments, the antibody molecule is a fully human single domain antibody.

[0032] In some embodiments, the heavy chain variable region (or the single domain antibody) comprises an amino acid sequence set forth in SEQ ID NO:10, 11 or 12, or the heavy chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:10, 11 or 12, and is capable of specifically binding to GPRC5D.

[0033] In some embodiments, the CAR comprises, in order from N-terminus to C-terminus, the extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR further comprises a signal peptide at the N-terminus. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO:17.

[0034] In some embodiments, the CAR further comprises a hinge region between the extracellular antigen binding domain and the transmembrane domain. In some embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO:19. In some embodiments, the transmembrane region comprises the amino acid sequence set forth in SEQ ID NO:21.

[0035] In some embodiments, the intracellular signaling domain comprises a 4-1BB intracellular domain and a CD3 ζ intracellular domain. In some embodiments, the 4-1BB intracellular domain comprises the amino acid sequence set forth in SEQ ID NO:23. In some embodiments, the CD3ζ intracellular domain comprises the amino acid sequence set forth in SEQ ID NO:25.

[0036] In some embodiments, the end of the CAR is further linked to a safety switch for CAR-T cell clearance. In some embodiments, a safety switch is linked to the C-terminus of the CAR. In some embodiments, the safety switch comprises tEGFR, or a fusion protein with suicide capabilities.

[0037] In some embodiments, a truncated EGFR molecule (tEGFR) is linked to the C-terminus of the CAR. In some embodiments, the CSF2RA signal peptide is further linked to the N-terminus of tEGFR.

[0038] In some embodiments, the tEGFR comprises the amino acid sequence set forth in SEQ ID NO:31. In some embodiments, the CSF2RA signal peptide comprises the amino acid sequence set forth in SEQ ID NO:29.

[0039] In some embodiments, the safety switch is linked to the CAR via a self-cleaving polypeptide. In some embodiments, the self-cleaving polypeptide comprises the amino acid sequence set forth in SEQ ID NO:27 or 33.

[0040] In some embodiments, the CAR has an amino acid sequence set forth in SEQ ID NO:35, 37, or 39, or the amino acid sequence of the CAR has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:35, 37, or 39.

[0041] According to another aspect, the present disclosure provides a bispecific chimeric antigen receptor (CAR), the extracellular antigen-binding domain of which comprises (i) an antibody molecule or antigen-binding fragment thereof that targets GPRC5D, and (ii) an antibody molecule or antigen-binding fragment thereof that targets a second target, wherein the single domain antibody heavy chain variable region (V HH ) and the complete anti-scFv (variable region) are as described above, and said second antibody molecule is selected from CD3, BCMA or a combination thereof.

[0042] According to another aspect, the present specification further provides an isolated nucleic acid molecule encoding the above-mentioned CAR. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in any one of SEQ ID NOs:13, 14, 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.

[0043] According to another aspect, the present disclosure further provides an expression vector comprising the above-described nucleic acid molecule. In some embodiments, the expression vector is selected from a plasmid, a retroviral vector, and a lentiviral vector.

[0044] According to another aspect, the present specification further provides a host cell comprising the above-mentioned CAR, nucleic acid molecule or expression vector. In some embodiments, the host cell is an immune cell. In some embodiments, the host cells comprise T cells or NK cells, preferably 293T cells.

[0045] According to another aspect, the present specification further provides an engineered immune cell expressing the above-mentioned CAR. In some embodiments, the modified immune cells comprise immune cells, preferably T cells or NK cells. In some embodiments, the modified immune cells comprise autologous or allogeneic cells.

[0046] According to another aspect, the present specification further provides a pharmaceutical composition comprising the above-mentioned cells and a pharma- ceutically acceptable carrier. According to another aspect, the present specification further provides a use of the above-mentioned CAR, nucleic acid molecule, expression vector, host cell, or modified immune cell in the preparation of a medicament for preventing or treating a GPRC5D-related disease.

[0047] In some embodiments, the GPRC5D-related disease is cancer or an autoimmune disease, preferably a plasma cell malignancy disease such as multiple myeloma, or a B-cell malignancy disease such as Hodgkin's lymphoma or non-Hodgkin's lymphoma.

[0048] According to another aspect, the present specification further provides a method for preventing or treating a GPRC5D-associated disease, comprising administering an effective amount of the modified immune cell or pharmaceutical composition to a subject in need thereof to treat the same. In some embodiments, the method further comprises inhibiting activity of the engineered immune cell or the pharmaceutical composition by administering to the subject an EGFR antibody.

[0049] In some embodiments, the GPRC5D-related disease is cancer or an autoimmune disease, preferably a plasma cell malignancy disease such as multiple myeloma, or a B-cell malignancy disease such as Hodgkin's lymphoma or non-Hodgkin's lymphoma.

[0050] Based on the highly specific expression profile of GPRC5D, its potential safety, and the fact that BCMA therapy has already achieved good therapeutic effects, this application has developed a CAR-T product targeting GPRC5D for the treatment of refractory, relapsed and multiple myeloma. The GPRC5D binding domain was developed by screening a humanized single-domain antibody library, and has a relatively simple structure and low immunogenicity. [Brief description of the drawings]

[0051] [Figure 1]The results of enzyme-linked immunosorbent assay (ELISA) of some of the panned phage monoclonals with target and control antigens are shown. [Figure 2A] This shows the flow cytometry analysis results of CHO-K1-GPRC5D cell line stained with different antibodies. Human GPRC5D PE-conjugated Antibody stained is the staining result using a commercially available antibody for flow cytometry, Fluorescein (FITC) AffiniPure Goat Anti-Human IgG stained is a negative control stained with only fluorescently labeled antibody, and Positive benchmark stained / Fluorescein (FITC) AffiniPure Goat Anti-Human IgG stained is the staining result using a positive control antibody and a fluorescently labeled antibody. [Figure 2B] The results of flow cytometry analysis of the binding of some phage monoclonals to CHO-K1-GPRC5D and CHO-K1 cells are shown. [Diagram 3] The results of enzyme-linked immunosorbent assay analysis of multiple unrelated antigens at the phage level for the screened phage monoclonals are shown. Negative phage control is a negative control phage antibody clone, Anti-M13 phage mouse / anti-mouse HRP Ab is a negative antibody control with only primary and secondary antibodies added, anti-mouse HRP Ab is a negative antibody control with only secondary antibodies added, anti-human IgG HRP Ab is a negative antibody control with only secondary antibodies added, anti-his tag HRP Ab is a positive antibody control to detect the antigen tag, and Positive Benchmark 1 is a positive antibody control for the target antigen. [Figure 4]Flow cytometry analysis of the screened phage monoclonals binding to several different GPRC5D positive and negative cell lines at the phage level. Negative phage control is a negative control phage antibody clone, Positive Benchamrk1 Ab is a positive antibody for the target antigen, and FITC anti-human IgG Ab is a negative control with only secondary antibody added. [Diagram 5] The results of enzyme-linked immunosorbent assay analysis of multiple unrelated antigens at the protein level of the screened phage monoclonals are shown. Anti-human IgGHRP Ab is a negative antibody control with only secondary antibody added, anti-his tagHRP Ab is a positive antibody control detecting the antigen tag, and Positive Benchamrk1 is a positive antibody control for the target antigen. [Figure 6] Flow cytometry analysis of the screened phage monoclonals binding to multiple different GPRC5D positive and negative cell lines at the protein level is shown. Positive Benchmark1 Ab is the positive antibody for the target antigen, and FITC anti-human IgG Ab is the negative control with only the secondary antibody added. [Figure 7] 1 shows the results of an experiment in which FACS was used to detect binding between a monoclonal antibody and CHO-K1-GPRC5D cells, which highly express a target antigen. [Figure 8] The results of an experiment in which FACS was used to detect the binding of the corresponding monoclonal antibodies to monkey mouse-derived highly expressing GPRC5D cells (ie, CHOS-macaque GPRC5D (FIG. 8A) and CHOS-mouse GPRC5D (FIG. 8B)), respectively, are shown. [Figure 9]A schematic diagram of the structure of GPRC5D CAR vector construction is shown. CAR contains an extracellular signal peptide (SP), ScFv (VL-linker-VH) or single domain (VHH) of the target antigen GPRC5D binding domain, a hinge region and a transmembrane region (CD8ahinge+TM) between the cell membrane and the extracellular binding domain, a 4-1BB costimulatory molecule, a CD3ζ intracellular domain, a T2A self-cleaving peptide, and a truncated tEGFR. [Figure 10] The expression status of tEGFR in each CAR-T cell group is shown. [Figure 11] The killing results of each CAR-T cell group against various target cells (8226-luc, U266-luc, MM1.s-luc, H929-luc, CCRF-luc, Raji-luc) are shown. [Figure 12] The in vitro proliferation status of each CAR-T after multiple stimulation of target cells (8226) is shown. [Figure 13] The results of the CD107a degranulation effect of tumor cells derived from different tissues on each group of CAR-T cells are shown. [Figure 14] Figure 14 shows the antitumor efficacy of CAR-T cells corresponding to different clones in H929-luc tumor-bearing NCG mice. Figure 14A shows the time course of photon counts after reinfusion of CAR-T cells, Figure 14B shows the survival rate of mice reinfused with CAR-T cells, and Figure 14C shows the percentage change in mouse lymphocytes of T cells after reinfusion of CAR-T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Unless otherwise explained, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0053] An antibody refers to an immunoglobulin secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (polypeptides, viruses, bacteria, etc.), which are accordingly called antigens. The basic structure of a classical antibody molecule is a tetramer consisting of two identical heavy chains and two identical light chains. Based on conservative differences in amino acid sequences, the heavy and light chains are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxy terminus. The variable regions of one heavy chain and one light chain interact with each other to form an antigen-binding site (Fv). Among the variable regions, certain regions are more variable in the composition and sequence of amino acid residues than other regions (framework regions, FR) in the variable region, and are called hypervariable regions (HVRs), which are actually the important sites where antibodies bind to antigens. These hypervariable region sequences are complementary to antigenic determinants, and are therefore also called complementarity-determining regions (CDRs). Each heavy chain and light chain has three complementarity determining regions, designated HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, respectively.

[0054] An "antigen-binding fragment" of an antibody molecule refers to an amino acid fragment of the antibody molecule that is involved in specific binding to an antigen, such as Fab, Fab', and (Fab')2. Single-chain antibodies and single-domain antibodies that have antigen-binding ability are single peptide-chain antibody molecules and can be considered as "antigen-binding fragments" of classical antibody molecules.

[0055] "Fc fragment" refers to the stem region of a Y"-shaped antibody molecule, i.e., the fragment crystallizable (Fc) region includes the second and third constant domains of the heavy chain (CH2 and CH3 domains). The antibody Fc region can be obtained by hydrolysis of the antibody molecule with a protease (such as papain). In some instances, the Fc region may include a hinge, CH2, and CH3. If the Fc region includes a hinge, it can mediate dimerization between two Fc-containing polypeptides. The Fc fragment may be derived from IgG, IgM, IgD, IgE, or IgA. In some instances, the Fc region is derived from IgG1, IgG2, IgG3, or IgG4. "Fc fragment" also includes mutant Fc fragments derived from wild-type Fc fragments that have been modified while still maintaining their effector functions. "Mutant Fc fragments" include an amino acid sequence having at least one amino acid modification on the amino acid sequence of the wild-type Fc fragment. In some examples, the variant Fc fragment has at least one amino acid substitution compared to the parent Fc fragment (wild-type Fc fragment), for example, about 1 to about 10 amino acids in the parent Fc fragment are substituted, and preferably about 1 to about 5 amino acids are substituted. In some examples, the variant Fc fragment Fc region has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the parent Fc fragment. The effector function of the "Fc fragment" may include binding to an Fc receptor, binding to Clq, and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), mediation of phagocytosis, and the like.

[0056] Single chain antibodies (single chain fragment variable, scFv) are composed of the heavy and light chain variable regions of an antibody linked into a single peptide chain by a short peptide. By correctly folding, the variable regions from the heavy and light chains form an Fv segment through non-covalent interactions, so that scFv better retains its affinity activity for the antigen.

[0057] "Single domain antibody (sdAb)" or "V HH The term "antibody" refers to an antibody molecule that has antigen-binding ability and contains a heavy chain variable region without a light chain. From the structure, a single domain antibody can also be considered as an antigen-binding fragment of an antibody molecule. It was first discovered in camelids, and then researchers discovered more single domain antibodies with antigen-binding ability by screening antibody libraries (such as phage display libraries). Single domain antibodies have advantages over regular antibody molecules (e.g., classical tetrameric antibody molecules) or antigen-binding fragments thereof, including, but not limited to, smaller molecular weight, easier to reach tissues or sites that regular antibody molecules have difficulty in reaching when used in humans, or can contact epitopes in proteins or polypeptides that regular antibody molecules have difficulty in reaching, high stability, and resistance to temperature and pH changes and the action of denaturants and proteases, etc.

[0058] A "fully human antibody" refers to an antibody in which both the variable region and the constant region (if present) are derived from human germline immunoglobulin sequences. Fully human antibodies can be obtained by various techniques, including phage antibody library technology, single B cell clone technology, transgenic mouse technology (e.g., using transgenic mice into which human germline immunoglobulin genes have been introduced and from which the mouse's own germline immunoglobulin genes have been removed), and the like. Compared with animal-derived antibodies (e.g., mouse-derived antibodies), fully human antibodies have the advantage of being less immunogenic and more safe when used in human patients.

[0059] A "bispecific antibody" refers to an antibody molecule that has two different binding sites that can recognize and bind two different antigens, respectively. For example, one binding site of the bispecific antibody may be used to bind to an immune cell (e.g., a T cell) and the other binding site may be used to bind to a tumor cell, thereby enhancing the killing effect of the immune cell against the tumor cell and reducing side effects such as off-target toxicity. Such dual-function antibodies generally have a higher therapeutic effect as drugs to treat tumors than monoclonal antibody drugs. Similarly, antibody molecules can be engineered to contain multiple different binding sites to generate "trispecific antibodies," "tetraspecific antibodies," and the like. As used herein, "multispecific antibodies" encompass such bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and the like.

[0060] "Immunoconjugate" refers to a GPRC5D-binding peptide disclosed herein linked to a therapeutic agent, such as a cytotoxic agent, a drug (e.g., an immunosuppressant), or a radiotoxic agent.

[0061] A "fusion protein" refers to a protein molecule that is composed of at least two different peptide segments and is artificially created (e.g., by genetic engineering techniques). These peptide segments do not occur in nature or in the same protein molecule. Common examples of fusion proteins that include antibody fragments include antibody-cytokine fusion proteins, antibody-cytotoxin proteins (also called immunotoxins), enzyme-labeled antibodies used in immunoassays, chimeric antigen receptors (CARs), etc.

[0062] "Target" or "specifically binds" means that a molecule (e.g., an antibody or antigen-binding fragment thereof) has a higher binding affinity for another molecule (such as a tumor cell surface antigen) compared to other molecules present at the same time in the environment. "Target" or "specifically binds" does not exclude that the molecule may have binding affinity for more than one molecule, for example, a bispecific antibody may have high affinity for two different antigens.

[0063] A "binding complex" refers to a complex formed between a molecule and its binding target, including both. Examples of binding complexes include antigen-antibody complexes, ligand-receptor complexes, and protein dimers. The mechanisms that form binding complexes mainly involve non-covalent forces such as hydrogen bonds, van der Waals bonds, and ionic bonds.

[0064] GPRC5D is a G protein-coupled receptor C5 family subtype D, belonging to the orphan receptors, and a seven-transmembrane protein. An orphan receptor refers to a receptor that has a clear structural similarity to other already identified receptors, but whose endogenous ligand has not yet been discovered. GPRC5D is highly expressed on the surface of primary multiple myeloma cells, and its expression in normal tissues is restricted to the hair follicle region. Studies have shown that GPRC5D in 65% of multiple myeloma patients has an expression threshold of more than 50%, making GPRC5D a potential target for treating multiple myeloma (MM).

[0065] "Disease state" refers to the presence or absence of a disease, the severity of the disease, the stage, separation, progression, and the like of the disease. A GPRC5D-binding peptide refers to a polypeptide that binds GPRC5D. For example, the GPRC5D-binding peptide is a single domain antibody that specifically targets GPRC5D. A "peptide segment" refers to a polypeptide fragment, which is a short amino acid sequence, for example, about 2 to 20 amino acids in length, and is typically a portion of a polypeptide or protein.

[0066] EC50 (concentration for 50% of maximal effect) refers to the concentration that causes 50% of the maximal effect. In FACS, when used to indicate the ability of an antibody molecule to bind to the corresponding antigen on a cell, it may refer to the concentration of the antibody molecule that produces half of the maximum fluorescence intensity. The lower the EC50 value, the higher the binding affinity to the antigen on the cell.

[0067] The term "purification tag" refers to an amino acid sequence that is expressed together with a protein or polypeptide of interest in the form of a fusion protein used to purify the protein or polypeptide of interest, and includes, but is not limited to, His6 tag, Flag tag, MBP (maltose binding protein) tag, GST (glutathione S-transferase) tag, SUMO (small ubiquitin related modifier), etc. These tags can be removed by enzymatic cleavage after purification, or can be used with a tag (e.g., His6 tag) without affecting the normal function of the protein or polypeptide of interest.

[0068] A "detectable tag" refers to a sequence of amino acids or other chemical groups attached to a protein or polypeptide that is used to indicate the presence or amount of the protein or polypeptide in a sample or to track the location of the protein or polypeptide within the body or cells of a subject. Examples of detectable tags include various enzymes that can be used in immunoassays, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), fluorophores (FAM, FITC, etc.) or fluorescent proteins (GFP, etc.), radioisotopes ( 3 H, 14 C. 35 When the detectable tag is an enzyme, the presence or amount of a protein or polypeptide bound to the enzyme can be determined by the enzymatic activity of the enzyme.

[0069] The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, including, but not limited to, peptides, oligopeptides, dimers, trimers and multimers composed of amino acid residues. Both full-length proteins and fragments thereof are encompassed in this sense. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation and similar modifications. Furthermore, for the purposes of the present invention, "polypeptide" refers to a protein containing modifications to the native sequence, such as deletions, additions and substitutions (which are usually conservative in practice), so long as the protein retains the desired activity. These modifications may be deliberate, as by site-directed mutagenesis, or may be accidental, such as by mutation of the host producing the protein or by errors due to PCR amplification.

[0070] As used herein, "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably and refer to a nucleotide polymer. Such nucleotide polymers may contain natural and / or non-natural nucleotides, including (but are not limited to) DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0071] The term "vector" refers to a nucleic acid molecule (e.g., a nucleic acid, a plasmid, or a virus, etc.) that can be engineered to contain a polynucleotide of interest (e.g., a coding sequence for a polypeptide of interest) or that can replicate in a host cell. A vector may include one or more of the following components: an origin of replication, one or more regulatory sequences (e.g., a promoter and / or enhancer) that control the expression of the polynucleotide of interest, and / or one or more selectable marker genes (antibiotic resistance genes and genes useful in colorimetric assays, e.g., β-galactose). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0072] "Host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. A host cell may be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells, fungal cells, such as yeast, plant cells, and insect cells. Non-limiting exemplary mammalian cells include (but are not limited to) NSO cells, 293 and CHO cells, and their derived cells, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in terms of morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations. A host cell further includes cells transfected in vivo with a nucleic acid molecule or expression vector provided herein.

[0073] "Treatment" refers to a procedure performed to obtain a beneficial or desired clinical outcome for a subject. As used herein, "treatment" encompasses a variety of therapeutic procedures, including administration of any possible drug to a subject, surgery, radiation, etc. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the extent of disease, prevention or delay of disease spread (e.g., metastasis, e.g., to the lungs or lymph nodes), prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of disease status, inhibition of disease or disease progression, delay of disease progression, and remission (whether partial or complete remission). The methods provided herein encompass any one or more of these therapeutic aspects. In accordance with the above, "treatment" does not require complete elimination or complete remission of all symptoms of a disorder or disease.

[0074] The term "therapeutically effective amount" refers to an amount of active compound sufficient to induce in a subject the biological or medical response desired by the clinician. The "therapeutically effective amount" of the fusion protein of the present invention may be determined by one of skill in the art depending on factors such as the route of administration, the weight, age, condition of the subject, etc. For example, a typical daily dose may range from 0.01 mg to 100 mg or more of active ingredient per kg of body weight.

[0075] The term "pharmaceutical acceptable carrier" as used in reference to pharmaceutical compositions refers to a solid or liquid diluent, filler, antioxidant, stabilizer, and other substance that can be safely administered, which is suitable for administration to humans and / or animals without undue adverse side effects and is suitable for maintaining the activity of the drug or active agent therein. Depending on the route of administration, a variety of different vectors well known in the art can be administered, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer, emulsifiers, isotonic saline, and / or pyrogen-free water. The pharmaceutical compositions provided herein can be formulated into clinically acceptable dosage forms, such as powders, injections, and the like. The pharmaceutical compositions of the present invention can be administered to a subject using any suitable route, for example, orally, intravenously, intramuscularly, subcutaneously, intraperitoneally, rectally, sublingually, or by inhalation, transdermally, and the like.

[0076] "Subject" refers to an animal, for example, but not limited to, a mammal, including but not limited to humans, rodents, monkeys, felines, canines, equines, bovines, hogs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sport animals, and mammalian pets. A subject may be male or female, and may be of any age, including children, juveniles, adolescents, adults, and geriatric subjects. In some examples, a subject refers to an individual who needs treatment for a disease or disorder. In some examples, a subject to be treated may be a patient who has a disorder related to the treatment or is at risk of developing the disorder. In certain examples, a subject is a human, such as a human patient. The term is generally used interchangeably with "patient", "subject to be detected", "subject to be treated", and the like.

[0077] As used herein, "population" generally refers to a healthy population. When performing a specific analysis for a disease, "population" can also refer to individuals who do not suffer from the disease but have other diseases. Also, a portion of individuals can be designated as a "population" according to characteristics such as age, smoking status, alcoholism status, and individual health status. "Normal GPRC5D content" in a population can be determined by measuring a sufficient number of individuals.

[0078] "Biological sample" means a quantity of a substance derived from a living or formerly living organism, including (but not limited to) blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0079] The term "sequence identity" (also called "sequence identity"), when referring to amino acid or nucleotide sequences, refers to the amount of the degree of identity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Generally, before calculating the percentage of identity between two amino acid or nucleotide sequences, sequence alignment is performed and gaps (if any) are introduced. Two sequences are considered to be identical or matched at an aligned position if the amino acid residue or base in the two sequences is identical at that position, and are considered to be identical or mismatched at that position if the amino acid residue or base in the two sequences is different. In some algorithms, the number of matched positions is divided by the total number of positions in the alignment window to obtain the sequence identity. In other algorithms, the number of gaps and / or the length of the gap are also taken into account. For the purposes of the present invention, the publicly available alignment software BLAST (available at the web page ncbi.nlm.nih.gov) can be used to obtain optimal sequence alignments and calculate sequence identity between two amino acid or nucleotide sequences by using default settings.

[0080] Chimeric antigen receptors (CARs), also called chimeric T cell receptors and chimeric immune receptors, are engineered protein receptor molecules that can confer desired specificity, such as the ability to bind to a specific tumor antigen, to immune effector cells. Chimeric antigen receptors generally consist of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In most cases, the antigen-binding domain is a scFv sequence that is responsible for the recognition and binding of a specific antigen. The intracellular signaling domain generally includes a signaling domain derived from an immunoreceptor tyrosine-based activation motif (ITAM), such as the CD3ζ molecule, which is responsible for the activation of immune effector cells and produces a killing effect. In addition, chimeric antigen receptors can also include a signal peptide at the amino terminus that is responsible for the intracellular localization of the nascent protein, as well as a hinge region between the antigen-binding domain and the transmembrane domain. In addition to the signaling domain, the intracellular signaling domain may further include a co-stimulatory domain, such as that derived from the 4-1BB molecule.

[0081] "CAR-T cells" refers to T cells expressing a CAR, and are generally obtained by transducing T cells with an expression vector encoding a CAR. Common expression vectors are viral vectors, such as lentiviral expression vectors. Chimeric antigen receptor modified T cells (CAR-T) have the ability of specific targeted killing activity and sustained proliferation without being restricted by major histocompatibility complexes. In addition to T cells, expression vectors encoding a CAR can also be used to transform other lymphocytes, such as NK cells, to obtain targeted killer cells expressing the CAR.

[0082] "Self-cleaving peptide" refers to a short peptide that can undergo ribosomal skipping rather than protease hydrolysis to achieve the function of the cleaved protein, and may include T2A, F2A, P2A, and the like. The "CSF2RA signal peptide", i.e., colony stimulating factor 2 receptor subunit alpha signal peptide, can induce the expression of newly synthesized proteins such as EGFRt on the surface of CAR-T cells.

[0083] "EGFRt" or "tEGFR" are used interchangeably herein and refer to a gene encoding a truncated human epidermal growth factor receptor polypeptide or its coding product, lacking the distal membrane EGF-binding domain and the cytoplasmic signaling tail, but retaining the extracellular epitope recognized by anti-EGFR antibodies. EGFRt can be used as a non-immunogenic selection tool and tracking marker with genetically modified cell functions. Herein, it can act, on the one hand, as a marker molecule for CAR-T cells, and, on the other hand, can be used to eliminate CAR-T cells in vivo, if necessary, for example, by the ADCC pathway mediated by EGFR antibodies (e.g., cetuximab) (see US8802374B2), i.e., as a safety switch upon clinical transformation.

[0084] In addition to use with tEGFR, the CAR-T cells provided herein are also contemplated for use in combination with other mechanisms of safety switches. For example, the CARs provided herein can be engineered to control CAR activity using a chemical dimerization inducer (CID). In one example, the complete CARs provided herein can be expressed as separate fusion proteins (e.g., CD3ζ intracellular domain is expressed separately from other portions) and the complete CAR can be reconstituted in cells using a dimerization mechanism based on FKBP / FRB binding domains and rapamycin (or a rapamycin analog), thereby controlling the integrity and activity of the CAR depending on whether rapamycin is present (see, for example, the description in U.S. Pat. No. 11,084,880). Furthermore, when a self-cleaving peptide is used to allow for the co-expression of a fusion protein with suicide capabilities, CAR-T cell activity can be controlled by causing degradation of the CAR molecule or CAR-T cell death. Examples of this embodiment include degradation of the CAR molecule using PROTAC technology and the use of proteins such as caspase-9 that have the ability to induce apoptosis. [8] .

[0085] Brief description of amino acid and nucleotide sequences A description of the amino acid and encoding nucleic acid sequences of several antibodies or antigen-binding fragments (e.g., single domain antibodies) and CARs and related polypeptides used herein (particularly in the Examples) is provided below.

[0086] SEQ ID NO:1 is the amino acid sequence of CDR1 of clone #18, i.e., CDR1 of the extracellular antigen-binding domain of GPRC5D CAR 2290. SEQ ID NO:2 is the amino acid sequence of CDR2 of clone #18, i.e., the CDR2 of the extracellular antigen-binding domain of GPRC5D CAR 2290. SEQ ID NO:3 is the amino acid sequence of the CDR3 of the #18 clone, i.e., the CDR3 of the extracellular antigen-binding domain of GPRC5D CAR 2290.

[0087] SEQ ID NO:4 is the amino acid sequence of CDR1 of clone #39, i.e., CDR1 of the extracellular antigen-binding domain of GPRC5D CAR 2436. SEQ ID NO:5 is the amino acid sequence of CDR2 of clone #39, i.e., the CDR2 of the extracellular antigen-binding domain of GPRC5D CAR 2436. SEQ ID NO:6 is the amino acid sequence of the CDR3 of the #39 clone, i.e., the CDR3 of the extracellular antigen-binding domain of GPRC5D CAR 2436.

[0088] SEQ ID NO:7 is the amino acid sequence of CDR1 of the #41 clone, i.e., CDR1 of the extracellular antigen-binding domain of GPRC5D CAR 2438. SEQ ID NO:8 is the amino acid sequence of CDR2 of the #41 clone, i.e., the CDR2 of the extracellular antigen-binding domain of GPRC5D CAR 2438. SEQ ID NO:9 is the amino acid sequence of the CDR3 of the #41 clone, i.e., the CDR3 of the extracellular antigen-binding domain of GPRC5D CAR 2438.

[0089] SEQ ID NO:10 is the amino acid sequence of the heavy chain variable region of clone #18, i.e., the sequence of the extracellular antigen-binding domain of GPRC5D CAR 2290. SEQ ID NO:11 is the amino acid sequence of the heavy chain variable region of clone #39, i.e., the sequence of the extracellular antigen-binding domain of GPRC5D CAR 2436. SEQ ID NO:12 is the amino acid sequence of the heavy chain variable region of the #41 clone, i.e., the sequence of the extracellular antigen-binding domain of GPRC5D CAR 2438.

[0090] SEQ ID NO:13 is the heavy chain variable region coding sequence of clone #18, i.e., the coding sequence of the extracellular antigen-binding domain of GPRC5D CAR 2290. SEQ ID NO:14 is the coding sequence of the heavy chain variable region of clone #39, i.e., the coding sequence of the extracellular antigen-binding domain of GPRC5D CAR 2436. SEQ ID NO:15 is the coding sequence of the heavy chain variable region of the #41 clone, i.e., the coding sequence of the extracellular antigen-binding domain of GPRC5D CAR 2438.

[0091] SEQ ID NO:16 is the coding nucleic acid sequence of CD8α signal peptide. CD8α signal peptide can be used to direct newly synthesized CAR to the cell surface. The signal peptide in mature CAR may be excised, and therefore may not contain the signal peptide. SEQ ID NO:17 is the amino acid sequence of the CD8α signal peptide.

[0092] SEQ ID NO:18 is the coding nucleic acid sequence of the CD8α hinge region. The CD8α hinge region can be used to link the extracellular antigen binding domain and the transmembrane domain in the CAR. SEQ ID NO:19 is the amino acid sequence of the CD8α hinge region.

[0093] SEQ ID NO:20 is the coding nucleic acid sequence of the CD8α transmembrane domain. SEQ ID NO:21 is the amino acid sequence of the CD8α transmembrane domain.

[0094] SEQ ID NO:22 is the coding nucleic acid sequence for the 4-1BB intracellular domain. SEQ ID NO:23 is the amino acid sequence of the 4-1BB intracellular domain.

[0095] SEQ ID NO:24 is the coding nucleic acid sequence for the CD3 zeta intracellular signaling domain. SEQ ID NO:25 is the amino acid sequence of the CD3 zeta intracellular signaling domain.

[0096] SEQ ID NO:26 is the coding nucleic acid sequence for the self-cleaving peptide T2A. SEQ ID NO:27 is the amino acid sequence of the self-cleaving peptide T2A. Due to the presence of the self-cleaving peptide, the proteins (CAR and EGFRt) on both sides of the self-cleaving peptide are separated from each other during translation. For convenience of explanation, the CAR that contains multiple elements as a fusion protein can be considered to also contain T2A and EGFRt.

[0097] SEQ ID NO:28 is the coding nucleic acid sequence for the CSF2RA signal peptide. SEQ ID NO:29 is the amino acid sequence of the CSF2RA signal peptide. It is located upstream of EGFRt and can be used to direct newly synthesized EGFRt to the cell surface. The CSF2RA signal peptide can be cleaved off from EGFRt.

[0098] SEQ ID NO:30 is the coding nucleic acid sequence of EGFRt. SEQ ID NO:31 is the amino acid sequence of EGFRt.

[0099] SEQ ID NO:32 is a coding nucleic acid sequence for the self-cleaving peptide P2A, and the self-cleaving peptide P2A can be selected to replace T2A as the linker peptide between CAR and EGFRt. SEQ ID NO:33 is the amino acid sequence of the self-cleaving peptide P2A.

[0100] SEQ ID NO:34 is the coding sequence of GPRC5D CAR 2290(#18). SEQ ID NO:35 is the amino acid sequence of GPRC5D CAR 2290(#18).

[0101] SEQ ID NO:36 is the coding sequence for GPRC5D CAR 2436 (#39). SEQ ID NO:37 is the amino acid sequence of GPRC5D CAR 2436 (#39).

[0102] SEQ ID NO:38 is the coding sequence of GPRC5D CAR 2438(#41). SEQ ID NO:39 is the amino acid sequence of GPRC5D CAR 2438 (#41).

[0103] The sequences shown in SEQ ID NOs:34 to 39 are only the codes or amino acid sequences for the CAR structure and do not include the EGFRt element. Furthermore, the above clones #18, #39 and #41 are examples of specific single domain antibodies targeting GPRC5D for CAR construction provided by the present application.

[0104] GPRC5D-binding peptide The present specification provides a GPRC5D binding peptide that specifically binds to GPRC5D. The GPRC5D binding peptide binds to GPRC5D molecules with relatively high binding affinity. The GPRC5D may be a fusion protein linked to other polypeptides, such as GPRC5D-VLP, or may be a GPRC5D membrane protein expressed on the cell surface. For example, as described in the following examples, the binding ability of the GPRC5D binding peptide to GPRC5D may be measured by a measurement method such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry fluorescence sorting technology (FACS). In addition, it may be measured by other protein interaction measurement methods known in the art, such as surface plasmon resonance (SPR) biolayer interference (BLI) technology.

[0105] In some embodiments, the GPRC5D-binding peptide is an antibody molecule or an antigen-binding fragment thereof. Preferably, the GPRC5D-binding peptide is a single domain antibody. As described in the Examples herein, the GPRC5D-binding peptides provided herein are obtained from screening a human phage antibody library and are fully human single domain antibodies.

[0106] Single domain antibodies are antibodies that contain only a heavy chain variable region, examples of which include (but are not limited to) heavy chain antibodies, naturally occurring light chain depleted antibodies, single domain antibodies derived from classical four chain antibodies and engineered antibodies. Single domain antibodies may be derived from any species, including but not limited to mouse, human, camel, llama, alpaca, vicuna, guanaco, shark, goat, rabbit, and / or cow.

[0107] Provided herein are the CDR sequences of three GPRC5D-binding peptides, the sequences shown in SEQ ID NOs: 1-3, 4-6, and 7-9, respectively. Based on the CDR sequences of the GPRC5D-binding peptides (single domain antibodies) provided herein, those skilled in the art can construct each polypeptide construct having GPRC5D-binding ability, including using framework regions (FRs) from different antibody molecules in combination with these CDR sequences. These framework regions include natural framework region sequences from human antibodies or animal (e.g., mouse, rat, sheep, camel, etc.) antibodies. These framework regions may also include sequence variants of framework regions generated by sequence modification of natural framework regions. By combining the CDR sequences provided herein with different framework region sequences to form heavy chain variable regions and measuring the binding ability to GPRC5D, a polypeptide construct that specifically binds to GPRC5D can be easily obtained.

[0108] In some embodiments, the heavy chain variable region of a GPRC5D-binding peptide (single domain antibody) provided by the present invention comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99% or even 100% sequence identity) to the sequence shown in SEQ ID NO:10.

[0109] In some embodiments, the heavy chain variable region of the GPRC5D binding peptide (single domain antibody) provided by the present invention comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99% or even 100% sequence identity) to the sequence shown in SEQ ID NO:11.

[0110] In some embodiments, the heavy chain variable region of the GPRC5D binding peptide (single domain antibody) provided by the present invention comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99% or even 100% sequence identity) to the sequence shown in SEQ ID NO:12.

[0111] As will be understood by those skilled in the art, based on the specific sequences provided herein, by substituting, deleting, or adding a small number of amino acids and verifying or screening the binding ability or biological activity of the resulting product with the corresponding antigen GPRC5D, corresponding mutants of the antibody molecule targeting GPRC5D provided herein can be obtained, and these mutants should also be included in the scope of the present invention. For example, the antibody molecule or single domain antibody provided herein can have at least one and no more than 10, for example, no more than 5, 4, 3, 2, or 1 amino acid changes in its full length or variable region sequence or CDR sequence.

[0112] It is contemplated that the GPRC5D-binding peptides described herein may include conservative amino acid substitutions. Conservative amino acid substitutions may generally be described as the replacement of an amino acid residue with another amino acid residue of similar chemical structure, and have little or no effect on the function, activity or other biological properties of the polypeptide. Conservative amino acid substitutions are well known in the art. Conservative substitutions may be, for example, the replacement of one amino acid in the following groups (a) to (e) with another amino acid in the same group: (a) small aliphatic non-polar or weakly polar residues: Ala, ser, Thr, pro, and Gly; (b) polar negatively charged groups and their (uncharged) amides: Asp, Asn, Glu, and Gin; (c) polar positively charged groups: His, Arg, and Lys; (d) large aliphatic non-polar residues: Met, Leu, lie, Val, and CYS; (e) aromatic groups: Phe, Tyr, and Trpo.

[0113] The polypeptide construct may further comprise one or more GPRC5D binding peptides, which are directly linked in tandem or linked in tandem via a linker sequence. In some embodiments, the polypeptide construct comprises two identical GPRC5D binding peptides. In some embodiments, the polypeptide construct comprises two GPRC5D binding peptides with at least one amino acid difference in the amino acid sequence. In some embodiments, the polypeptide construct comprises at least one GPRC5D binding peptide provided herein and at least one other GPRC5D binding peptide. In some embodiments, the polypeptide construct comprises two different GPRC5D binding peptides provided herein. In some embodiments, the polypeptide construct comprises three different GPRC5D binding peptides provided herein.

[0114] The linker sequence may be a naturally occurring linker, a synthetic linker or a combination of both. Particularly suitable linker sequences mainly comprise amino acid residues selected from glycine (Gly), serine (Ser), alanine (Ala) and threonine (Thr). By way of example, the linker may comprise at least 75%, e.g. at least 80%, at least 85% or at least 90% (calculated based on the total number of residues present in the peptide linker) of amino acid residues selected from Gly, ser, Ala and Thr. The linker may also consist exclusively of Gly, Ser, Ala and / or Thr residues. In some embodiments, the linker comprises 1-25 glycine residues, 5-20 glycine residues, 5-15 glycine residues or 8-12 glycine residues. In some embodiments, suitable peptide linkers typically comprise at least 50% glycine residues, e.g. at least 75% glycine residues. In some embodiments, the peptide linker comprises only glycine residues. In some examples, the peptide linker may comprise, for example, (GS) n and n is an integer of 1 to 20, for example.

[0115] Fusion protein containing GPRC5D-binding peptide The above polypeptide construct containing multiple GPRC5D-binding peptides belongs to a fusion protein containing GPRC5D-binding peptides. Furthermore, the GPRC5D-binding peptide can form a fusion protein with another polypeptide.

[0116] In some embodiments, the GPRC5D-binding peptide can be linked to an Fc fragment to form a fusion protein. The Fc fragment can be located at the C-terminus and N-terminus of the GPRC5D-binding peptide. Preferably, the Fc fragment can be located at the C-terminus of the GPRC5D-binding peptide. The fusion protein formed by the GPRC5D-binding peptide and the Fc fragment has the ability to specifically bind to GPRC5D and has the effector function of the Fc fragment, such as complement-dependent cytotoxicity (CDC), mediation of antibody-dependent cellular cytotoxicity (ADCC), mediation of phagocytosis, etc. Furthermore, fusion with the Fc fragment increases the half-life of the GPRC5D-binding peptide in the body, thereby extending the administration interval when the GPRC5D-binding peptide is used as a therapeutic drug. Non-limiting fusion protein constructs comprising GPRC5D-binding peptides may include, but are not limited to, the forms VHH1-Fc, VHH1-VHH2-Fc and VHH1-VHH2-VHH3-Fc, where VHH refers to the GPRC5D-binding peptide (single domain antibody) and VHH1, VHH2 and VHH3 may be the same or different.

[0117] In some embodiments, the GPRC5D-binding peptide can be linked to a protein tag to form a fusion protein. The protein tag can include a purification tag and a detectable tag. The purification tag can include, but is not limited to, His6 tag, Flag tag, MBP tag, GST tag, SUMO tag, etc. The detectable tag can be used to indicate the presence or content of the GPRC5D-binding peptide in a sample, or can be used to track the location information of the GPRC5D-binding peptide in a subject or cell. Examples of detectable tags include various enzymes that can be used in immunoassays, such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP), and fluorescent proteins such as GFP. Due to the specific binding ability of the GPRC5D-binding peptide to GPRC5D, the amount of the GPRC5D-binding peptide can be determined by the amount of the detectable tag bound to the GPRC5D-binding peptide, thereby determining the content of GPRC5D in the sample.

[0118] In other embodiments, the GPRC5D-binding peptides can be linked to cytokines or therapeutic proteins to form fusion proteins, in which case the therapeutic effect of the cytokine or therapeutic protein can be achieved by targeted delivery of the cytokine or therapeutic protein to specific tissues or cells (e.g., tumor tissues expressing GPRC5D) via specific binding of the GPRC5D-binding peptide to GPRC5D.

[0119] Multispecific antibodies containing GPRC5D-binding peptides In some embodiments, the present disclosure provides a multispecific binding peptide comprising at least one domain (or functional unit) that binds to GPRC5D and one or more additional binding domains, which may bind to a second antigen or protein other than GPRC5D.

[0120] In some embodiments, the present disclosure provides a multispecific binding peptide comprising at least two domains that bind to GPRC5D, the at least two domains that bind to GPRC5D having differences in amino acid sequence and each binding to a different epitope on GPRC5D.

[0121] In some embodiments, the present disclosure provides a multispecific binding peptide comprising at least two domains that bind to GPRC5D and one or more additional binding domains, wherein the at least two domains that bind to GPRC5D have amino acid sequence differences and each binds to a different epitope on GPRC5D, and the one or more additional binding domains may bind to a second antigen or protein other than GPRC5D.

[0122] In some embodiments, the binding domain of GPRC5D is a single domain antibody as provided herein, and the one or more additional binding domains may be a single domain antibody, single chain antibody or other antigen-binding fragment.

[0123] In some embodiments, the second antigen is a tumor-associated antigen (TAA) or a tumor microenvironment-associated antigen (TMEAA). In some embodiments, the second antigen is an immunomodulatory antigen, wherein the antigen is associated with enhancing or inhibiting a signaling pathway in an immune cell. In some embodiments, the second antigen is a component of the T cell receptor complex, e.g., a T cell surface molecule such as CD3 (including the gamma, delta, epsilon, zeta, and eta chains).

[0124] Preferably, said multispecific binding peptide is a bispecific antibody molecule. In some embodiments, the multispecific binding peptide further comprises an Fc fragment. The presence of an Fc fragment can favor the formation of multimers of the binding domains and provide associated effector functions.

[0125] Immune complex (or antibody complex) containing GPRC5D-binding peptide The present specification provides a complex comprising at least one GPRC5D-binding peptide that specifically binds to GPRC5D provided herein and one or more other functional moieties. The other moieties may be chemical groups, for example therapeutic agents such as cytotoxic drugs, or may be tracers. In some examples, the moieties may be targeting moieties, small molecule drugs (e.g., non-polypeptide drugs less than 500 Da), toxins, cell growth inhibitors, cytotoxic drugs, immunosuppressants, radioactive agents suitable for diagnostic purposes, radioactive metal ions for therapeutic purposes, etc.

[0126] In some embodiments, the immunoconjugate is an antibody drug conjugate (ADC) comprising one or more GPRC5D-binding peptides provided herein and a therapeutic agent, which is cytotoxic, inhibits cell proliferation, or provides some therapeutic benefit. In some examples, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzymatically active toxin or fragment thereof from a bacterial, fungal, plant, or animal source), or a radioisotope (i.e., a radioconjugate). In some examples, the antibody drug conjugates provided by the present invention allow for targeted delivery of the drug moiety to the tumor. In some cases, this results in targeted killing of tumor cells.

[0127] In some examples, the therapeutic agent includes, for example, daunomycin, doxorubicin, methotrexate, vindesine, maytansinoids, etc. In some examples, the therapeutic agent has intracellular activity. In some examples, the immune complex that binds to GPRC5D is internalized, and the therapeutic agent has the ability to block cellular protein synthesis activity, block nucleic acid synthesis activity, and cause cell growth arrest or cell death.

[0128] In some embodiments, an immunoconjugate comprises one or more GPRC5D-binding peptides provided herein and a tracer. The immunoconjugate can be used for research or diagnostic purposes, for example, for in vivo cancer detection. The tracer can generate a detectable signal directly or indirectly. For example, the tracer can be 3 H, 14 C. 32 P, 35 S, 123 The tracer may be a radioisotope such as I, a fluorescent isothiocyanate, a fluorescent (fluorophore) or chemiluminescent (chromophore) compound such as rhodamine or fluorescein, an imaging agent, or a metal ion. In some embodiments, the tracer is a radioactive atom for scintigraphic studies, e.g. 99 Tc or 123I, or spin labels for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI), e.g. 89 Zr, 123 I, 19 F, 13 C. 15 N, 17 It is O. 89 Zr can be complexed with a variety of metal chelators and attached to antibodies, for example for PET imaging.

[0129] The binding of the GPRC5D-binding peptide to other functional moieties may be covalent or non-covalent. An example of a non-covalent bond is via the Biotin-Avidin System. An example of a covalent bond is via various chemical linkers, including peptide linkers, cleavable linkers, or non-cleavable linkers. Exemplary linker components include 6-maleimidocaproyl (MC), maleimidopropioyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and N-succinimidyl (4-carboxy-acetyl)aminobenzoate (SIAB).

[0130] In some embodiments, the linker may include amino acid residues. Exemplary amino acid linker moieties include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Exemplary dipeptides include valine-citrulline (vc or val-cit), alanine-phenylalanine (afa-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycyl-glycyl-glycine (gly-gly-gly). Amino acid residues that comprise the amino acid linker moiety include naturally occurring residues as well as non-naturally occurring amino acid analogs such as citrulline. Amino acid linker moieties can be designed and optimized for their selectivity for enzymatic cleavage by specific enzymes such as tumor-associated proteases, cathepsins B, C and D, plasmin proteases, etc.

[0131] Conjugates of GPRC5D-binding peptides with cytotoxic drugs can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCI), active esters (such as disuccinimidyl substrates), aldehydes (such as glutaraldehyde), bisazide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0132] Pharmaceutical compositions containing GPRC5D-binding peptides and methods of treatment The GPRC5D-binding peptides disclosed herein, as well as fusion proteins, multispecific antibody molecules, and immunoconjugates comprising the GPRC5D-binding peptides (which can be formulated into pharmaceutical compositions with pharma- ceutically acceptable carriers), may be used to administer to subjects for the prevention or treatment of tumors. The effective amount for such use may depend on the severity of the disease and the overall state of the patient's own immune system. Dosage regimens also vary depending on the disease and the condition of the subject, and generally range from a single bolus or continuous infusion to multiple doses per day (e.g., every 4-6 hours). A clinician skilled in the art can readily determine whether a particular subject is a candidate for such treatment, for example, by using clinical tests, physical examinations, and the subject's family history.

[0133] In some embodiments, the GPRC5D-binding peptides disclosed herein and fusion proteins, multispecific antibody molecules, and immunoconjugates containing the GPRC5D-binding peptides may be administered in combination with one or more other drugs (e.g., anti-tumor agents).

[0134] In some embodiments, the disease or disorder to be treated is a tumor, preferably multiple myeloma.The GPRC5D-binding peptide disclosed herein (as well as the fusion protein, multispecific antibody molecule, immunoconjugate containing the GPRC5D-binding peptide) can be used by any suitable method or route, optionally in combination with other anti-tumor agents.The administration route can be, for example, oral, intravenous, intraperitoneal, subcutaneous or intramuscular.

[0135] The nucleic acid molecules encoding the GPRC5D-binding peptides provided herein, expression vectors containing the nucleic acid molecules, and host cells transfected with the nucleic acid molecules or expression vectors alone may also be used for the above-mentioned therapeutic purposes through various methods. For example, in the case of expression vectors, they can be introduced into subjects through gene therapy means known in the art to express the target protein or polypeptide (GPRC5D-binding peptide) and achieve therapeutic purposes.

[0136] Detection, diagnostic or therapeutic kits containing GPRC5D-binding peptides The GPRC5D binding peptide provided herein, as well as other forms of molecules (such as fusion proteins or bispecific antibody molecules) that contain the GPRC5D binding peptide, can specifically bind to GPRC5D in sample.By measuring the amount of GPRC5D binding peptide-GPRC5D complex formed, or the amount of GPRC5D binding peptide in the GPRC5D binding peptide-GPRC5D complex formed, the content (or presence) of GPRC5D in sample can be easily determined.

[0137] As explained above, for this purpose, the GPRC5D-binding peptides provided herein may be linked to various detection tags, facilitating detection by various means, including but not limited to bioluminescence, fluorescence, radioactive labeling, the amount of product produced by enzyme reaction, etc. By measuring the content of GPRC5D in a sample and comparing it with the normal content of GPRC5D in a normal population, the disease state or severity of the subject from which the sample was provided can be determined. By repeatedly detecting the change in the content of GPRC5D over time during the course of treatment of a subject, it can also be determined whether the treatment measures are effective, thereby providing a basis for changing the treatment plan.

[0138] The GPRC5D-binding peptides provided herein and other forms of molecules (e.g., fusion proteins or bispecific antibody molecules) that contain the GPRC5D-binding peptides can be placed in containers to form detection, diagnostic or treatment kits. These containers can be in the form of boxes, ampoules, vials, tubes, bags, or other suitable containers known in the art. These containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding drugs. If necessary, instructions for use are also provided with the containers. The instructions can generally include information on how to use the GPRC5D-binding peptides or compositions that contain the GPRC5D-binding peptides to treat or prevent tumors (e.g., multiple myeloma), such as a description of a therapeutic agent (e.g., GPRC5D-binding peptides), a dosing regimen for treating or preventing neoplasms (e.g., multiple myeloma), and / or cautions, warnings, indications, contraindications, side effects, animal pharmacology, clinical studies, and / or reference materials. The instructions may be printed directly on the container (if present), or may be provided as a label affixed to the container, or as a separate paper, pamphlet, card, or folded piece of literature provided in or with the container.

[0139] The GPRC5D-binding peptides (or referred to as antibody molecules targeted to GPRC5D) provided herein are fully human single domain antibodies that have binding affinity for GPRC5D similar to or better than a control antibody.

[0140] Chimeric antigen receptors (CARs) The chimeric antigen receptor (CAR) provided herein is an artificially constructed chimeric protein that comprises an extracellular antigen binding domain that specifically binds to GPRC5D, a transmembrane domain, and one or more intracellular signaling domains.The characteristics of these CARs include the ability to redirect T cell specificity and reactivity to cells that express GPRC5D in a non-MHC-restricted manner.Non-MHC-restricted GPRC5D recognition allows T cells or NK cells that express the disclosed CARs to recognize antigens independently of antigen processing.

[0141] The intracellular signaling domain may include a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. The T cell receptor signaling domain may include an intracellular domain of a T cell receptor, such as the intracellular portion of a CD3ζ protein. The costimulatory signaling domain is an intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an effective response of lymphocytes to antigens.

[0142] 1. Extracellular antigen-binding domain In some embodiments, the CAR provided herein comprises an extracellular antigen-binding domain that specifically binds to GPRC5D.For example, the antigen-binding domain can be an scFv, which can specifically bind to the heavy chain variable region and the light chain variable region of any antibody or its antigen-binding fragment linked by a peptide linker.As another example, for the construction of a CAR using a single domain antibody that specifically binds to GPRC5D, the extracellular antigen-binding domain can comprise the heavy chain variable region from the single domain antibody, but does not comprise any light chain variable region.

[0143] In some embodiments, the extracellular antigen-binding domain of the CAR provided herein comprises a heavy chain variable region from clone #18, wherein the sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of #18 are as follows: the sequence of HCDR1 is GGSFSGYY (SEQ ID NO:1); The sequence of HCDR2 is INHSGST (SEQ ID NO:2); The sequence of HCDR3 is ARARRYGGRTRFDP (SEQ ID NO:3).

[0144] In some embodiments, the extracellular antigen-binding domain of the CAR provided herein comprises a heavy chain variable region from clone #39, wherein the sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of #39 are as follows: the sequence of HCDR1 is GFIFSSYG (SEQ ID NO:4); the sequence of HCDR2 is ISSSGDYT (SEQ ID NO:5); The sequence of HCDR3 is ARMSFRRYDH (SEQ ID NO:6).

[0145] In some embodiments, the extracellular antigen-binding domain of the CAR provided herein comprises a heavy chain variable region from clone #18, wherein the sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of #41 are as follows: the sequence of HCDR1 is GFSFSGYI (SEQ ID NO:7); the sequence of HCDR2 is TSSSGTET (SEQ ID NO:8); The sequence of HCDR3 is ARYYSKYGRSYHVDS (SEQ ID NO:9).

[0146] In some embodiments, the extracellular antigen-binding domain of the CAR provided herein comprises the heavy chain variable region from clone #18 (SEQ ID NO: 10) or any functional variant thereof. In other embodiments, the extracellular antigen-binding domain of the CAR provided herein comprises the heavy chain variable region from clone #39 (SEQ ID NO:11) or any functional variant thereof.

[0147] In other embodiments, the extracellular antigen-binding domain of the CAR provided herein comprises the heavy chain variable region from the #41 clone (SEQ ID NO: 12) or any functional variant thereof. A functional variant herein may include insertions, deletions, or substitutions of several amino acid residues compared to its parent sequence (the VH amino acid sequence provided herein) and substantially retains the ability to specifically bind to GPRC5D.

[0148] In some embodiments, a functional variant may include insertion, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues compared to its parent sequence, while the binding affinity of the parent sequence to GPRC5D remains at least 50%, 60%, 70%, 80%, 90% or even higher than that of the parent sequence. As known to those skilled in the art, the sequence of the VH framework region may be changed to a large extent, as long as the spatial position of the corresponding CDR sequence is maintained. For example, the sequence of the framework region in the VH amino acid sequence may be changed, for example, by substituting the framework regions of different antibody species for each other, to obtain a functional variant that substantially retains the antigen binding ability. Furthermore, functional mutants that still have antigen-binding ability can be obtained by altering a small number of residues in the CDR sequences, for example, one, two or three amino acid residues, and detecting the ability of the altered antibody molecule to bind to the corresponding antigen.

[0149] Conservative amino acid substitutions of functionally similar amino acids are well known to those of skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T) 2) Aspartic acid (D), glutamic acid (E) 3) Asparagine (N), Glutamine (Q) 4) Arginine (R), Lysine (K) 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V) 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0150] The extracellular antigen-binding domain of the CAR provided herein comprises two or more of the above Vs having GPRC5D-binding ability. HH The extracellular antigen-binding domain or a portion thereof may include two or more tandemly linked V fragments. They may be directly linked in tandem or may be linked in tandem via a peptide linker. HH The use of fragments helps to facilitate recognition and binding of target molecules (e.g., GPRC5D) by the constructed CAR. Preferably, each of these VH fragments binds to a different epitope on GPRC5D.

[0151] CAR can include a signal peptide sequence, for example, at the N-terminus of the antigen-binding domain. Any suitable signal peptide sequence can be used. In one embodiment, the signal peptide sequence is CD8α signal peptide. Although the signal peptide sequence can promote the expression of CAR on the cell surface, the presence of the signal peptide sequence in the expressed CAR is not essential for the CAR to perform its function. When the CAR is expressed on the cell surface, the signal peptide sequence can be cleaved from the CAR. Thus, in some embodiments, the CAR lacks a signal peptide sequence.

[0152] Between the antigen binding domain and the transmembrane domain of the CAR there may be a hinge region (or spacer domain) comprising a polypeptide sequence. The hinge region may comprise up to 300 amino acids, preferably 10-100 amino acids. In one embodiment, the CAR provided herein comprises a CD8α protein hinge region.

[0153] 2. Transmembrane domain A CAR can include a transmembrane domain fused to the extracellular domain of the CAR, hi one embodiment, a transmembrane domain that is naturally associated with other elements of the CAR (such as a bridging region or an intracellular signaling domain) is used.

[0154] The transmembrane domain may be of natural or synthetic origin. If of natural origin, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains for use in the disclosed CARs may include at least the transmembrane domains of the α, β or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain may be synthetic, in which case it comprises primarily hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan and valine occur at each end of the synthetic transmembrane domain.

[0155] Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, can form the link between the transmembrane domain and the intracellular T cell signaling domain and / or the T cell costimulatory domain of the CAR. Exemplary linker sequences include one or more glycine-serine doublets. In some embodiments, the transmembrane domain comprises a transmembrane domain of a T cell receptor, such as the CD8α transmembrane domain (SEQ ID NO:21).

[0156] 3. Intracellular domain The intracellular region of the CAR contains one or more intracellular T cell signaling domains and is responsible for activating at least one normal effector function of the T cell expressing the CAR. Exemplary T cell signaling domains are provided herein and known to those of skill in the art. In CAR, the entire intracellular T cell signaling domain can be used, but in many cases it is not necessary to use the entire chain. In terms of using a truncated portion of the intracellular T cell signaling domain, the truncated portion may be used instead of the complete chain, as long as it transmits the relevant T cell effector function signal.

[0157] Examples of intracellular T cell signaling domains used in CARs include the cytoplasmic continuum of the T cell receptor (TCR) and costimulatory molecules that act synergistically to initiate signaling following antigen receptor binding, as well as any derivatives or variants of these sequences, and any synthetic sequences that have the same function.

[0158] The T cell receptor signaling domain regulates the activation of the T cell receptor complex in a stimulatory or inhibitory manner. The CARs disclosed herein may include a signaling motif called an immunoreceptor tyrosine-based activation motif or ITAM, and may include a cytoplasmic signaling sequence that acts in a stimulatory manner. Examples of ITAM-containing primary cytoplasmic signaling sequences that may be included in the disclosed CARs include intracellular domains from CD3zeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CDS, CD22, CD79a, CD79b, and CD66d proteins. In some embodiments, the cytoplasmic signaling molecule in the CAR includes an intracellular T cell signaling domain from CD3zeta.

[0159] The intracellular domain of the CAR provided herein may include a CD3 zeta chain portion and an intracellular costimulatory signaling domain. The costimulatory signaling domain may include an intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an effective response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0160] In some embodiments, the CAR may include a CD3ζ signaling domain, a CD8 signaling domain, a CD28 signaling domain, a 4-1BB signaling domain, or a combination of two or more thereof. In one embodiment, the intracellular domain includes a CD3ζ signaling domain and a CD28 signaling domain. In another embodiment, the cytoplasmic domain includes a CD3ζ signaling domain and a 4-1BB signaling domain. In yet another embodiment, the cytoplasmic domain includes a CD3ζ signaling domain and a CD28 and CD137 signaling domain. One skilled in the art can change the order of one or more T cell signaling domains on the CAR as needed. The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention can be linked to each other in a random or specified order. Alternatively, a short polypeptide linker, preferably 2-10 amino acids in length, can form the linkage. A glycine-serine doublet provides a particularly suitable linker. Additionally, there may be a spacer domain comprising a polypeptide sequence between the signaling domain and the transmembrane domain of the CAR. The spacer domain may comprise up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0161] 4. Further explanation of CAR Based on the CAR provided herein (as a parent CAR), one skilled in the art can modify it, for example by fusing it to other polypeptides or by retaining only fragments of the CAR provided herein, and these fusion molecules or fragments have at least some of the biological activity of the parent CAR, such as the activity of recognizing target cells (e.g., tumor cells expressing GPRC5D), or the activity of detecting, treating or preventing a disease.

[0162] The CARs provided herein can further comprise additional amino acids at the amino or carboxy terminus, or at both termini, that are not present in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the CAR or functional portion, such as target cell recognition, cancer detection, cancer treatment or prevention, etc. More desirably, the additional amino acids have enhanced biological activity compared to the biological activity of the parent CAR.

[0163] Further provided are functional variants of the CARs described herein, which have substantial or significant sequence identity or similarity with the parent CAR, and the functional variant retains the biological activity of the CAR as the variant. Functional variants include variants such as those of the CARs described herein (parent CARs) that retain the ability to recognize target cells to a similar extent, the same extent, or a greater extent than the parent CAR. With reference to the parent CAR, the functional variant may have, for example, at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identity in amino acid sequence with the parent CAR.

[0164] A functional variant may, for example, comprise the amino acid sequence of a parent CAR with at least one conservative amino acid substitution. Alternatively, a functional variant may further comprise the amino acid sequence of a parent CAR with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent CAR.

[0165] The CARs provided herein may include synthetic amino acids to replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, a-amino-n-decanoic acid, homoserine, S-acetamidomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-monoglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglyc ... Carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-monocarboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-monomethyl-lysine, N',N'-dibenzyl-lysine, 6-monohydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, oc-monoaminocycloheptanecarboxylic acid, -(2-amino-2-norbornane)-monocarboxylic acid, γ-diaminobutyric acid, α,β-diaminopropanoic acid, homophenylalanine, α-tert-butylglycine, and the like.

[0166] The CARs provided herein may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via a disulfide bond, or converted to an acid addition salt, and / or optionally dimerized or multimerized, or conjugated.

[0167] Methods for making chimeric antigen receptors, T cells containing such receptors, and their uses (e.g., for treating cancer) are known in the art. For example, the nucleic acid molecules encoding the disclosed chimeric antigen binding receptors may be included in an expression vector (e.g., lentiviral vector) that is used for expression in host cells, such as T cells, to make the disclosed CARs. In some embodiments, the method for using the chimeric antigen receptor includes isolating T cells from a subject, transforming the T cells with an expression vector (e.g., lentiviral vector) that encodes the chimeric antigen receptor, and administering the engineered T cells expressing the chimeric antigen receptor to the subject for treatment, e.g., for treating a tumor in the subject.

[0168] Nucleic Acid Molecules and Expression The present specification provides the coding nucleic acid sequence that codes each component that constructs CAR. These nucleic acid sequences may be prepared by any suitable method, including, for example, cloning, amplification, or direct chemical synthesis. Chemical synthesis produces single-stranded oligonucleotides. This can be converted into double-stranded DNA by hybridization to a complementary sequence or by polymerization with DNA polymerase using the single strand as a template. Those skilled in the art recognize that chemical synthesis of DNA is generally limited to sequences of about 100 bases, but longer sequences can be obtained by linking shorter sequences.

[0169] Exemplary nucleic acids may be prepared by cloning techniques. Examples of suitable cloning and sequencing techniques, as well as sufficient explanations to guide the skilled artisan through many cloning practices, are well known. Nucleic acids may also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), self-sustained sequence replication system (3SR). Various cloning methods, host cells, and in vitro amplification methods are well known to those skilled in the art.

[0170] In some embodiments, the nucleic acid molecule encodes the CAR provided herein for expression in T cells to generate chimeric antigen receptor T cells. The nucleic acid molecule encoding the chimeric antigen binding receptor may be included in a vector (e.g., a lentiviral vector) for expression in a host cell, such as a T cell. Exemplary cells include T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and regulatory T cells.

[0171] The nucleic acid molecules can be expressed in recombinantly engineered cells, such as bacteria, plant, yeast, insect, and mammalian cells. Antibodies, antigen-binding fragments, HH (For single domain antibodies) alone or H and V L It can be expressed as a chain or as a fusion protein.

[0172] To generate scFv, H and V L The DNA fragment encoding the V can be operably linked to another fragment encoding a flexible linker, e.g., another fragment encoding the amino acid sequence (GlY-ser) , thereby H and V L The sequence is V L and V H The domains are expressed as a contiguous single-chain protein linked by a flexible linker.

[0173] Those of skill in the art are familiar with the numerous expression systems used to express proteins, including E. coli, other bacterial hosts, yeast, and a variety of higher eukaryotic cells such as COS, CHO, HeLa, and myeloma cell lines.

[0174] To produce a CAR, the host cell is preferably a mammalian cell, such as a human cell. In some embodiments, the host cell may be a peripheral blood lymphocyte (PBL) or peripheral blood mononuclear cell (PBMC), or a T cell. The T cell may be any T cell, for example, a cultured T cell, for example, a primary T cell, or a T cell from a cultured T cell line, such as Jurkat, SupTl, or a T cell obtained from a mammal. If obtained from a mammal, the T cell may be obtained from a variety of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cell may also be enriched or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. The T cell may be any type of T cell, may be at any developmental stage, and may be CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells (e.g., Th1 and Th2 cells, CD8 + These include, but are not limited to, T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, and the like. T cells include CD8 + T cells or CD4 + It may be a T cell.

[0175] The nucleic acid encoding the CAR or its components described herein can be modified without reducing its biological activity. For example, some modifications can be made to facilitate cloning or expression. Such modifications are well known to those skilled in the art and include, for example, stop codons, methionine added to the amino terminus to provide an initiation site, and additional amino acids (e.g., polyhistidine) located on either end to create conveniently located restriction sites to facilitate purification steps. In addition to recombinant methods, the CAR or its components of the present disclosure can also be constructed in whole or in part using standard peptide synthesis, which is well known in the art.

[0176] Pharmaceutical compositions and medical uses A CAR provided herein or a host cell expressing a CAR (e.g., a T cell or an NK cell) can specifically recognize and bind to a target cell expressing GPRC5D, e.g., a tumor cell.

[0177] Thus, in some embodiments, the present disclosure provides pharmaceutical compositions for preventing or treating diseases associated with GPRC5D expression, comprising a host cell expressing a CAR provided herein, particularly a CAR-T cell, and a pharma- ceutical acceptable carrier.These pharmaceutical compositions can be formulated into dosage forms suitable for the mode of administration, such as injections.

[0178] In other embodiments, the present disclosure provides a method for treating a disease associated with GPRC5D expression, comprising administering to a subject in need thereof an effective amount of a CAR-T cell or a pharmaceutical composition comprising a CAR-T cell provided herein. In some embodiments, the mode of administration is intravenous injection, such as intravenous injection, intramuscular administration, such as intramuscular injection, or in situ administration at the tumor site, etc.

[0179] In another embodiment, the present specification provides a use of the CAR-T cells in the preparation of a medicament for treating a disease associated with GPRC5D expression. In some embodiments, the disease associated with GPRC5D expression is a tumor, such as a plasma cell malignancy disease (eg, multiple myeloma) or a B cell malignancy (eg, Hodgkin's lymphoma or non-Hodgkin's lymphoma), or an autoimmune disease.

[0180] In some embodiments, the CAR-T cells or pharmaceutical compositions herein can be administered to a subject to prevent or treat a tumor. In these applications, the CAR-T cells or pharmaceutical compositions targeting target cells expressing GPRC5D are administered to a subject in a therapeutically effective amount to delay or inhibit tumor growth or metastasis, reduce tumor volume, or suppress signs or symptoms of cancer. The therapeutically effective amount depends on the severity of the disease and the general health of the patient. The therapeutically effective amount is an amount that provides a subjective alleviation of symptoms or an objectively identifiable improvement as noted by a clinician or other qualified observer. In one embodiment, the therapeutically effective amount is an amount necessary to inhibit tumor growth, inhibit metastasis, reduce tumor volume, or an amount effective to reduce signs or symptoms of a tumor. The therapeutically effective amount of the administered agent may vary depending on the desired effect and the subject being treated. In some examples, the therapeutic amount is an amount that eliminates or reduces the tumor burden in the patient, or prevents or reduces metastatic cell growth, or alleviates tumor symptoms.

[0181] Any administration method, including local administration and systemic administration, can be used for the disclosed CAR-T cells or pharmaceutical compositions. For example, local, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal and subcutaneous administration can be used. The particular mode of administration and dosing regimen will be selected by the attending physician, taking into account the specifics of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic).

[0182] The administration of the CAR-T cells or pharmaceutical compositions provided herein may be accompanied by the administration of other anti-cancer or anti-angiogenic agents or therapeutic treatments (e.g., surgical removal of the tumor or radiation therapy). For example, the subject may receive one or more additional therapies or therapeutic agents before or after administration of a therapeutically effective amount of the CAR-T cells. For example, the additional therapies may include, but are not limited to, the use of chemotherapeutic agents, anti-angiogenic agents, or combinations thereof. In another example, at least a portion of the tumor is surgically or otherwise removed or reduced in size or volume before administration of an effective amount of the CAR-T cells or pharmaceutical compositions provided herein.

[0183] Specific examples of other therapeutic agents that can be used include microtubule binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors.These agents (administered in therapeutically effective amounts) and treatments can be used alone or in combination.For example, any suitable anti-cancer or anti-angiogenesis agent can be administered in combination with the CAR-T cells or pharmaceutical compositions disclosed herein. Research Overview

[0184] The inventors used a large-scale phage antibody library to screen all-human GPRC5D-specific antibodies, evaluated the specificity of these antibodies at the phage level and protein level by enzyme-linked immunosorbent assay (ELISA) and fluorescence activated cell sorting (FACS) experiments, then evaluated the affinity of the screened antibodies for binding to cells by FACS measurement, constructed these antibody molecules on second-generation CAR lentivirus expression vectors, transfected them into T cells, and initially evaluated the killing function of these antibody molecules by in vitro cell killing experiments. Finally, three fully human antibody clones with good specificity and very high killing function were obtained.

[0185] Through panning of recombinant GPRC5D protein and GPRC5D protein / cell (CHO-K1-GPRC5D), the inventors selected a total of 768 monoclonals using different antibody libraries for primary screening with enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) detection, of which 216 clones specifically bound to GPRC5D-VLP protein and GPRC5D expression positive cell CHO-K1-GPRC5D, but did not bind to control protein VLP and GPRC5D expression negative cell CHO-K1. After sequencing, 35 different monoclonal sequences were obtained. Next, we subjected these 35 antibodies to flow cytometry analysis (FACS) identification with various GPRC5D positive (CHO-K1-GPRC5D, MM1S) and negative cell lines (CHO-K1, Jurkat), enzyme-linked immunosorbent (ELISA) identification with various unrelated antigens (VLP, BAFFR-his-Bio (BAFFR-his-Bio), IL10 -Bio, SA) and GPRC5D protein (GPRC5D-VLP), and three clones showed good binding and specificity in both various cell lines and protein antigens. The acquisition of these clones laid the foundation for the subsequent development of all-human GPRC5D CAR-T products or antibody drugs.

[0186] The present invention uses a fully human phage library to perform antibody screening and directly obtain fully human monoclonal antibodies. Compared with traditional hybridoma technology, it avoids the difficult humanization step of mouse antibodies, and fully human antibodies are less immunogenic than humanized mouse antibodies, and have better potential in the development of antibody drugs or CAR-T.

[0187] Through a fully human phage library, antibody clones (including single domain clones #18, #39, #41, etc.) that specifically bind to cell surface GPRC5D antigen are obtained, and then these clones and the control antibody benchmark1 (the variable region sequence is obtained from GC5B596 in Chinese Patent Application Publication CN109715667A) are constructed into second-generation CAR structures, and then lentivirus packaging is performed and T cell lines are transfected to screen for fully human GPRC5D antibody clones and candidate CAR-T molecules that have better functions than the control benchmark1 at the CAR-T cell line level in terms of activation and killing of target cell lines, stimulating proliferation of target cell lines, etc.

[0188] During the development process, phage-level antibody screening / specificity identification can be used to quickly and efficiently screen specific antibody clones, followed by CAR-T function testing to select the optimal candidate antibodies, thereby optimizing the antibody screening process for CAR-T development, thereby ensuring the quality of development while improving the efficiency of research and development.

[0189] The following table shows the single domain heavy chain variable region sequences that were specifically studied, as well as the amino acid sequences and coding sequences of the other components used in CAR construction. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0190] In vitro functional validation of CAR-T cells Experimental objectives and principles: Specific antibody clones targeting GPRC5D protein were enriched from a phage antibody library by affinity panning, and then screened and identified to obtain clones that bind to GPRC5D-positive target cells. However, these clones need to be constructed into second-generation CARs and their function in CAR-T cells needs to be further confirmed. To this end, the inventors produced these cloned lentiviral vectors and transduced T cells to produce CAR-T cells. Then, the in vitro biological efficacy of CAR-T cells was evaluated by CD107a degranulation assay and in vitro cytotoxicity assay, and the CAR function of the published antibody sequence (Benchmark1) was evaluated at the same time. Through these CAR-T level functional verifications, the inventors finally selected candidate single-chain antibody clones with ideal efficacy and safety for future CAR-T product development.

[0191] Working Example Example 1. Enrichment of specific antibody clones targeting GPRC5D protein from a phage antibody library by affinity panning Appropriate negative and positive panning strategies are used to enrich for the desired specific antibody clones from the phage antibody library.

[0192] Phage antibody library construction The phage antibody libraries constructed by the present inventors include a natural library, a semi-synthetic library, and a single domain library. The semi-synthetic phage antibody library used together with the natural library solves the problem that the natural library may lack GPRC5D high affinity antibody clones. The single domain phage antibody library is an antibody library consisting of only amino acids in the variable region of a heavy chain antibody, and although its molecular weight is only 12 to 15 kDa, it has the same or higher specificity and affinity as conventional antibodies. Furthermore, single domain antibodies have attracted attention due to their characteristics such as stable physical and chemical properties, high affinity, ease of production by recombinant expression, and ease of combination with other target or epitope antibodies.

[0193] GPRC5D protein panning GPRC5D-VLP was used as a positive panning protein and VLP protein was used as a negative panning protein to perform multiple rounds of panning to obtain a phage pool enriched for the desired antibody clone. The experimental steps are briefly described below.

[0194] 1) Antigen coating: Antigen GPRC5D-VLP is diluted to 10 μg / mL in clean coating buffer (PBS), 100 μL of working solution is added to each well, panned every 6 wells, and allowed to bind overnight at 4°C. Dilute the antigen VLP protein to 10 μg / mL in clean coating buffer (PBS), add 100 μL of working solution to each well, pan every six wells, and bind overnight at 4 °C, the negative panning antigen is denoted as negative plate, and the positive panning antigen is denoted as positive plate.

[0195] 2) Blocking: Turn the antigen upside down and remove any remaining liquid in the well with absorbent paper. Add 250 μL of 3% BSA-PBS to block, and leave to block at room temperature for 2 hours. 3) Phage library (5x10) incubated with control antigen VLPs 12By adding a 50 μl PBS containing phage particles, phage antibody clones that non-specifically bind to the VLP are removed.

[0196] 4) After incubation, the supernatant is transferred to a positive plate to which the target antigen GPRC5D-VLP is bound, and incubation is continued to allow the phage to bind to the target antigen. 5) Wash the magnetic beads with washing solution to wash away unbound phages. 6) Elute the positive phages from the target antigen with the elution solution, and neutralize them by adding a neutralizing solution.

[0197] 7) The eluted phages are reinfected with the host bacteria XL1-blue, and the recovered phages are amplified. A small amount of the sample is left over for gradient dilution, which is then used to infect the host bacteria, and the number of recovered phages is calculated by applying it to an Amp-resistant plate. 8) Repeat steps 1) to 6) until a significant increase in phage recovery (number of eluted phages / number of input phages) is observed, typically performing three rounds of panning. The enriched phage pool can be used for subsequent monoclonal selection and ELISA / FACS screening.

[0198] Main materials and reagents: Fully human phage antibody libraries, including natural libraries, semi-synthetic libraries, and single domain libraries. helper phage KO7, Thermo / Invitrogen, 18311019; Human GPRC5D Protein-VLP, Kactus, GPR-HM05P; VLP protein, Kactus, order High binding ELSIA plate, Costar,#3590 Blocking solution: PBS+3% BSA Washing solution: PBS + 0.1% Tween20 Eluent: 0.2M Glycine, pH2.2 Neutralizing solution: 1M Tris, pH9.1

[0199] Test Results: Using different antibody libraries, a significant increase in recovery was observed for each of the three rounds of protein panning (Table 1), demonstrating that antibody clones were effectively enriched. Table 1. Protein panning experiment results [Table 2]

[0200] It can be seen that after three rounds of panning, all the different antibody libraries were enriched (the recovery rate of the third round was significantly improved compared to the previous rounds). However, the specific binding clones screened by this panning strategy were few, and in the subsequent FACS experiment, they were poorly bound to CHO-K1-GPRC5D cells that highly express GPRC5D antigen, i.e., they cannot recognize the native GPRC5D antigen on the cell surface. Therefore, in the subsequent experiment, the inventors used the method of panning alternately on proteins and cells to enrich the specific antibody clones from the phage antibody library that can bind to both GPRC5D protein and native GPRC5D on the cell surface. Table 2 shows the results of using the combined panning of recombinant GPRC5D protein and CHO-K1-GPRC5D / CHO-K1 cell line. As can be seen from the recovery rates, all four pannings were enriched and could be used for the next step of monoclonal selection. Table 2. Protein + cell panning experiment results [Table 3]

[0201] Example 2. Screening of specific clones from enriched phage pools using ELISA and FACS Purpose and principle: The phage pool enriched by the affinity panning step contains phage antibodies with various characteristics, such as specific clones, non-specific clones, and negative clones. To obtain specific clones, the present inventors need to isolate monoclonals, package monoclonal phage, and perform primary screening of a large number of monoclonals by ELISA and FACS to select monoclonals that specifically bind to both GPRC5-VLP protein and GPRC5D-positive cell line CHO-K1-GPRC5D. The specific monoclonals are further subjected to DNA sequencing to determine the unique antibody sequence contained therein.

[0202] In the ELISA primary screening, those that only bind to GPRC5D-VLP and do not bind to control antigen VLP were identified as specific clones.FACS primary screening was performed using GPRC5D high expression positive cell line CHO-K1-GPRC5D and GPRC5D negative cell line CHO-K1, and those that only bind to cell CHO-K1-GPRC5D and do not bind to CHO-K1 cells were identified as specific clones.Through both ELISA and FACS primary screening, the present inventors obtained candidate antibodies that can bind to recombinantly expressed GPRC5D protein and recognize the native state GPRC5D molecule on the cell surface, and then subjected to further screening.

[0203] The brief steps of the ELISA primary screening experiment are as follows. 1) Monoclonal phages are cultured and packaged in deep well 96-well plates. 2) GPRC5D-VLPs are diluted to 10 μg / mL in PBS and added to high-binding microplates at 100 μL / well and coated overnight at 4° C. 3) Discard the coating solution, add 250 μL of blocking solution to each well, and block at room temperature for 2 hours.

[0204] 4) Wash the plate twice with 250 μL of washing solution. 5) Add 100 μL of phage supernatant from step 1) to the wells coated with the target antigen and allow to bind at room temperature for 2 h. 6) Wash the plate four times with 250 μL of washing solution.

[0205] 7) Add mouse anti-M13 primary antibody diluted 1:2000, 100 μL / well, and incubate at room temperature for 45 minutes. 8) Wash the plate four times with 250 μL of washing solution. 9) Add HRP Donkey anti-mouse IgG diluted 1:2000, 100 μL / well, and incubate at room temperature for 45 min.

[0206] 10) Wash the plate six times with 250 μL of washing solution. 11) Add 100 μL of TMB color substrate and allow to develop for 10 minutes. 12) Add 100 μL of 2MH2SO4 to stop the reaction and read the results on a microplate reader.

[0207] The brief steps of a FACS primary screening experiment are as follows. 1) Monoclonal phages are cultured and packaged in deep well 96-well plates. 2) CHO-K1-GPRC5D and CHO-K1 cells were washed twice with PBS and then diluted with PBS to 1x10 7 / mL and dispense 50 μL into a 96-well deep well plate. 3) Add 50 μL of packaged monoclonal phage to each well, mix evenly, and then allow to bind at 4° C. for 2 h.

[0208] 4) Wash twice with 200 μL of PBS. 5) Add mouse anti-M13 primary antibody diluted at 1:2000 (100 μL / well), mix evenly by pipetting, and incubate at room temperature for 45 minutes. 6) Wash twice with 200 μL of PBS.

[0209] 7) Add 1:300 diluted FITC horse anti mouse IgG (H+L) (100 μL / well), mix evenly by pipetting, and then incubate at room temperature for 45 minutes. 8) Wash twice with 200 μL of PBS and finally resuspend the cells in 200 μL of PBS. 9) The fluorescence intensity of the sample FITC channel was measured using a flow cytometer and the results were analyzed.

[0210] Main materials and reagents: Helper phage KO7, Thermo / Invitrogen, 18311019 Streptavidin, Pierce, 21125 Human GPRC5D Protein-VLP, Kactus, GPR-HM05P; BiotinylatedHuman IL-10 Protein, Kactus, IL1-HM410B; Biotinylated Recombinanthuman BAFFR Protein, Kactus, BAF-HM40RB; High binding ELSIA plate, Costar, #3590

[0211] Corning 96 Well Clear Round Bottom TC-Treated Microplate, Costar, #3799 Blocking solution: PBS+3% BSA Washing solution: PBS + 0.1% Tween20 Soluble one-component TMB substrate solution, Tiangen, PA-107-02 Anti-M13 Bacteriophage Coat Protein g8p antibody, abcam, ab9225

[0212] HRP Goat anti-mouse IgG(minimal x-reactivity) Antibody, Biolegend, 405306 HRP Donkey anti-human IgG (minimal x-reactivity) Antibody, Biolegend,410902 HRP Conjugated AntiHis-Tag Mouse Monoclonal Antibody,CWBIO, CW0285

[0213] FITChorse anti mouse-IgG(H+L), Vector, FI2000 H_GPRC5D CHO-K1 Cell Line Clone, Shanghai Genechem, GM-C03857 Human GPRC5D PE-conjugated Antibody,RD,FAB6300RP Fluorescein (FITC) AffiniPure Goat Anti-Human IgG, Fcγ fragment specific,Jackson ImmunoReseach, 109-095-190

[0214] Experimental results: After the enrichment, the monoclonals are randomly selected from the phage antibody pool and packaged into phages, and then the binding of the monoclonal phages to GPRC5D-VLP protein and control protein VLP is detected by phage ELISA to find phage antibody clones specific to GPRC5D. The ELISA results of some clones are shown in Figure 1. As can be seen from the figure, clones A1, A2, A4, A7 and A8 strongly bind to the target antigen GPRC5D (GPRC5D-VLP) and do not bind to the control antigen VLP, and have good specificity. Clones A3, A5 and A6 bind to both the target antigen and the control antigen, and do not meet the requirements for specific binding. Negative phage control is a negative control phage antibody clone that does not bind to either the target antigen or the control antigen, Anti-M13 phage mouse / anti-mouse HRP Ab is a negative antibody control with only the primary and secondary antibodies added, Anti-mouse HRP Ab is a negative antibody control with only the secondary antibody added, Anti-human IgG HRP Ab is a negative antibody control with only the secondary antibody added, they do not bind to either the target antigen or the control antigen. Anti-his tag HRP Ab detects the positive antibody control of the antigen tag, binds to the antigen of the his tag, and indicates that the coated antigen is bound to the plate. Positive Benchamrk1 is a positive antibody of the target antigen, binds to the target antigen and does not bind to the control antigen.

[0215] The results of the FACS primary screening of antibody clones corresponding to ELISA are shown in Figure 2. Figure 2A shows the results of staining CHO-K1-GPRC5D cell line with commercially available flow cytometry antibody Human GPRC5D PE-conjugated antibody and Positive benchmark1 (the variable region sequence is obtained from GC5B596 in China Patent Application Publication CN109715667A), and the results show that all of the cell lines can bind to GPRC5D, indicating that the CHO-K1-GPRC5D cell line is a cell line positive for GPRC5D expression. In Figure 2B, the A1, A2, A3, A4, A5, A6 and A7 clones bind to CHO-K1-GPRC5D but not to the CHO-K1 cell line, and are specific clones. The A8 clone does not bind to either of the two cell lines, and is a negative clone. Negative phage control is a negative control phage antibody clone that does not bind to either the target antigen or the control antigen, and Positive Benchmark1 Ab (GC5B596) is a positive antibody for the target antigen that binds to the high-expressing cell line CHO-K1-GPRC5D but does not bind to the control negative cell line CHO-K1.

[0216] Through ELISA detection and FACS primary screening, the inventors obtained a total of 216 clones that were double positive by ELISA and FACS and had good specificity, then sequenced the obtained 216 clones with double positive and good specificity, and obtained 35 different monoclonal sequences after sequencing, and then the monoclonals with the 35 different sequences were further subjected to FACS identification of multiple cell lines and ELISA identification of multiple antigens to detect the binding specificity of the candidate clones. In the following examples, three single domain antibody clones (clone numbers 18, 39 and 41) were selected from the above to carry out the experiments, and their HCDR sequences, heavy chain variable region (VH) sequences and VH coding nucleic acid sequences are as shown above.

[0217] Example 3. Further characterization of monoclonal specificity at the phage level by ELISA and FACS Experimental purpose and principle: The antibody used for treatment is required to have very good target specificity, bind only to the target antigen, and not to any unrelated antigen. On the other hand, there are differences (isomers or mutants) in the amino acid sequence of the same antigen in different cell lines, or the ligands that bind are different, and it is necessary to investigate whether the antibody of the present inventors can bind to all of the cells positive for various target proteins. In order to further analyze the specificity and generality of these single clones and find the best candidate clones, the present inventors further evaluated the specificity of the primary screening clones by enzyme-linked immunosorbent assay (ELISA) and flow cytometry.

[0218] Further characterization of monoclonal specificity by ELISA using multiple unrelated antigens In this experiment, we used the target antigen GPRC5D-VLP antigen and various GPRC5D unrelated antigens to react with these monoclonal phage antibodies, and analyzed whether these clones have any non-specific binding to other GPRC5D unrelated antigens. From this experiment, we obtained several clones with excellent specificity.

[0219] Experimental method: Same as ELISA primary screening. Main materials and reagents: [Table 4]

[0220] Test Results: Antibodies used in therapy must have excellent target specificity. To further analyze the specificity of these monoclonal antibodies, the present inventors used enzyme-linked immunosorbent assay (ELISA) to characterize the multiple clones obtained in Example 2 for various antigens. The results are shown in Figure 3, where Negative phage control is a negative control phage antibody clone that does not bind to either the target antigen or the control antigen, Anti-M13 phage mouse / anti-mouse HRP Ab is a negative antibody control with only the primary antibody and the secondary antibody added, Anti-mouse HRP Ab is a negative antibody control with only the secondary antibody added, which does not bind to either the target antigen or the control antigen, and Positive Benchmark1 Ab (GC5B596) is a positive antibody control for the target antigen (GPRC5D-VLP), which binds to the target antigen and does not bind to the control antigen. Anti-human IgGHRP Ab is a negative antibody control with only secondary antibody added, and anti-hisHRP Ab is a positive antibody control that detects the antigen tag, binding to the antigen in the his tag and indicating that the coated antigen was bound to the plate. Clones 18, 39, and 41 all bound to the GPRC5D antigen, but did not bind to the four unrelated antigens, indicating that clones 18, 39, and 41 bound to the GPRC5D antigen and had good specificity.

[0221] Further characterization of monoclonal specificity using different cell lines at the phage level by FACS In this experiment, we used GPRC5D positive and negative cell lines to react with these monoclonal phage antibodies, and analyzed whether these clones can bind to GPRC5D antigen on different cell lines, and whether they have any non-specific binding to other cell lines that do not express GPRC5D. From this experiment, we obtained several clones with excellent specificity.

[0222] Experimental method: Same as FACS primary screening. Main materials and reagents: CHO-K1-GPRC5D cell line, GPRC5D-positive cell line; MM1S cell line, GPRC5D positive cell line; CHO-K1 cell line, GPRC5D negative cell line; Jurkat cell line, GPRC5D negative cell line; The remaining reagents were the same as in the FACS primary screening.

[0223] Test Results: Antibodies used in therapy must have excellent target specificity. To further analyze the specificity of these monoclonal antibodies, we used enzyme-linked immunosorbent assay and flow cytometry to characterize the only clone obtained in Example 2 in more antigens and cell lines. The results are shown in Figure 4, where Negative phage control is a negative control phage antibody clone. Clones 18, 39, and 41 bound to two GPRC5D-positive cell lines CHO-K1-GPRC5D and MM1S, but did not bind to two GPRC5D-negative cell lines CHO-K1 and Jurkat, and had good specificity; Positive Benchamrk1 Ab (GC5B596) was a positive antibody for the target antigen, bound to the high-expression cell line CHO-K1-GPRC5D, but did not bind to the control negative cell line CHO-K1; and FITC anti-human IgG Ab was a negative control with only the secondary antibody added, and did not bind to either of both cell lines.

[0224] Example 4. Further characterization of the binding specificity of monoclonals at the protein level by ELISA and FACS Experimental purpose and principle Specific antibody clones targeting GPRC5D antigen were enriched from phage antibody library by affinity panning, screened and identified to obtain clones that bind to GPRC5D antigen, but their binding ability and specificity needed to be further confirmed after converting the antibody molecules expressed from prokaryotic system into those expressed from eukaryotic system. To this end, we prepared IgG expression plasmids of these clones and purified Protein A into antibodies by transient transfection of CHOS cells expression. Then, the binding specificity of monoclonals was further identified by ELISA and FACS.

[0225] Further characterization of binding specificity at the protein level by ELISA and at the monoclonal antibody level Main materials and reagents: It is the same as a phage-level ELISA reagent.

[0226] Test Results: In order to further analyze whether these monoclonal antibodies still retain the binding ability and specificity of the original antibodies after being expressed as IgG format antibodies in eukaryotic cell systems, the inventors expressed the three clones obtained in Example 3 by CHOS, purified Protein A into IgG format antibodies, and identified them by enzyme-linked immunosorbent assay (ELISA) with multiple antigens. The results are shown in Figure 5. Positive Benchmark1 Ab is a positive antibody for the target antigen (GPRC5D-VLP), binds to the target antigen, and does not bind to the control antigen. Anti-human IgGHRP Ab is a negative antibody control with only secondary antibody added, and anti-hisHRP Ab is a positive antibody control to detect the antigen tag, binds to the antigen of the his tag, indicating that the coated antigen was bound to the plate. Clones 18, 39, and 41 bind to the GPRC5D antigen and do not bind to the four unrelated antigens, indicating that clones 18, 39, and 41 bind to the GPRC5D antigen and have good specificity.

[0227] Further characterization of binding specificity at the protein level by FACS and at the monoclonal antibody level Main materials and reagents: Fluorescein (FITC) AffiniPure Goat Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoReseach, 109-095-190.

[0228] Test Results: To further analyze whether these monoclonal antibodies still retain the binding ability and specificity of the original antibodies after being expressed as IgG format antibodies in eukaryotic cell systems, the inventors expressed the three clones obtained in Example 3 in CHOS and purified Protein A into IgG format antibodies, which were identified in more antigens and cell lines by applying enzyme-linked immunosorbent and flow cytometry. The results are shown in Figure 6. Clones 18, 39, and 41 bound to both GPRC5D-positive cell lines, CHO-K1-GPRC5D and MM1S, but did not bind to the two GPRC5D-negative cell lines, CHO-K1 and Jurkat, demonstrating good specificity. Positive Benchamrk1 Ab (GC5B596) is a positive antibody for the target antigen, which bound to the high-expression cell line, CHO-K1-GPRC5D, but did not bind to the control negative cell line, CHO-K1. FITC anti-human IgG Ab is a negative control to which only the secondary antibody was added, and did not bind to either of the two cell lines.

[0229] Example 5. Testing the binding ability of monoclonal antibodies to high-expressing cell lines by FACS The degree of affinity between GPRC5D antibody molecules and antigens may have a significant impact on the killing effect and survival time of CAR-T or antibody drugs in the patient's body. Because it is difficult to obtain purified antigens for the target GPRC5D, the inventors adopted the FACS binding method to analyze the half effective concentration (Ec50) of the antibody molecules, providing important information for the development process.

[0230] The brief steps of a FACS binding experiment are as follows. 1) Preparation of antibodies at different concentrations: Anti-GPRC5D IgG was diluted 5-fold in sequence starting from 300 nM using PBS, and then diluted down to 0.00384 nM to obtain a total of eight concentrations, to prepare the binding ability of the antibody to CHO-K1-GPRC5D cells. 2) Wash CHO-K1-GPRC5D cells twice with PBS and then resuspend them in 1x10 cells in PBS. 7 / mL and dispense 50 μL into a 96-well deep well plate.

[0231] 3) Add 50 μL of diluted antibody to each well, mix evenly, and then bind at 4° C. for 2 h. 4) Wash twice with 200 μL of PBS. 5) Add 1:300 diluted Alexa FluorR 647 AffiniPure Goat Anti-Human IgG (100 μL / well), mix evenly by pipetting, and then incubate at room temperature for 45 min.

[0232] 6) Wash twice with 200 μL of PBS and finally resuspend the cells in 200 μL of PBS. 7) Measure the fluorescence intensity of the sample in the Alexa FluorR 647 channel using a flow cytometer. 8) Analyze the binding constants via Graphpad Prism software.

[0233] Main materials and reagents: CHO-K1-GPRC5D cell line, GPRC5D-positive cell line; Alexa FluorR 647 AffiniPure Goat Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoReseach, 109-605-008.

[0234] Experimental Results Affinity refers to the strength with which a single molecule binds to its ligand, and generally, the strength of the interaction between two molecules can be evaluated and ranked by measuring the binding ability to positive cell lines by FACS. The smaller the Ec50 value, the greater the affinity of the antibody to its target. As shown in Table 3 and Figure 7, Benchmark1, Clone 18, Clone 39 and Clone 41 all bind to the GPRC5D overexpressing cell line CHO-K1-GPRC5D, Clone 18 has a slightly higher affinity than Benchamrk1, and a higher affinity than Clone 39 and Clone 41. Table 3. Affinity measurement results [Table 5]

[0235] Example 6. FACS detection of cross-species cross-reactivity of monoclonal antibodies To investigate the cross-species properties of the three monoclonal antibodies identified in the study, we detected their binding to mouse and monkey-derived GPRC5D target antigens. Because the GPRC5D antigen has a complex structure and it is difficult to obtain purified antigens, we constructed overexpression cell lines from monkeys and mice and confirmed the cross-species properties of the candidate antibodies by FACS analysis. The brief steps of the FACS binding experiment are the same as in Example 4.

[0236] Main materials and reagents: CHOS-mouseGPRC5D cell line, a positive cell line derived from GPRC5D-overexpressing mice; CHOS-macaque GPRC5D cell line, a positive cell line derived from monkeys overexpressing GPRC5D; Alexa FluorR 647 AffiniPure Goat Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoReseach, 109-605-008; Fluorescein (FITC) AffiniPure Goat Anti-Human IgG (H+L), Jackson ImmunoReseach, 109-095-008.

[0237] Test Results: GPRC5D antigen has a complex structure, and it is difficult to obtain purified antigen. We overexpressed mouse-derived GPRC5D protein and monkey-derived GPRC5D protein on CHOS cells, respectively. The cross-characteristics in monkeys and mice were confirmed by detecting the binding of candidate antibodies and overexpressing cell lines. As shown in Figure 8A, clones 18 and 41 all bound to CHOS-macaque GPRC5D, i.e., GPRC5D monkey-derived positive cell line, clone 39 had weak cross-reaction with Benchamrk1 monkey, and as shown in Figure 8B, clones 18, 41, 39 and benchmark1 all bound to CHOS-mouse GPRC5D cell line, i.e., GPRC5D mouse-derived positive cell line. Clone 18 and Benchamrk1 bound more strongly to mouse GPRC5D cell line than clone 39 and clone 41.

[0238] Example 7. Detection of CAR-T cell surface EGFRt expression The function of the candidate clone on CAR-T cells is verified. Based on the candidate antibody sequence, the inventors constructed a CAR vector with the structure shown in FIG. 9, the CAR structure includes CD8α signal peptide, VHH or scFv, CD8α hinge region, CD8α transmembrane region, 4-1BB costimulatory molecule and CD3ζ, and the truncated EFGR molecule (EGFRt) can be connected with T2A to be used as a safety switch during clinical transformation. Then, lentivirus packaging is performed on the CAR vector, and the EGFRt expression status is detected 5 to 7 days after transfection of the CAR lentivirus into T cells (FIG. 10). Since EGFRt is co-expressed with the CAR molecule, it can be used as an indirect detection indicator of the T cell surface distribution of the CAR molecule, and does not affect the structure and function of the CAR.

[0239] The simple experimental steps for CAR-T / EGFRt expression detection are as follows: 1) 1×10 6 Remove CAR-T / T cells / well, wash once with PBS, centrifuge at 300g for 5 min, and discard the supernatant. 2) Resuspend the cell pellet in 100 μL of PBS, add 5 μL each of APC-CD5 antibody and APC-EGFR antibody, and incubate at 4° C. in the dark for 15 minutes. 3) Wash twice with PBS and centrifuge at 300g for 5 minutes. 4) Resuspend in 200 μL of PBS and detect by flow cytometry.

[0240] Main materials and reagents: APC anti-human EGFR Antibody, Clone AY13, BioLegend, Cat. No.352906; Fetal bovine serum (FBS), Gibco, Cat. No. 10099141. Test Results: As shown in FIG. 10, on day 7, T cells in each group were detected to express EGFR, all of which were ≧30%.

[0241] Example 8. CD107a degranulation experiment Experimental principle and purpose: CD107a is a marker for intracellular microvesicles. When microvesicles carrying granzymes fuse with the cell membrane, CD107a on the cell membrane increases, and when their recovery is blocked by monesin (purchased from BioLegend), it can quantitatively reflect the release intensity of microvesicles. When CAR-T is stimulated by a target antigen on a target cell, it causes the release of granzymes, and the activation status of T cells can be determined by detecting the increase in CD107a by flow cytometry.

[0242] The simple experimental steps for CD107a degranulation are as follows. 1) CAR-T cells and target cells to be measured were centrifuged at room temperature at 300g for 5 min, the supernatant was discarded, and then 2x10 cells were cultured in 1640 medium + 10% FBS. 6 Resuspend at 37.5 cells / mL. 2) Add 100 μL of the CAR-T cells to be measured and 100 μL of the target cells to a 96-well plate and mix evenly. 3) Add 1.5 μL of PE / Cy7 anti-human CD107a antibody and 0.2 μL of monensin to the cells in each well, then place in a cell incubator and incubate at 37°C, 5% CO2 for 4 hours.

[0243] 4) After incubation, centrifuge at 300 g for 5 minutes at 4°C, discard the supernatant, and wash the cells twice with 200 μL of PBS. 5) Resuspend the cells in 100 μL of PBS, add 1.5 μL each of BV421 anti-human CD8 and APC anti-human EGFR antibodies, mix thoroughly, and then incubate on ice in the dark for 20 minutes. 6) After incubation, wash the cells three times with 200 μL of PBS, resuspend in 100 μL of PBS, and then detect by flow cytometry.

[0244] Main materials and reagents: Target cells U266-luc (GPRC5D+), 8226-luci (GPRC5D+), MM.1S-luci (GPRC5D+), Raji-luc (GPRC5D-), CCRF-luc (GPRC5D-), Molt4-luc (GPRC5D-), Jurkat-luc (GPRC5D-); Fetal bovine serum (FBS), Gibco, Cat. No. 10099141; Monensin, BioLegend, Cat. No. 420701; PE / Cy7 mouse anti-human CD107a, BD, Cat. No. 561348; BV421 mouse anti-human CD8, BD, Cat. No. 301036; APC anti-human EGFR Antibody, Clone AY13, BioLegend, Cat. No.35290.

[0245] Test Results: CAR-T cells were obtained by lentivirus transduction, and the CAR-T cells were cultured in vitro for 9-12 days before the CD107a degranulation experiment. The CAR-T cells and target cells to be detected were incubated with monensin and CD107a antibody for 4 h, and the cell density of both the CAR-T cells and target cells was 2×10 5 cells / mL. The samples were then labeled with CD8 and EGFR antibodies, and then detected by flow cytometry. The Flowjo software was used to analyze the data, and a live cell gate (P1) was selected from the scatter plot, and cell debris was removed. The cells in the P1 gate were analyzed to select a single dispersed cell gate (P2). Next, CD8-positive cells were further selected in the P2 gate (P3). Finally, the proportion of CD107a-positive cells in cells positively stained with EGFR antibodies (i.e., CAR-positive cells) was analyzed in the P3 gate. The analysis results are shown in Table 4, and GPRC5D CAR-T showed a significant increase in degranulation levels only after co-incubation with GPRC5D-positive cells, but there was no significant increase in degranulation levels after co-incubation with negative cell lines. Among them, the degranulation levels of GPRC5D-positive cells of clone numbers 18, 39, and 41 were high, and all of them were higher than Benchmark1.

[0246] Table 4. Results of CD107a degranulation effects of different target cells on different clones of CAR-T cells. [Table 6]

[0247] Example 9. In vitro cell killing experiments Experimental objectives and principles: In vitro cell killing experiments are performed to evaluate the antigen-specific killing ability of GPRC5D CAR-T cells using U266, MM1.s, 8226, and H929 as GPRC5D positive target cells, and Raji and CCRF cells as GPRC5D negative target cells. Here, the above target cells are obtained by lentiviral transduction to obtain target cells that stably express firefly luciferase, so that the activity of luciferase in the sample can reflect the number of target cells. CAR-T cells and target cells are co-incubated and cultured. When target cells are killed by CAR-T cells, luciferase is released and rapidly inactivated (firefly luciferase half-life is about 0.5 hours). If target cells are not killed or inhibited by CAR-T cells, more luciferase will be produced as target cell proliferation and luciferase expression continue. Therefore, luciferase activity can be used to detect the killing of target cells by CAR-T.

[0248] The brief experimental steps for in vitro cell killing are as follows. 1) Centrifuge the above target cells at room temperature at 500 g for 5 min, discard the supernatant, and then resuspend the cells in 1640 + 10% FBS medium at 1x10 5 Resuspend at 37.9 cells / mL and add 100 μL of target cells to each well of a 96-well plate. 2) Based on the CAR positive rate and effector-to-target ratio of the CAR-T sample to be measured, add 100uL of CAR-T cells to each well of a 96-well plate, mix evenly with the target cells, and then place in a carbon dioxide incubator and incubate for 24h. 3) Using a luciferase detection kit, detect the luciferase activity in each well sample.

[0249] Main materials and reagents: GPRC5D positive target cells U266-luc, MM1.s-luc 8226-luc, H929-luc GPRC5D-negative target cells Raji-luc, CCRF-luc; Steady-Glo Luciferase Assay System, Promega, Cat. No. E2520.

[0250] Test Results: CAR-T cell samples and a certain number of target cells (1x10 4 (1000 cells / mL) are mixed at different effector-to-target ratios (E:T) and then co-incubated for 24 h, and then the luciferase activity (RLU) in the samples is detected. Luciferase activity can reflect the number of target cells in a sample, so the change in luciferase activity in a sample can give the killing / inhibition ability of CAR-T cells against target cells. The lower the luciferase activity reading (RLU), the more the target cells are killed.

[0251] As shown in Figure 11, all CAR-T cell samples were able to effectively kill GPRC5D-positive target cells. Among them, the killing of clones 18, 39, and 41 was higher than that of Benchmark 1 in both 8226-luc and U266-luc. Negative cells Raji were killed to a certain extent by Benchmark 1. Clones 18, 39, and 41 did not have non-specific killing of negative cells. Based on CD107a degranulation experiments and in vitro cytotoxicity experiments, clones 18, 39, and 41 were all superior to benchmark 1 in terms of in vitro function. Below, we will further verify and evaluate the CAR cytokines and in vitro proliferation ability of these three clones.

[0252] Example 10. Measurement of cytokine expression on CAR cells Experimental purpose and principle U266, MM1.s, 8226, and H929 are used as GPRC5D positive target cells, and Raji and CCRF cells are used as GPRC5D negative target cells. GPRC5D CAR-T cells and target cells are incubated, and the expression of IFN-r and TNFa in CAR cells is measured by flow cytometry.

[0253] The simple experimental steps are as follows. 1) CAR-T cells and target cells to be measured were each centrifuged at room temperature at 300g for 5 min, the supernatant was discarded, and then 8x10 cells were cultured in 1640 medium + 10% FBS. 6 Resuspend at 37.5 cells / mL. 2) Add 50 μL of the CAR-T cells to be measured and 50 μL of the target cells to each 96-well plate and mix thoroughly. 3) Add 50 μL of a premix of monensin and brefeldin A (corresponding to 0.2 μL / well of each) to the cells in each well, then place in a cell incubator and incubate at 37° C., 5% CO 2 for 4 hours.

[0254] 4) After incubation, centrifuge at 300 g for 5 minutes at 4°C, discard the supernatant, and wash the cells twice with 200 μL of PBS. 5) Resuspend the cells in 100 μL of PBS, add 1 μL each of BV421 anti-human CD8 and APC anti-human EGFR antibodies, mix thoroughly, incubate on ice in the dark for 20 minutes, and wash twice with 200 μL of PBS. 6) Add 100 μL of fixative to each well, incubate at 4°C for 20 minutes, and wash twice with 1x wash buffer. 7) Add 50 μL of PE-TNFα and PE-Cy7-INFγ antibody buffer (1.5 μL each), wash twice with 1x wash buffer at 4° C. for 20 minutes, resuspend in 100 μL of PBS, and then detect by flow cytometry.

[0255] Main materials and reagents: Target cells U266-luc (GPRC5D+), 8226-luc(GPRC5D+), MM.1S-luc (GPRC5D+)H929-luc (GPRC5D+) Raji-luc (GPRC5D-), CCRF-luc (GPRC5D-) Fetal bovine serum (FBS), Gibco, Cat. No. 10099141; Monensin, BioLegend, Cat. No. 420701; PE / Cy7 mouse anti-human CD107a, BD, Cat. No. 561348; BV421 mouse anti-human CD8, BD, Cat. No. 301036; APC anti-human EGFR Antibody, Clone AY13, BioLegend, Cat. No.35290 PE anti-human TNF-a Biolegend, Cat. No.502909 PE-Cy7 anti-human IFN-r Biolegend, Cat. No. 506578.

[0256] Experimental Results Analyze with Flowjo software, select live cell gate (P1) from the scatter plot, and remove cell debris. Analyze cells in P1 gate to select single dispersed cell gate (P2). Then, further select CD8 positive cells in P2 gate (P3). Finally, analyze the proportion of TNF-α / IFN-r positive cells in cells stained positive with TNF-α antibody or IFN-r (i.e., CAR positive cells) in P3 gate. The analysis results are shown in Table 5, and GPRC5D CAR-T showed a significant increase in the levels of TNF-α and IFN-r only after co-incubation with GPRC5D positive cells, but there was no significant increase in the levels of TNF-α and IFN-r after co-incubation with negative cell lines. Among them, the levels of TNF-a and IFN-r of GPRC5D positive cells of clone numbers 18, 39, and 41 were high, and all were higher than Benchmark1. At the same time, the three clones had uniform expression of TNF-α and IFN-r in each positive target cell, and Benchmark1 had low secretion of these two cytokines under stimulation by U266 and H929.

[0257] Table 5. Percentage of cytokine IFNγ-positive cells and TNFα cells in CAR-T cells after co-incubation of CAR-T cells with multiple target cells (8226-luc, U266-luc, MM1.s-luc, H929-luc, CCRF-luc, Raji-luc) in CAR. [Table 7]

[0258] Example 11. Repeated stimulation proliferation experiments Experimental objectives and principles: Mitomycin-treated target cells 8226 were mixed with different groups of GPRC5D CAR-T cells, and after multiple stimulations, the CAR-T cells and target cells were co-incubated and cultured, thereby determining the proliferation ability after multiple stimulations by different CAR-T target cells.

[0259] The simple experimental steps are as follows. 1) 8226 7×10 6 The cells are harvested and centrifuged at 300 g for 5 min at room temperature. 2) The density of the complete medium is 0.2 x 10 6 The solution was adjusted to cells / mL, 5 μL of Mitomycin mother solution (1 μg / μL) was added, mixed uniformly, and then cultured for 24 h before use. 3) After 24 h of treatment, the 8226-Mitomycin cells were taken, centrifuged at 300 g to exchange the liquid, washed six times with PBS, and resuspended in CTS medium and counted to obtain a density of 6 × 10 6 Adjust to cells / mL and prepare for use.

[0260] 4) 3 x 10 each 5Take 100 CAR-T cells and transfer them to a 24-well plate. Add 50 μL of 8226-Mitomycin cells to each well of CAR-T to make the effector-to-target ratio E:T = 1:1. Refill with CTS complete medium until the final volume of the culture is 500 μL, mix evenly, culture at 37 degrees and 5% CO2 for 96 h, count, and treat the target cells (CCRF-CEM) with Mitomycin again. Repeat the above steps to create an amplification curve.

[0261] Main materials and reagents: Mitomycin C, MCE, Cat.No. HY-113061; Target cells: 8226. Test Results: As shown in Table 6 and Figure 12, all four sets of CAR-T samples proliferated efficiently after repeated stimulation, and after 7 stimulations of target cells, the CAR-T cell proliferation ability of clone numbers 18, 39, and 41 was greater than that of Benchmark 1. The proliferation ability of CAR-T cells after stimulation by target cells is closely related to the long-term prognosis of patients, therefore, it is believed that CAR clones 2290 (#18), 2436 (#39), and 2438 (#41) have greater long-term proliferation ability and the potential to eliminate tumor cells.

[0262] Table 6. In vitro proliferation status of each CAR-T after multiple stimulation of target cells (8226) [Table 8]

[0263] Example 12. Measuring the ability of organ-derived tumors to stimulate CAR cell degranulation CD107a is a marker for intracellular microvesicles. When microvesicles carrying granzymes fuse with the cell membrane, CD107a on the cell membrane increases, and when their recovery is blocked by monesin (purchased from BioLegend), it can quantitatively reflect the release intensity of microvesicles. When CAR-T is stimulated by a target antigen on a target cell, it causes the release of granzymes, and the activation status of the target cell can be determined by detecting the increase in CD107a by flow cytometry.

[0264] The simple experimental steps for CD107a degranulation are as follows. 1) CAR-T cells and target cells to be measured were centrifuged at room temperature at 300g for 5 min, the supernatant was discarded, and then 2x10 cells were cultured in 1640 medium + 10% FBS. 6 Resuspend at 37.5 cells / mL. 2) Add 100 μL of the CAR-T cells to be measured and 100 μL of the target cells to a 96-well plate and mix evenly. 3) 1.5 μL of PE / Cy7 anti-human CD107a antibody and 0.2 μL of monensin were added to the cells in each well, and the wells were then placed in a cell incubator (37° C., 5% CO2) and incubated for 4 hours.

[0265] 4) After incubation, centrifuge at 300 g for 5 minutes at 4°C, discard the supernatant, and wash the cells twice with 200 μL of PBS. 5) Resuspend the cells in 100 μL of PBS, add 1.5 μL each of BV421 anti-human CD8 and APC anti-human EGFR antibodies, mix thoroughly, and then incubate on ice in the dark for 20 minutes. 6) After incubation, wash the cells three times with 200 μL of PBS, resuspend in 100 μL of PBS, and then detect by flow cytometry.

[0266] Main materials and reagents: Target cells 8226, 293T, A375, HepG2, Nalm6, NCHI460, OVCAR-3, Raji, HCT116, Panc-1, MDA-MB-468, KATOIII Fetal bovine serum (FBS), Gibco, Cat. No. 10099141; Monensin, BioLegend, Cat. No. 420701; PE / Cy7 mouse anti-human CD107a, BD, Cat. No. 561348; BV421 mouse anti-human CD8, BD, Cat. No. 301036; APC anti-human EGFR Antibody, Clone AY13, BioLegend, Cat. No.35290.

[0267] Test Results: CAR-T cells were obtained by lentivirus transduction, and the CAR-T cells were cultured in vitro for 9-12 days before the CD107a degranulation experiment. The CAR-T cells and target cells to be detected were incubated with monensin and CD107a antibody for 4 h, and the cell density of both the CAR-T cells and target cells was 2×10 6cells / mL. The samples were then labeled with CD8 and EGFR antibodies, and then detected by flow cytometry. The samples were analyzed using Flowjo software, and a live cell gate (P1) was selected from the scatter plot, and cell debris was removed. The cells in the P1 gate were analyzed to select a single dispersed cell gate (P2). Next, CD8-positive cells were further selected in the P2 gate (P3). Finally, the proportion of CD107a-positive cells in cells stained positively with EGFR antibodies (i.e., CAR-positive cells) was analyzed in the P3 gate. The analysis results are shown in Figure 13, and GPRC5D CAR-T of clones 18, 39, and 41 showed a significant increase in the degranulation level only after co-incubation with GPRC5D-positive cells, but there was no significant increase in the degranulation level after co-incubation with cell lines of organ-derived tumor cells. It was shown that GPRC5D CAR-T cells are less likely to be activated by normal tissue organ cells, and have a high level of safety.

[0268] The above in vitro experiments demonstrated that the CAR-T cells of candidate clones 18, 39, and 41 could effectively recognize the GPRC5D antigen on tumor cells and produce effective killing, and the in vitro negative cells were verified to have no non-specific activation and killing activity, which is expected to be highly safe in clinical practice. Repeated stimulation experiments showed that the three candidate clones were efficiently amplified under target antigen stimulation in vitro and had long-term effective potential to kill tumor cells.

[0269] Example 13. Antitumor drug efficacy experiment in NCG tumor-bearing mice The antitumor efficacy of the three candidate clones in mice will be further verified. The inventors have identified a tumor cell line, H929-Luc, which expresses firefly luciferase (ffLuc), to be able to express firefly luciferase normally, but H929 itself is a naturally occurring cell line that highly expresses GPRC5D. H929-1uc cells will be inoculated into immunodeficient NCG mice via the tail vein, and after tumors are formed in the mice, candidate clone CAR-T cells will be inoculated to verify their antitumor activity.

[0270] Easy test flow is as follows: A cell line, H929-luc, that overexpresses firefly luciferase (ffLuc) is generated, and one that can normally express firefly luciferase is identified. The cells are grown to logarithmic growth phase and then harvested. Forty-eight NCG mice were purchased, housed for 1 week, and injected with H929-luc cells via the tail vein, each at 5 × 10 6 Inject cells / 200 μL of PBS and observe the general condition of the mice daily after injection. On day 10, the mice are grouped and injected via the tail vein with CAR-T cells (4E+06 CAR+ cells / mouse), control non-transfected Mock-T cells (2E+07 cells / mouse), and PBS buffer, respectively. Imaging observations are performed periodically, the mice are weighed, and the general condition of the mice is observed and recorded, and the proliferation of T cells in the mouse blood is regularly measured.

[0271] Test Results: The efficacy of the mouse in vivo experiment is shown in Figure 14. As shown in Figure 14A, PBS, mockT, and benmark1 continued to increase the tumor burden after reintroduction of CAR cells, and clone 41 had a slightly lower tumor burden than PBS and mockT, but had no significant inhibitory effect on tumor growth. The tumor burden of clones 39 and 18 was significantly inhibited. However, clone 18 mice gradually died on the 7th day after reinjection, which is expected to be highly toxic to mice and have low clinical safety, and the inhibitory effect is not clear because only the remaining one mouse was evaluated for the end point photon count. The tumor inhibitory effect of clone 39 was significant, and the survival rate of the mice was high (Figures 14A and 14B). Figure 14C shows the status of T cell proliferation in the blood of mice, and mockT and benmark1 T cell proliferation was not significant, while clone 39 maintained a sustained and efficient expansion in vivo that was significantly better than clone 41 and clone 18. In conclusion, clone 39 has the highest in vivo antitumor efficacy and is also highly safe in mice, making it an excellent candidate clone for late-stage clinical development.

[0272] The nucleotide and amino acid sequences of the Benchmark 1 antibody herein are as follows: Benchmark1 nucleotide sequence (SEQ ID NO:40) GGATCCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACAGCTTCACCGGCTACACCATGAACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCCTGATCAACCCCTACAACAGCGACACCAACTACGCCCAGAAGCTGCAGGGCCGCGTGACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAGCTGCGCAGCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCGTGGCCCTGCGCGTGGCCCTGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCAAGGCCAGCCAGAACGTGGCCACCCACGTGGGCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCGCCTGATCTACAGCGCCAGCTACCGCTACAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAACCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACCGCTACCCCTACACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGTCTAGA

[0273] Benchmark1 Amino Acid Sequence (SEQ ID NO: 41) GSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIKSR

[0274] References: 1. Shah U A, Mailankody S. Emerging immunotherapies in multiple myeloma. BMJ (online), 2020, 370:m3176 2. Soekojo C Y, Ooi M, Mel S D, et al. Immunotherapy in Multiple Myeloma. Cells, 2020, 9 (3):601. 3. Feng, D, Sun, J. Overview of anti-BCMA CAR-T immunotherapy for multiple myeloma and relapsed / refractory multiple myeloma. Scand J Immunol. 2020; 92: e12910.

[0275] 4. https: / / ash.confex.com / ash / 2022 / webprogram / Paper157988.html 5. https: / / www.fda.gov / drugs / resources-information-approved-drugs / fda-approves-teclistamab-cqyv-relapsed-or-refractory-multiple-myeloma 6. https: / / ash.confex.com / ash / 2022 / webprogram / Paper159707.html

[0276] 7. Nikhil C. Munshi, Larry D. Anderson, Jr., Nina Sha et al. Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma. N Engl J Med 2021; 384:705-716 8. CARVYKTI TM Prescribing Information. Horsham, PA: Janssen Biotech, Inc.

[0277] 9. Smith EL, et al. GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells. Sci Transl Med. 2019 Mar. 27;11(485) 10. Kodama T, Yu K , Nakai W , et al. Anti-GPRC5D / CD3 Bispecific T-Cell-Redirecting Antibody for the Treatment of Multiple Myeloma. Molecular Cancer Therapeutics, 2019, 18(9):molcanther.1216.2018.

[0278] 11. Kodandaram Pillarisetti, et. al. A T-cell-redirecting bispecific G-protein-coupled receptor class 5 member D x CD3 antibody to treat multiple myeloma. Blood. 2020 Apr 9; 135(15): 1232-1243. 12. Sham Mailankody, et al. GPRC5D-Targeted CAR T Cells for Myeloma. N. Engl. J. Med. 2022;387:1196-206.

Claims

1. A GPRC5D-binding peptide comprising a heavy chain variable region of an antibody molecule, wherein HCDR1, HCDR2, and HCDR3 in the heavy chain variable region are HCDR1 is GGSFSGYY (SEQ ID NO: 1), HCDR2 is INHSGST (SEQ ID NO: 2), Combination 1) in which the sequence of HCDR3 is ARARRYGGRTRFDP (SEQ ID NO: 3); HCDR1 is GFIFSSYG (SEQ ID NO: 4), The sequence of HCDR2 is ISSSGDYT (SEQ ID NO:5), Combination 2) in which the sequence of HCDR3 is ARMSFRRYDH (SEQ ID NO: 6); and HCDR1 is GFSFSGYI (SEQ ID NO: 7), The sequence of HCDR2 is TSSSGTET (SEQ ID NO:8), 3), in which the sequence of HCDR3 is ARYYSKYGRSYHVDS (SEQ ID NO:9).

2. The GPRC5D-binding peptide of claim 1 , which is an antibody molecule or an antigen-binding fragment thereof.

3. The GPRC5D-binding peptide according to claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO: 10, 11 or 12, or the heavy chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 10, 11 or 12, and is capable of specifically binding to GPRC5D.

4. The GPRC5D-binding peptide according to any one of claims 1 to 3, which is a single domain antibody molecule.

5. The GPRC5D-binding peptide according to any one of claims 1 to 4, which is a fully human antibody molecule.

6. The GPRC5D-binding peptide according to any one of claims 1 to 5, wherein the binding ability to cells expressing GPRC5D on their surface is such that the EC50 value detected by FACS is 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, or 5 nM or less.

7. A fusion protein comprising at least one GPRC5D-binding peptide according to any one of claims 1 to 6.

8. The fusion protein according to claim 7, comprising at least two GPRC5D-binding peptides according to any one of claims 1 to 6.

9. The fusion protein according to claim 7 or 8, wherein the HCDR1, HCDR2 and HCDR3 of the two GPRC5D binding peptides are each selected from the different combinations listed in claim 1.

10. one of the two GPRC5D-binding peptides comprises an amino acid sequence as shown in SEQ ID NO: 10 or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence as shown in SEQ ID NO: 10, and can specifically bind to GPRC5D; the other comprises an amino acid sequence as shown in SEQ ID NO: 11 or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence as shown in SEQ ID NO: 11, and can specifically bind to GPRC5D; one of the two GPRC5D-binding peptides comprises an amino acid sequence as shown in SEQ ID NO: 10 or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence as shown in SEQ ID NO: 10, and can specifically bind to GPRC5D; the other comprises an amino acid sequence as shown in SEQ ID NO: 12 or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence as shown in SEQ ID NO: 12, and can specifically bind to GPRC5D; The fusion protein according to any one of claims 7 to 9, wherein one of the two GPRC5D-binding peptides comprises an amino acid sequence as shown in SEQ ID NO: 11 or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence as shown in SEQ ID NO: 11 and is capable of specifically binding to GPRC5D, and the other comprises an amino acid sequence as shown in SEQ ID NO: 12 or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence as shown in SEQ ID NO: 12 and is capable of specifically binding to GPRC5D.

11. The fusion protein according to any one of claims 7 to 10, comprising at least three of the GPRC5D binding peptides, wherein the HCDR1, HCDR2 and HCDR3 of the three of the GPRC5D binding peptides are each a combination of the three listed in claim 1.

12. The first of the three GPRC5D-binding peptides of the fusion protein comprises an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence as set forth in SEQ ID NO: 10, and can specifically bind to GPRC5D; the second comprises an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence as set forth in SEQ ID NO: 11, and can specifically bind to GPRC5D; and the third comprises an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence as set forth in SEQ ID NO: 12, and can specifically bind to GPRC5D. The fusion protein according to any one of claims 7 to 11, comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 12 and capable of specifically binding to GPRC5D.

13. The fusion protein according to any one of claims 7 to 12, further comprising an Fc fragment.

14. The fusion protein according to any one of claims 7 to 13, further comprising another binding peptide capable of specifically binding to an antigen different from GPRC5D.

15. The fusion protein according to any one of claims 7 to 14, wherein the further binding peptide is a single domain antibody or a single chain antibody.

16. The fusion protein of any one of claims 7 to 15, further comprising a detectable or purification tag.

17. The fusion protein according to any one of claims 7 to 16, wherein the detectable tag has enzymatic activity.

18. A nucleic acid molecule encoding the GPRC5D-binding peptide according to any one of claims 1 to 6 or the fusion protein according to any one of claims 7 to 17.

19. 19. The nucleic acid molecule of claim 18, comprising the nucleotide sequence shown in SEQ ID NO: 13, 14 or 15.

20. 20. An expression vector comprising the nucleic acid molecule of claim 18 or 19.

21. 21. A host cell comprising a nucleic acid molecule according to claim 18 or 19 or an expression vector according to claim 20.

22. A multispecific antibody molecule comprising at least a first functional portion and a second functional portion, wherein the first functional portion comprises a GPRC5D binding peptide described in any one of claims 1 to 6, and the second functional portion has a binding specificity different from that of the first functional portion.

23. The multispecific antibody molecule of claim 22, wherein the second functional portion has binding specificity for an immune cell.

24. The multispecific antibody molecule of claim 22 or 23, wherein the second functional portion has binding specificity for a T cell.

25. The multispecific antibody molecule according to any one of claims 22 to 24, wherein the second functional portion has binding specificity for CD3.

26. An immunoconjugate comprising a GPRC5D-binding peptide according to any one of claims 1 to 6 linked to a therapeutic agent.

27. The immunoconjugate of claim 26 , wherein the therapeutic agent is a drug.

28. 28. The immunoconjugate of claim 26 or 27, wherein the therapeutic agent is cytotoxic.

29. The immunoconjugate of any one of claims 26 to 28, wherein the therapeutic agent is a radioisotope.

30. 1) A GPRC5D-binding peptide according to any one of claims 1 to 6, A fusion protein according to any one of claims 7 to 17, A multispecific antibody molecule according to any one of claims 22 to 25, or An immunoconjugate according to any one of claims 26 to 29, and 2) A pharmaceutical composition comprising a pharma- ceutically acceptable carrier.

31. Use of a GPRC5D-binding peptide according to any one of claims 1 to 6, a fusion protein according to any one of claims 7 to 17, a multispecific antibody molecule according to any one of claims 22 to 25 or an immune complex according to any one of claims 26 to 29 in the preparation of a medicament for treating a GPRC5D-related disease.

32. 32. The use according to claim 31 , wherein the GPRC5D-related disease is cancer or an autoimmune disease, said cancer being preferably a plasma cell malignant disease such as multiple myeloma, or a B-cell malignant disease such as Hodgkin's lymphoma or non-Hodgkin's lymphoma.

33. A method for treating a GPRC5D-related disease comprising administering to a subject in need thereof an effective amount of a GPRC5D-binding peptide according to any one of claims 1 to 7, a fusion protein according to any one of claims 7 to 17, a multispecific antibody molecule according to any one of claims 22 to 25, an immunoconjugate according to any one of claims 26 to 29 or a pharmaceutical composition according to claim 30.

34. 34. The method of claim 33, wherein the GPRC5D-related disease is cancer or an autoimmune disease.

35. 35. The method of claim 34, wherein the cancer is a plasma cell malignancy or a B cell malignancy.

36. 36. The method of claim 35, wherein the plasma cell malignancy is multiple myeloma and the B cell malignancy is Hodgkin's lymphoma or non-Hodgkin's lymphoma.

37. A method for detecting the content of GPRC5D in a biological sample, comprising: 1) contacting said biological sample with a GPRC5D-binding peptide according to any one of claims 1 to 6, a fusion protein according to any one of claims 7 to 17 or a multispecific antibody molecule according to any one of claims 22 to 25 to form a binding complex with said GPRC5D; and step 2) determining the amount of GPRC5D in the biological sample based on the amount of binding complex produced in step 1).

38. A method for detecting the content of GPRC5D in a biological sample, comprising: 1) linking a GPRC5D binding peptide according to any one of claims 1 to 6 to a detectable tag; 2) contacting the GPRC5D binding peptide having the detectable tag with the biological sample; and 3) determining the content of GPRC5D in the biological sample by detecting the amount of the detectable tag.

39. A detection kit comprising a GPRC5D-binding peptide according to any one of claims 1 to 6, a fusion protein according to any one of claims 7 to 17, or a multispecific antibody molecule according to any one of claims 22 to 25.

40. A pharmaceutical kit comprising a GPRC5D-binding peptide according to any one of claims 1 to 6, a fusion protein according to any one of claims 7 to 17, or a multispecific antibody molecule according to any one of claims 22 to 25.

41. 1. A method for identifying a GPRC5D-related disease in a subject, comprising: 1) determining the content of GPRC5D in a biological sample derived from the subject using the detection kit according to claim 39; and 2) comparing the content of GPRC5D to a normal content of GPRC5D in a population to determine whether or not the GPRC5D-related disease is present or its status.

42. 42. The method of claim 41, wherein the GPRC5D-related disease is cancer or an autoimmune disease.

43. 43. The method of claim 42, wherein the cancer is a plasma cell malignancy or a B cell malignancy.

44. 44. The method of claim 43, wherein the plasma cell malignancy is multiple myeloma and the B cell malignancy is Hodgkin's lymphoma or non-Hodgkin's lymphoma.

45. A method for determining the therapeutic effect of a drug for treating a GPRC5D-related disease in a subject, comprising: 1) identifying a GPRC5D-related disease status of said subject using the method according to any one of claims 42 to 44; 2) treating the subject with the drug; and step 3) repeating step 1) to identify a change in the GPRC5D-related disease state in the subject, and determining the therapeutic efficacy of the drug based on the change.

46. 46. ​​The method of claim 45, wherein the GPRC5D-related disease is cancer or an autoimmune disease, said cancer preferably being a plasma cell malignant disease such as multiple myeloma, or a B-cell malignant disease such as Hodgkin's lymphoma or non-Hodgkin's lymphoma.

47. 47. The method of claim 46, wherein the cancer is a plasma cell malignancy or a B cell malignancy.

48. 48. The method of claim 47, wherein said plasma cell malignancy is multiple myeloma and said B cell malignancy is Hodgkin's lymphoma or non-Hodgkin's lymphoma.

49. The pharmaceutical composition according to claim 30 for treating a GPRC5D-related disease.

50. A chimeric antigen receptor (CAR), comprising one or more antibody molecules or antigen-binding fragments thereof, the extracellular antigen-binding domain of which targets GPRC5D, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the antibody molecule are The sequence of HCDR1 is GGSFSGYY (SEQ ID NO: 1); The sequence of HCDR2 is INHSGST (SEQ ID NO: 2), Combination 1) in which the sequence of HCDR3 is ARARRYGGRTRFDP (SEQ ID NO: 3); The sequence of HCDR1 is GFIFSSYG (SEQ ID NO: 4), The sequence of HCDR2 is ISSSGDYT (SEQ ID NO:5), Combination 2) in which the sequence of HCDR3 is ARMSFRRYDH (SEQ ID NO: 6); and The sequence of HCDR1 is GFSFSGYI (SEQ ID NO: 7), The sequence of HCDR2 is TSSSGTET (SEQ ID NO:8), HCDR3 sequence is ARYYSKYGRSYHVDS (SEQ ID NO: 9).

51. The CAR of claim 50, wherein the extracellular antigen-binding domain comprises two of the antibody molecules or antigen-binding fragments thereof linked in tandem, preferably the antibody molecules or antigen-binding fragments thereof are linked via a linker peptide.

52. The CAR of claim 51 , wherein the two antibody molecules or antigen-binding fragments thereof are the same or different.

53. The CAR of any one of claims 50 to 52, wherein the antibody molecule is a single domain antibody, preferably wherein the antibody molecule is a fully human single domain antibody.

54. The CAR according to any one of claims 50 to 53, wherein the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO: 10, 11 or 12, or the heavy chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 10, 11 or 12, and is capable of specifically binding to GPRC5D.

55. The CAR according to any one of claims 50 to 54, comprising, in order from the N-terminus to the C-terminus, the extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

56. The CAR according to any one of claims 50 to 55, further comprising a signal peptide at the N-terminus.

57. The CAR according to any one of claims 50 to 56, wherein the signal peptide comprises the amino acid sequence shown in SEQ ID NO:

17.

58. The CAR of any one of claims 50 to 57, further comprising a hinge region between the extracellular antigen-binding domain and the transmembrane domain.

59. The CAR according to any one of claims 50 to 58, wherein the signal peptide comprises the amino acid sequence shown in SEQ ID NO:

19.

60. The CAR of any one of claims 50 to 59, wherein the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:

21.

61. The CAR according to any one of claims 50 to 60, wherein the intracellular signaling domain comprises a 4-1BB intracellular domain and a CD3ζ intracellular domain.

62. The CAR according to any one of claims 50 to 61, wherein the 4-1BB intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 23, and / or the CD3ζ intracellular domain comprises the amino acid sequence shown in SEQ ID NO:

25.

63. The CAR of any one of claims 50 to 62, wherein the CAR has an amino acid sequence as set forth in SEQ ID NO: 35, 37 or 39, or the amino acid sequence of the CAR has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence as set forth in SEQ ID NO: 35, 37 or 39.

64. The CAR according to any one of claims 50 to 63, wherein the end of the CAR is further linked to a safety switch for CAR-T cell clearance, preferably the safety switch is linked to the C-terminus of the CAR, more preferably the safety switch comprises a truncated EGFR molecule (tEGFR) or a fusion protein having suicide capability.

65. The CAR according to any one of claims 50 to 64, wherein a self-cleaving polypeptide and tEGFR are further linked to the C-terminus of the CAR, preferably a CSF2RA signal peptide is further linked to the N-terminus of the tEGFR, more preferably the tEGFR comprises the amino acid sequence shown in SEQ ID NO: 31, and / or the CSF2RA signal peptide comprises the amino acid sequence shown in SEQ ID NO:

29.

66. 66. The CAR of any one of claims 50 to 65, wherein the safety switch is linked to the CAR via a self-cleaving polypeptide, preferably wherein the self-cleaving polypeptide comprises the amino acid sequence shown in SEQ ID NO: 27 or 33.

67. The CAR of any one of claims 50 to 66, wherein the CAR is a bispecific CAR, and the extracellular antigen-binding domain further comprises a second antibody molecule, or an antigen-binding fragment thereof, that targets a second target, and the second target is selected from CD3, BCMA, or a combination thereof.

68. An isolated nucleic acid molecule encoding the CAR of any one of claims 50 to 67.

69. 69. The nucleic acid molecule of claim 68, comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 13, 14, 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38.

70. 70. An expression vector comprising the nucleic acid molecule of claim 69.

71. 71. The expression vector of claim 70, selected from a plasmid, a retroviral vector, and a lentiviral vector.

72. A host cell comprising the CAR according to any one of claims 50 to 67, the nucleic acid molecule according to claim 68 or 69, or the expression vector according to claim 70 or 71.

73. A modified immune cell expressing the CAR of any one of claims 50 to 67.

74. 74. The modified immune cell of claim 73, which is an immune cell, preferably an autologous or heterologous immune cell, more preferably said immune cell is a T cell or a NK cell.

75. A pharmaceutical composition comprising a cell according to any one of claims 72 to 74 and a pharma- ceutically acceptable carrier.

76. Use of a CAR according to any one of claims 50 to 67, a nucleic acid molecule according to claim 68 or 69, an expression vector according to claim 70 or 71, or a cell according to any one of claims 72 to 74, in the preparation of a medicament for preventing or treating a GPRC5D-associated disease.

77. 77. The use according to claim 76, wherein the GPRC5D-related disease is cancer or an autoimmune disease, said cancer being preferably a plasma cell malignant disease such as multiple myeloma, or a B-cell malignant disease such as Hodgkin's lymphoma or non-Hodgkin's lymphoma.

78. A method for preventing or treating a GPRC5D-related disease, comprising administering an effective amount of a cell according to any one of claims 72 to 74 or a pharmaceutical composition according to claim 75 to a subject in need thereof for treatment.

79. 79. The method of claim 78, further comprising inhibiting activity of said immune effector cells or said pharmaceutical composition by administering to said subject an EGFR antibody.

80. 80. The method of claim 78 or 79, wherein the GPRC5D-related disease is cancer or an autoimmune disease, said cancer preferably being a plasma cell malignant disease such as multiple myeloma, or a B-cell malignant disease such as Hodgkin's lymphoma or non-Hodgkin's lymphoma.

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