GPRC5D single-domain antibody B11

Abstract

The invention provides a GPRC5D single-domain antibody B11, and the GPRC5D single-domain antibody B11 has relatively strong binding activity and reaction specificity on GPRC5D. The invention provides an amino acid sequence of a single-domain antibody, an antibody derivative, a biological material and application thereof. The single-domain antibody provided by the invention can be used for constructing a chimeric antigen receptor T cell, and the chimeric antigen receptor T cell has stronger killing power on a target cell.

Description

[0001] The present application is a divisional application, the parent application information is as follows: the title of the invention is GPRC5D single domain and humanized antibody thereof; the original application date is November 8, 2024, and the original application number is 202411591311X. TECHNICAL FIELD

[0002] The present application relates to the field of genetic engineering and antibody technology, in particular to GPRC5D single domain antibody B11. BACKGROUND

[0003] Multiple myeloma (MM) is a malignant plasma cell disease, and its tumor cells originate from plasma cells in bone marrow, and plasma cells are cells developed to the final functional stage of B lymphocytes. Therefore, multiple myeloma can be classified into the scope of B lymphocyte lymphoma. WHO classifies it as one of B cell lymphomas, called plasma cell myeloma / plasmacytoma. Its characteristics are abnormal proliferation of bone marrow plasma cells accompanied by excessive production of monoclonal immunoglobulin or light chain (M protein), and a small number of patients can be non-secretory MM without M protein. Multiple myeloma is often accompanied by multiple osteolytic lesions, hypercalcemia, anemia, and kidney damage. Because the production of normal immunoglobulin is inhibited, various bacterial infections are prone to occur.

[0004] At present, the main treatment method for MM is BCMA (B cell maturation antigen) target therapy, but for MM patients with negative or low expression of BCMA, relapse still occurs after receiving targeted BCMA CAR-T cell therapy, and there is a problem of target escape. In order to alleviate the problem of relapse mediated by BCMA escape, it is crucial to find a better specific MM target (such as GPRC5D) or simultaneously target other antigen targets (such as CD3, CD19, CD38, GPRC5D, etc.), and GPRC5D is likely to become a popular candidate target for treating MM.

[0005] GPRC5D (G protein-coupled receptor C5 family subtype D) is an atypical class C orphan G protein-coupled receptor, which belongs to 7 transmembrane proteins. In recent years, GPRC5D has become an extremely attractive target for the treatment of multiple myeloma (MM). This is mainly because GPRC5D is highly expressed on the surface of multiple myeloma cells, while its expression in normal tissues is very limited. Therefore, the present application aims to provide an antibody sequence targeting GPCR5D and its application in CAR-T and immune cell construction, thereby providing a new treatment idea for patients who are ineffective or relapse to existing therapies. SUMMARY

[0006] In order to make up for the shortcomings of the prior art, the purpose of the present application is to provide a GPRC5D single domain antibody B11, its antibody derivatives and applications.

[0007] To achieve the above object, the present application adopts the following technical solutions:

[0008] The first aspect of the present application provides a single-domain antibody of GPRC5D, which comprises HCDR1-3 of a heavy chain variable region shown in SEQ ID NO: 4, and the sequence of the HCDR1-3 is determined according to any one of Kabat, IMGT, Chothia, AbM, Contact numbering system.

[0009] In the context of the present application, the term "antibody" is used in the broadest sense and explicitly covers single-domain antibodies, monoclonal antibodies, polyclonal antibodies, human-derived antibodies, chimeric antibodies and multispecific antibodies (e.g. bispecific antibodies) formed from at least two complete antibodies, as long as they exhibit the desired biological activity.

[0010] In the present application, the term "single-domain antibody" (sdAb) is also called VHH antibody or camelid antibody, which is an artificially designed antibody molecule, a kind of natural light chain missing heavy chain antibody (HCAb) found in camelids, such as llamas, dromedaries and chondrichthyes, such as sharks and skates, which includes two constant regions (CH2 and CH3), a hinge region and a heavy chain variable region (VHH), and then a single-domain antibody containing only one heavy chain variable region, i.e. VHH antibody, is cloned. The crystal structure of VHH antibody is an ellipse with a size of 4 nm x 2.5 nm x 3 nm, and the molecular weight is only 1 / 10 of that of ordinary antibody, about 12-14 kDa, which is the smallest complete antigen binding fragment, so it is also called nanobody.

[0011] In the present application, the term "CDR" refers to the "hypervariable region" or "complementarity determining region" of an antibody, there are 3 CDRs in both heavy chain variable region and light chain variable region, which together form the antigen binding site of the antibody, and can form a precise complement with the antigenic determinant in spatial structure. Examples of other antibody fragments include Fab, Fab', F(ab')2, Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from different antibody fragments. The positions of CDRs in the variable region are marked differently according to different numbering systems, numbering schemes include IMGT, Chothia, Kabat, etc. The IMGT numbering scheme is based on the amino acid sequence alignment of the germline V gene; the Chothia numbering scheme is based on the crystal structure; the Kabat numbering scheme defines the variability parameter as the number of different amino acids at a given position divided by the frequency of the most amino acids at that position. In some embodiments, the sequences of HCDR1-3 of the heavy chain variable region of the antibody described in the present application can be obtained based on the full-length sequence of the heavy chain variable region according to the Kabat, IMGT, Chothia, AbM or Contact numbering system definition, and the CDR sequences defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system are also within the scope of the present application.

[0012] Further, the amino acid sequences of the HCDR1-3 are shown as SEQ ID NO: 1-3.

[0013] Further, the single domain antibody comprises a heavy chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 4 or consists of SEQ ID NO: 4.

[0014] The second aspect of the present application provides an antibody derivative, the antibody derivative comprises any one of the following: 1) the single domain antibody of the first aspect of the present application and a detectable label coupled thereto; 2) a pharmaceutical composition comprising the single domain antibody of the first aspect of the present application and a pharmaceutically acceptable carrier.

[0015] In some embodiments, the detectable label comprises at least one of a radioisotope, a metal nanomaterial, a fluorescein, a biotin, an avidin, a biotin / avidin protein complex, a biotin / avidin protein complex, a chromophore, an electron-dense substance, and an enzyme.

[0016] In the present application, the term "pharmaceutical composition" refers to a composition comprising at least one biologically active compound. The pharmaceutical compositions described in the present application can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. In some embodiments, oral administration is preferred. The pharmaceutical compositions of the present application can contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In certain cases, pharmaceutical acids, bases or buffers can be used to adjust the pH of the formulation to improve the stability of the formulated compound or its administration dosage form. The term parenteral as used in the present application includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions described in the present application can be administered to a subject by any route which enables delivery of the active ingredients to the intended site of action. The pharmaceutical compositions of the present application can also be co-administered with other therapeutic, prophylactic, palliative, and / or alleviating agents for multiple myeloma. Other therapeutic, prophylactic, palliative, and / or alleviating agents for multiple myeloma can be administered simultaneously with, before, or after the primary active ingredient (e.g., the antibody of the first aspect of the present application), even in the same composition. Other therapeutic agents can also be administered separately or in a different dosage form from the primary active ingredient.

[0017] In the present application, the term "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not itself induce the production of antibodies to the individual receiving the composition, and that can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers. Such carriers are well known in the art. The pharmaceutically acceptable carrier in the pharmaceutical composition can include fluids such as water, saline, glycerol, and ethanol. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like can also be present in such vehicles.

[0018] The third aspect of the present application provides a chimeric antigen receptor, which comprises the single-domain antibody of the first aspect of the present application.

[0019] Further, the chimeric antigen receptor further comprises an Fc sequence, an SP export signal, a hinge region, a transmembrane region, a costimulatory signal domain, and an intracellular signal transduction region.

[0020] Further, the Fc sequence is shown in SEQ ID NO: 35.

[0021] Further, the sequence of the SP export signal is shown in SEQ ID NO: 34.

[0022] In some embodiments, the hinge region is selected from the hinge regions of the following molecules: CD8, 4-1BB, IgG1, IgG4, PD-1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof.

[0023] Furthermore, the hinge region is CD8 Hinge.

[0024] Furthermore, the amino acid sequence of the CD8 Hinge is shown in SEQ ID NO: 37.

[0025] In some embodiments, the transmembrane region is selected from the transmembrane regions of the following molecules: CD8, 4-1BB, IgG1, IgG4, PD-1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof.

[0026] Furthermore, the transmembrane region is CD8 TM.

[0027] Furthermore, the amino acid sequence of the CD8 TM is shown in SEQ ID NO: 38.

[0028] In some embodiments, the costimulatory signaling domain is selected from the costimulatory signaling domains of the following molecules: 4-1BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8α, CD8β, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278 and variants thereof.

[0029] Furthermore, the co-stimulatory signaling domain is 4-1BB.

[0030] Furthermore, the amino acid sequence of 4-1BB is shown in SEQ ID NO: 39.

[0031] In some embodiments, the intracellular signal transduction region is selected from the intracellular signal transduction region of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD278, CD21, CD22, FcεRI, FcRγ, FcRβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d and their variants.

[0032] Furthermore, the intracellular signal transduction region is CD3ζ.

[0033] Further, the amino acid sequence of the CD3 zeta is set forth in SEQ ID NO: 40.

[0034] In some embodiments, the chimeric antigen receptor further comprises a suicide gene, a 2A peptide.

[0035] Further, the suicide gene comprises EGFRt.

[0036] Further, the amino acid sequence of the EGFRt is set forth in SEQ ID NO: 43.

[0037] Further, the suicide gene further comprises a signal peptide of the EGFRt.

[0038] Further, the amino acid sequence of the signal peptide of the EGFRt is set forth in SEQ ID NO: 42.

[0039] Further, the 2A peptide is T2A.

[0040] Further, the amino acid sequence of the T2A is set forth in SEQ ID NO: 41.

[0041] Further, the chimeric antigen receptor further comprises a signal peptide.

[0042] Further, the amino acid sequence of the signal peptide is set forth in SEQ ID NO: 36.

[0043] In the present application, the term "binds" or "binds to" refers to a selective binding interaction with an antigen and can be distinguished from non-desired or non-specific interactions. The ability of an antigen binding site to bind to a particular antigen can be determined by enzyme linked immunosorbent assay (ELISA) or other conventional binding assays known in the art.

[0044] In the present application, the modified antibody sequences are also within the scope of the present application. The term "modified" refers to any form of modification to the amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to the replacement of one or more amino acids in the original amino acid sequence with different amino acids. The term "deletion" refers to the reduction of one or more amino acids in the original amino acid sequence. The term "insertion" or "addition" refers to a change in the amino acid sequence resulting in the addition of one or more amino acids compared to the original amino acid sequence.

[0045] In the present application, the antibody sequence modified to have 80% and above identity with the amino acid sequence of the antibody of the first aspect of the present application also falls within the protection scope of the present application. The term "identity" also referred to as "homology" refers to an amino acid sequence having at least 80% sequence identity with the sequence provided in the present application. In order to determine the sequence identity, sequence alignment can be performed by various means known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, Megalign (DNASTAR), Snapgene or DNAMAN software, etc. Those skilled in the art can determine the appropriate parameters for alignment, including any algorithm required to achieve optimal alignment of the full length sequences compared.

[0046] In certain embodiments, those skilled in the art can change the combination category and sequence of the signal peptide, the cytoplasmic hinge region and transmembrane domain, the costimulatory domain and the intracellular immunoreceptor tyrosine activation motif according to the actual situation or needs, regardless of the form of change, as long as the chimeric antigen receptor has the CDR sequence of the heavy chain variable region or the heavy chain variable region sequence of the single domain antibody provided in the present application, which all fall within the protection scope of the present application.

[0047] The fourth aspect of the present application provides a nucleic acid molecule encoding the single domain antibody of the first aspect of the present application, the antibody derivative of the second aspect of the present application and / or the chimeric antigen receptor of the third aspect of the present application.

[0048] In the present application, the term "nucleic acid molecule" refers to any polymer form of either ribonucleotides or deoxyribonucleotides of any length. Typically, a nucleic acid molecule is a coding sequence, which as used herein refers to a DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into an mRNA and translated into a polypeptide in a host cell. The coding sequence is bounded by the start codon at the 5' (amino) terminus and the translation stop end codon at the 3' (carboxy) terminus. The coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even recombinant DNA sequences. A transcription termination sequence will typically be located 3' to the coding sequence.

[0049] Further, the nucleic acid molecule comprises the base sequence encoding HCDR1-3 in the heavy chain variable region of the single domain antibody of the first aspect of the present application as shown in SEQ ID NO: 45. And / or, the nucleic acid molecule comprises the base sequence of the chimeric antigen receptor as shown in SEQ ID NO: 56.

[0050] Further, the base sequence encoding HCDR1-3 is as shown in SEQ ID NO: 61-63.

[0051] Further, the nucleic acid molecule further comprises a base sequence encoding the Fc sequence as shown in SEQ ID NO: 84.

[0052] Further, the nucleic acid molecule further comprises a base sequence encoding the SP membrane egress signal as shown in SEQ ID NO: 83.

[0053] Further, the nucleic acid molecule further comprises a base sequence encoding the CD8 Hinge as shown in SEQ ID NO: 86.

[0054] Further, the nucleic acid molecule further comprises a base sequence encoding the CD8 TM as shown in SEQ ID NO: 87.

[0055] Further, the nucleic acid molecule further comprises a base sequence encoding the CD3 zeta as shown in SEQ ID NO: 89.

[0056] Further, the nucleic acid molecule further comprises a base sequence encoding the 4-1BB as shown in SEQ ID NO: 88.

[0057] Further, the nucleic acid molecule further comprises a base sequence encoding the EGFRt as shown in SEQ ID NO: 92.

[0058] Further, the nucleic acid molecule further comprises a base sequence encoding the signal peptide of the EGFRt as shown in SEQ ID NO: 91.

[0059] Further, the nucleic acid molecule further comprises a base sequence encoding the T2A as shown in SEQ ID NO: 90.

[0060] Further, the nucleic acid molecule further comprises a base sequence encoding the signal peptide as shown in SEQ ID NO: 85.

[0061] The fifth aspect of the present application provides a vector comprising the nucleic acid molecule of the fourth aspect of the present application.

[0062] In the present application, a vector comprising the nucleic acid molecule of the present application is provided. The term "vector" refers to an artificial construct that is capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. The vector of the present application can be a plasmid vector, a viral vector, etc. In some embodiments, the vector refers to a linear or circular nucleic acid molecule comprising the nucleic acid of the present application operably linked to other segments that provide for autonomous replication in a recombinant host cell, or an expression cassette according to the nucleic acid molecule. "Operably linked" means that the nucleic acid sequence of interest is linked to regulatory sequences in a manner that allows expression of the nucleotide sequence, e.g., in an in vitro transcription / translation system or in a host cell into which the vector has been introduced.

[0063] The sixth aspect of the present application provides a recombinant host cell comprising the nucleic acid molecule of the fourth aspect of the present application or the vector of the fifth aspect of the present application.

[0064] In the present application, the term "recombinant host cell" refers to the host cell into which the vector of the present application is introduced, so that the host cell after recombination can transcribe the nucleic acid sequence of interest and / or translate the protein of interest.

[0065] The seventh aspect of the present application provides the use of the single-domain antibody of the first aspect of the present application, the antibody derivative of the second aspect of the present application, the chimeric antigen receptor of the third aspect of the present application, the nucleic acid molecule of the fourth aspect of the present application, the vector of the fifth aspect of the present application and / or the recombinant host cell of the sixth aspect of the present application, characterized in that the use comprises any one of the following: 1) use in constructing CAR-T cells targeting GPRC5D; 2) use in constructing a bi-specific antibody comprising targeting GPRC5D; 3) use in preparing a product for diagnosing, treating the progression of GPRC5D-positive cancer, and / or monitoring the stability of GPRC5D-positive cancer; 4) use in preparing a product for detecting GPRC5D protein.

[0066] In the present application, the term "progression" refers to the malignant change of the disease, including the growth, spread or metastasis of the lesion (such as tumor). In some embodiments, the relative increase in the sum of the diameters of all measured target lesions is at least 20% (if the baseline measurement is the smallest, the baseline value is taken as the reference); in addition, the absolute value of the increase in the sum of the diameters must be at least 5 mm (the appearance of one or more new lesions is also considered as disease progression).

[0067] In the present application, the term "regression" refers to the reduction or complete disappearance of tumor volume, whether spontaneous or as a result of treatment. In some embodiments, tumor regression is assessed by examining tissue samples to determine the extent of reduction in tumor cell population and changes in tumor structure.

[0068] In the present application, the term "stability" refers to the degree of reduction of the target lesion that does not reach the level of disease remission, or the degree of increase that does not reach the level of disease progression, which is between the two. The minimum sum of diameters can be used as a reference during the study.

[0069] In some embodiments, the bi-specific antibody comprising targeting GPRC5D can also target a T cell marker.

[0070] Further, the T cell marker includes CD3, CD19, CD38.

[0071] Further, the T cell marker is CD3.

[0072] Further, the GPRC5D-positive cancer includes multiple myeloma, plasma cell leukemia.

[0073] Further, the GPRC5D-positive cancer is multiple myeloma.

[0074] Further, the product includes a pharmaceutical composition, a kit, a nucleic acid chip, a nucleic acid membrane strip.

[0075] The eighth aspect of the present application provides a method for preparing the single-domain antibody of the first aspect of the present application, the antibody derivative of the second aspect of the present application, or the chimeric antigen receptor of the third aspect of the present application, which comprises artificial synthesis or genetic engineering technology.

[0076] The genetic engineering technology refers to culturing the recombinant host cell of the sixth aspect of the present application under conditions suitable for expression of the antibody.

[0077] Advantages and beneficial effects of the present application: the GPRC5D single-domain antibody B11 provided by the present application has strong binding activity and reaction specificity to GPRC5D. The single-domain antibody can be used to construct a chimeric antigen receptor (CAR) and a bivalent antibody (a bispecific antibody). The CAR constructed based on the antibody sequence can be transduced into T cells to create CAR-T cells targeting GPRC5D, which can be used to treat multiple myeloma. The bivalent antibody designed based on the B11 sequence can simultaneously target GPRC5D and T cells, thereby activating T cells and guiding them to attack tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0078] Figure 1 It is a mixed library sequence case diagram.

[0079] Figure 2 It is a schematic diagram of the construction structure of pcDNA3.4-IgG1Fc vector.

[0080] Figure 3 It is a GPRC5D antibody cell binding verification diagram.

[0081] Figure 4 It is a GPRC5D purified antibody affinity verification diagram.

[0082] Figure 5 It is a GPRC5D candidate purified antibody and CHO-S-Mouse-GPRC5D binding ability detection diagram.

[0083] Figure 6 It is a schematic diagram of the construction structure of CAR-T vector.

[0084] Figure 7 It is a GPRC5D candidate purified antibody CAR-T killing rate detection diagram. DETAILED DESCRIPTION

[0085] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art and do not constitute a limitation of the present invention in any way.

[0086] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. The following examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Simple modifications to the present invention based on the essence of the present invention fall within the scope of protection claimed in the present invention.

[0087] Example 1 Construction of phage display library, selection of antibodies and verification 1. Alpaca immunization Construction of phage display library Two alpacas were immunized for a total of four times. The immunogen was 293F-GPRC5D (human sequence), subcutaneous immunization, and the immune adjuvant was GERBU. After immunization, serum was isolated from the immunized alpacas, and after verification by ELISA and FACS, peripheral blood of the alpacas was collected, PBMC cells were isolated, RNA was extracted, and reverse transcribed into cDNA. A single-domain antibody cloning primer combination was used to amplify the VHH sequence from the cDNA sample and subcloned into the phage display vector pDisplay. The VHH sequence was electroporated into SS320 Escherichia coli competent cells to construct a single-domain antibody phage display library (mixed library of two alpacas). The mixed library sequence is as follows: Figure 1 A library was constructed from two alpacas, and 48 clones were sequenced. After removing two antibody sequences with one base missing and one empty sequence, the remaining sequences were all antibody sequences, indicating good diversity.

[0088] 2. Antibody Selection and Validation The above-mentioned library was panned using the panning protein / cells: GPRC5D-VLP protein / CHO-S-GPRC5D cell line and the negative screening protein / cells: VLP protein / CHO-S cell line. Rounds 1 and 3 were VLP panning, while rounds 2 and 4 were cell panning. Candidate clone sequences such as 1-B11, 2-D06, 1-G08, 1-G09, 2-A12, 2-B05, 2-B06, and 2-B08 (Table 1) were screened and constructed into the pcDNA3.4-IgG1Fc antibody expression vector (e.g., Figure 2The supernatant was combined with CHO-S-GPRC5D cells for binding verification, and effective binding clone antibodies were purified for FACS EC50 detection after antibody purification. The results showed that the candidate antibodies combined with CHO-S-GPRC5D cells, among which C07, F08, B05, A12 and B11 combined more strongly.

[0089] Table 1. Candidate antibody and CDR sequence list

[0090] 1) GPRC5D antibody cell binding verification: flow cytometry detection parameters: use 3x10 5 cells / well to detect cell number. The primary antibody is GPRC5D transfected supernatant (100 μl / well), among which the positive antibody (10 μg / ml, 100 μl / well); the secondary antibody is PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution), and the results are shown in Figure 3 .

[0091] 2) GPRC5D purified antibody affinity verification: flow cytometry detection parameters: use CHO-S-GPRC5D cells (3x105 / well). The primary antibody is GPRC5D target candidate antibody and positive antibody (30 μg / ml, 3-fold gradient dilution 11 points, 100 μl / well); the secondary antibody is PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution), and the results are shown in Figure 4 Table 2.

[0092] Table 2. Antibody affinity analysis table

[0093] 3) GPRC5D candidate purified antibody binding detection with CHO-S-Mouse-GPRC5D: use CHO-S cells overexpressing mouse GPRC5D 3x10 5Flow cytometry detection was performed using GPRC5D candidate antibody + positive antibody (10 μg / ml, 100 μl / well) as primary antibody, PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution) as secondary antibody. The results are shown in Figure 5 As shown, B05 and F08 candidate antibodies showed strong binding to CHO-S-Mouse-GPRC5D cells.

[0094] Example 2: Construction of CAR-T cells and verification of killing ability of candidate antibodies 1. Preparation of CAR lentivirus: the positive control CAR-T is BCMA replaced GPRC5D CAR (reference doi: 10.1158 / 2643-3230.BCD-20-0020), the sequence is shown in Table 3, and the sequence is synthesized to construct pCDH-EF1a lentiviral expression plasmid, and the structure is shown in Figure 6 The lentivirus system plasmid (pCDH-EF1a lentiviral expression plasmid, PsPAX2, pMD2.G three-plasmid system, mixed in a mass ratio of 3:2:1) was transfected into logarithmic growth phase adherent 293T cells, and the cell culture supernatant harvested 48-72 hours after transfection was concentrated and filtered to obtain CAR lentivirus stored at -80°C for standby.

[0095] Table 3. CAR-T system sequence table

[0096] 2. Construction of CAR-T cells: peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood, and T cells were separated using human CD3 / 28 magnetic beads, and virus transduction was performed within 72 hours of activation. After 24 hours of transduction, the liquid was changed and cultured to the 8th day, and the cells were collected by centrifugation and resuspended in physiological saline. Flow cytometry was used to identify T cell surface EGFRt molecules, and the positive rate was ensured to be greater than 30%, and less than which EGFR-PE primary antibody and PE magnetic beads were used for enrichment and sorting. According to the CAR-T positive proportion and target cells mixed (effector to target ratio 1:1 and 5:1), subsequent killing experiments were performed, and the killing rate was detected, and the results are shown in Figure 7 As shown.

[0097] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives examples, it should be understood that further improvements can be made to the application. In summary, according to the principle of the application, the present application is intended to include any changes, uses or improvements of the application, including changes made by using conventional techniques known in the art, which depart from the scope disclosed in the present application.

Claims

1. A single domain antibody against GPRC5D, characterized in that The single-domain antibody comprises HCDR1-3 of the heavy chain variable region shown in SEQ ID NO: 4, wherein the sequences of the HCDR1-3 are determined according to any one of the numbering systems of Kabat, IMGT, Chothia, AbM, and Contact; Preferably, the amino acid sequences of HCDR1-3 are as shown in SEQ ID NO: 1-3; Preferably, the single-domain antibody comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 4 or consists of it.

2. An antibody derivative, characterized in that The antibody derivative comprises any one of the following: 1) The single-domain antibody of claim 1 and a detectable label conjugated thereto; Preferably, the detectable label comprises at least one of a radioisotope, a metal nanomaterial, fluorescein, biotin, avidin, a biotin / avidin complex, a biotin / avidin complex, a chromophore, an electron-dense substance, and an enzyme; 2) A pharmaceutical composition comprising the single domain antibody of claim 1 and a pharmaceutically acceptable carrier.

3. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the single domain antibody of claim 1; Preferably, the chimeric antigen receptor further comprises an Fc sequence, an SP membrane export signal, a hinge region, a transmembrane region, a co-stimulatory signaling domain, and an intracellular signal transduction region; Preferably, the Fc sequence is shown in SEQ ID NO: 35; Preferably, the sequence of the SP membrane-exiting signal is shown as SEQ ID NO:

34. Preferably, the hinge region is selected from the hinge regions of the following molecules: CD8, 4-1BB, IgG1, IgG4, PD-1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof; Preferably, the hinge region is CD8 Hinge; Preferably, the amino acid sequence of the CD8 Hinge is shown in SEQ ID NO: 37; Preferably, the transmembrane region is selected from the transmembrane regions of the following molecules: CD8, 4-1BB, IgG1, IgG4, PD-1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof; Preferably, the transmembrane region is CD8 TM; Preferably, the amino acid sequence of the CD8 TM is as shown in SEQ ID NO: 38; Preferably, the costimulatory signaling domain is selected from the costimulatory signaling domains of the following molecules: 4-1BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8α, CD8β, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278 and variants thereof; Preferably, the costimulatory signaling domain is 4-1BB; Preferably, the amino acid sequence of 4-1BB is as shown in SEQ ID NO: 39; Preferably, the intracellular signal transduction region is selected from the intracellular signal transduction regions of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD278, CD21, CD22, FcεRI, FcRγ, FcRβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d and variants thereof; Preferably, the intracellular signal transduction region is CD3ζ; Preferably, the amino acid sequence of CD3ζ is shown in SEQ ID NO:

40.

4. The chimeric antigen receptor according to claim 3, characterized in that The chimeric antigen receptor further comprises a suicide gene and a 2A peptide; Preferably, the suicide gene comprises EGFRt; Preferably, the amino acid sequence of EGFRt is shown in SEQ ID NO: 43; Preferably, the suicide gene further comprises a signal peptide of EGFRt; Preferably, the amino acid sequence of the EGFRt signal peptide is shown in SEQ ID NO: 42; Preferably, the 2A peptide is T2A; Preferably, the amino acid sequence of T2A is shown in SEQ ID NO: 41; Preferably, the chimeric antigen receptor further comprises a signal peptide; Preferably, the amino acid sequence of the signal peptide is shown in SEQ ID NO:

36.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the single domain antibody of claim 1, the antibody derivative of claim 2, and / or the chimeric antigen receptor of any one of claims 3-4; Preferably, the nucleic acid molecule comprises the base sequence encoding HCDR1-3 in the heavy chain variable region of the single-domain antibody according to claim 1 as shown in SEQ ID NO: 45; and / or, the nucleic acid molecule comprises the base sequence of the chimeric antigen receptor as shown in SEQ ID NO: 56; Preferably, the base sequences encoding HCDR1-3 are shown in SEQ ID NO:61-63; Preferably, the nucleic acid molecule further comprises a base sequence encoding an Fc sequence as shown in SEQ ID NO: 84; Preferably, the nucleic acid molecule further comprises a base sequence encoding an SP membrane exit signal as shown in SEQ ID NO: 83; Preferably, the nucleic acid molecule further comprises a base sequence encoding CD8 Hinge as shown in SEQ ID NO: 86; Preferably, the nucleic acid molecule further comprises a base sequence encoding CD8 TM as shown in SEQ ID NO: 87; Preferably, the nucleic acid molecule further comprises a base sequence encoding CD3ζ as shown in SEQ ID NO: 89; Preferably, the nucleic acid molecule further comprises a base sequence encoding 4-1BB as shown in SEQ ID NO: 88; Preferably, the nucleic acid molecule further comprises a base sequence encoding EGFRt as shown in SEQ ID NO: 92; Preferably, the nucleic acid molecule further comprises a base sequence encoding a signal peptide of EGFRt as shown in SEQ ID NO:91; Preferably, the nucleic acid molecule further comprises a base sequence encoding T2A as shown in SEQ ID NO: 90; Preferably, the nucleic acid molecule further comprises a base sequence encoding a signal peptide as shown in SEQ ID NO:

85.

6. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 5.

7. A recombinant host cell, characterized in that The recombinant host cell comprises the nucleic acid molecule of claim 5 or the vector of claim 6.

8. Use of the single domain antibody according to claim 1, the antibody derivative according to claim 2, the chimeric antigen receptor according to any one of claims 3-4, the nucleic acid molecule according to claim 5, the vector according to claim 6 and / or the recombinant host cell according to claim 7, characterized in that: The application includes any of the following: 1) Application in the construction of CAR-T cells targeting GPRC5D; 2) Application in the construction of bivalent antibodies targeting GPRC5D; 3) Use in the preparation of products for diagnosing, treating, and / or monitoring the stability of GPRC5D-positive cancers; 4) Application in the preparation of products for detecting GPRC5D protein.

9. The use according to claim 8, characterized in that The bivalent antibody comprising the antibody targeting GPRC5D can also target a T cell marker; Preferably, the T cell markers include CD3, CD19, and CD38; Preferably, the T cell marker is CD3; Preferably, the GPRC5D-positive cancer includes multiple myeloma and plasma cell leukemia; Preferably, the GPRC5D-positive cancer is multiple myeloma; Preferably, the products include pharmaceutical compositions, kits, nucleic acid chips, and nucleic acid membrane strips.

10. A method for preparing the single domain antibody of claim 1, the antibody derivative of claim 2, or the chimeric antigen receptor of any one of claims 3-4, said method comprising artificial synthesis or preparation using genetic engineering techniques; The preparation by genetic engineering technology refers to culturing the recombinant host cell according to claim 7 under conditions suitable for expressing the antibody.

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