Antibody specifically targeting GPRC5D and application thereof

Abstract

The invention provides an antibody for specifically targeting GPRC5D, an application of the antibody, and a single-domain antibody F08 for specifically targeting GPRC5D, wherein the binding activity and the reaction specificity of the single-domain antibody F08 for the GPRC5D are relatively high. The invention also provides nucleic acid molecules encoding the single domain antibodies, vectors and recombinant host cells, as well as methods of producing the single domain antibodies and antibody derivatives. 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 an antibody specifically targeting GPRC5D and its application. 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 range 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 through BCMA (B cell maturation antigen) target point, but for MM patients with negative or low expression of BCMA, relapse still occurs after receiving targeted BCMA CAR-T cell treatment, and there is a problem of target escape. In order to alleviate the problem of relapse mediated by BCMA escape, it is the key to find a better specific MM target point (such as GPRC5D) or simultaneously target other antigen target points (such as CD3, CD19, CD38, GPRC5D, etc.), and GPRC5D is likely to become a popular candidate target point 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 multiple myeloma (MM) treatment. 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 single-domain antibody F08 specifically targeting GPRC5D, its derivatives, biological materials and applications.

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

[0008] The present application provides a single-domain antibody specifically targeting GPRC5D, comprising HCDR1-3 of a heavy chain variable region, wherein HCDR1 consists of SEQ ID NO: 30; HCDR2 consists of SEQ ID NO: 31; and HCDR3 consists of SEQ ID NO: 32.

[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. diabodies) formed from at least two intact antibodies, as long as they exhibit the desired biological activity.

[0010] In the present application, the term "single-domain antibody" (sdAb) is also known as VHH antibody or camelid antibody, which is an artificially designed antibody molecule, a kind of naturally light chain-deleted 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 antibodies, 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 three 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 the 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 antibodies 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, 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] In some embodiments, the heavy chain variable region comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 33.

[0013] In the present application, the term "binds" or "targets" refers to a selective binding to 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 conventional binding assays known in the art.

[0014] In the present application, the modified antibody sequences also fall within the scope of the present application. The term "modification" 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.

[0015] 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 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 are able to determine appropriate parameters for alignment, including any algorithm needed to achieve optimal alignment of the full length sequences being compared.

[0016] The second aspect of the present application provides a chimeric antigen receptor comprising the single domain antibody of the first aspect of the present application.

[0017] Further, the chimeric antigen receptor further comprises an Fc sequence, an SP membrane exit signal, a hinge region, a transmembrane region, a costimulatory signal domain, an intracellular signal transduction region.

[0018] Further, the Fc sequence is as shown in SEQ ID NO: 35.

[0019] Further, the sequence of the SP membrane exit signal is as shown in SEQ ID NO: 34.

[0020] Further, the hinge region is selected from the hinge region of CD8, 4-1BB, IgG1, IgG4, PD-1, CD28, CD34, OX40, CD3 epsilon, NKG2D, NKG2C, Fc epsilon RI gamma, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof.

[0021] Further, the hinge region is CD8 Hinge.

[0022] Further, the amino acid sequence of the CD8 Hinge is as shown in SEQ ID NO: 37.

[0023] Further, the transmembrane region is selected from the transmembrane region of CD8, 4-1BB, IgG1, IgG4, PD-1, CD28, CD34, OX40, CD3 epsilon, NKG2D, NKG2C, Fc epsilon RI gamma, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof.

[0024] Further, the transmembrane region is CD8 TM.

[0025] Further, the amino acid sequence of the CD8 TM is set forth in SEQ ID NO: 38.

[0026] Further, the co-stimulatory signaling domain is selected from the co-stimulatory signaling domain of 4-1BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8a, CD8b, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278, and variants thereof.

[0027] Further, the co-stimulatory signaling domain is 4-1BB.

[0028] Further, the amino acid sequence of the 4-1BB is set forth in SEQ ID NO: 39.

[0029] Further, the intracellular signaling region is selected from the intracellular signaling region of CD3zeta, CD3gamma, CD3delta, CD3epsilon, CD278, CD21, CD22, FcsRI, FcRg, FcRp, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d, and variants thereof.

[0030] Further, the intracellular signaling region is CD3zeta.

[0031] Further, the amino acid sequence of the CD3zeta is set forth in SEQ ID NO: 40.

[0032] In some embodiments, the chimeric antigen receptor further comprises a suicide gene, a 2A peptide.

[0033] Further, the suicide gene comprises EGFRt.

[0034] Further, the amino acid sequence of the EGFRt is set forth in SEQ ID NO: 43.

[0035] Further, the suicide gene further comprises a signal peptide of the EGFRt.

[0036] Further, the amino acid sequence of the signal peptide of the EGFRt is set forth in SEQ ID NO: 42.

[0037] Further, the 2A peptide is T2A.

[0038] Further, the amino acid sequence of the T2A is set forth in SEQ ID NO: 41.

[0039] Further, the chimeric antigen receptor further comprises a signal peptide.

[0040] Further, the amino acid sequence of the signal peptide is shown as SEQ ID NO: 36.

[0041] In certain embodiments, the skilled person 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, no matter what form of change, as long as the chimeric antigen receptor has the CDR sequence or the heavy chain variable region sequence of the heavy chain variable region of the single domain antibody provided by the present application, which belongs to the protection scope of the present application.

[0042] The third aspect of the present application provides an antibody derivative, which comprises the single domain antibody of the first aspect of the present application and a detectable label coupled thereto.

[0043] Further, 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.

[0044] The fourth aspect of the present application provides a nucleic acid molecule, which encodes the single domain antibody of the first aspect of the present application, the chimeric antigen receptor of the second aspect of the present application and / or the antibody derivative of the third aspect of the present application.

[0045] 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 shown as SEQ ID NO: 55. And / or, the nucleic acid molecule comprises the base sequence of the chimeric antigen receptor shown as SEQ ID NO: 60.

[0046] Further, the base sequence encoding HCDR1-3 is shown as SEQ ID NO: 80-82.

[0047] Further, the nucleic acid molecule further comprises the base sequence encoding the Fc sequence shown as SEQ ID NO: 84.

[0048] Further, the nucleic acid molecule further comprises the base sequence encoding the SP membrane signal shown as SEQ ID NO: 83.

[0049] Further, the nucleic acid molecule further comprises the base sequence encoding the CD8 Hinge shown as SEQ ID NO: 86.

[0050] Further, the nucleic acid molecule further comprises the base sequence encoding the CD8 TM shown as SEQ ID NO: 87.

[0051] Further, the nucleic acid molecule further comprises a base sequence encoding CD3 zeta as shown in SEQ ID NO: 89.

[0052] Further, the nucleic acid molecule further comprises a base sequence encoding 4-1BB as shown in SEQ ID NO: 88.

[0053] Further, the nucleic acid molecule further comprises a base sequence encoding EGFRt as shown in SEQ ID NO: 92.

[0054] Further, the nucleic acid molecule further comprises a base sequence encoding signal peptide of EGFRt as shown in SEQ ID NO: 91.

[0055] Further, the nucleic acid molecule further comprises a base sequence encoding T2A as shown in SEQ ID NO: 90.

[0056] Further, the nucleic acid molecule further comprises a base sequence encoding signal peptide as shown in SEQ ID NO: 85.

[0057] In the present application, the term "nucleic acid molecule" refers to any polymeric form of ribonucleotides or deoxyribonucleotides. In general, 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 mRNA and translated into a polypeptide in a host cell. The coding sequence is bounded by a start codon at the 5' (amino) terminus and a 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 sequences that are recombinant in origin. A transcription termination sequence will usually be located 3' to the coding sequence.

[0058] The fifth aspect of the present application provides a vector comprising the nucleic acid molecule of the fourth aspect of the present application.

[0059] 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 gene or sequence of interest in a host cell. The vector of the present application can be a plasmid vector, a viral vector, and the like. 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 the regulatory sequence(s) in a manner that allows for 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).

[0060] 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.

[0061] In the present application, the term "recombinant host cell" refers to the introduction of the vector of the present application into a host cell, so that the host cell after recombination can transcribe the nucleic acid sequence of interest and / or translate the protein of interest.

[0062] The seventh aspect of the present application provides the use of the single domain antibody of the first aspect of the present application, the chimeric antigen receptor of the second aspect of the present application, the antibody derivative 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, which use comprises any one of the following: 1) use in constructing a CAR-T cell targeting GPRC5D; 2) use in constructing a bi-specific antibody comprising a targeting GPRC5D; 3) use in preparing a product for diagnosing, treating the progression of a GPRC5D-positive cancer, and / or monitoring the stability of a GPRC5D-positive cancer; 4) use in preparing a product for detecting GPRC5D protein.

[0063] Further, the bi-specific antibody comprising a targeting GPRC5D can also target a T cell marker.

[0064] Further, the T cell marker comprises CD3, CD19, CD38.

[0065] Further, the T cell marker is CD3.

[0066] Further, the GPRC5D-positive cancer comprises multiple myeloma, plasma cell leukemia.

[0067] Further, the GPRC5D-positive cancer is multiple myeloma.

[0068] Further, the product comprises a kit, a nucleic acid chip, a nucleic acid membrane strip.

[0069] In the present application, the term "progression" refers to the malignant change of the disease, including the appearance of growth, spread or metastasis of the lesion (such as tumor). In some embodiments, the minimum value of the sum of the diameters of all measured target lesions throughout the entire experimental study is taken as the reference, and the relative increase in diameter is at least 20% (if the baseline measurement is the minimum, the baseline value is taken as the reference); in addition, the absolute value of the increase in diameter must be at least 5 mm (the appearance of one or more new lesions is also considered as disease progression).

[0070] 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.

[0071] In the present application, the term "stability" refers to the extent of reduction in target lesions that does not reach the level of disease remission, or the extent of increase that does not reach the level of disease progression, between the two.

[0072] Advantages and beneficial effects of the present application: The present application provides a single-domain antibody F08 that specifically targets GPRC5D, which has high binding activity and reaction specificity for GPRC5D. The single-domain antibody can be used to construct chimeric antigen receptors (CAR) and diabodies (bispecific antibodies). 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 diabody designed based on the F08 sequence can simultaneously target GPRC5D and T cells, thereby activating T cells and directing them to attack tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 is a mixed library sequence case diagram.

[0074] Figure 2 is a schematic diagram of the construction structure of pcDNA3.4-IgG1 Fc vector.

[0075] Figure 3 is a GPRC5D antibody cell binding verification diagram.

[0076] Figure 4 is a GPRC5D purified antibody affinity verification diagram.

[0077] Figure 5 is a GPRC5D candidate purified antibody and CHO-S-Mouse-GPRC5D binding ability detection diagram.

[0078] Figure 6 is a schematic diagram of the construction structure of CAR-T vector.

[0079] Figure 7 is a GPRC5D candidate purified antibody CAR-T killing rate detection diagram. DETAILED DESCRIPTION

[0080] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation of the application.

[0081] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation of the application.

[0082] Example 1 Construction of phage display library, screening of antibodies and verification 1. Llama immunization to construct phage display library Two llamas were immunized a total of 4 times, and the immunogen was 293F-GPRC5D (human sequence). The immunization was subcutaneous, and the immunization adjuvant was GERBU. After immunization, the sera of the immunized llamas were separated, and ELISA and FACS detection verification were performed, respectively. Then, the peripheral blood of the above llamas was collected, PBMC cells were separated, RNA was extracted, and cDNA was reverse transcribed. VHH sequence was amplified from the cDNA sample using a single-domain antibody cloning primer combination, and was subcloned into the phage display vector pDisplay. The SS320 E. coli competent cells were electroporated to construct a single-domain antibody phage display library (2 llamas mixed library). The mixed library sequence is shown in Table 1. Figure 1 Two llamas were mixed to construct the library, and 48 clones were sequenced. Two antibody sequences with one base missing and one empty sequence were removed, and the remaining sequences were all antibody sequences, with good diversity.

[0083] 2. Screening of antibodies and verification The above-mentioned library was screened using the following proteins / cell lines: GPRC5D-VLP protein / CHO-S-GPRC5D cell line, and VLP protein / CHO-S cell line. The first and third rounds were VLP screening, and the second and fourth rounds were cell screening. The candidate clone sequences 1-B11, 2-D06, 1-G08, 1-G09, 2-A12, 2-B05, 2-B06, and 2-B08 were screened (Table 1) and constructed into a pcDNA3.4-IgG1 Fc antibody expression vector (as shown in Table 1). After expression by 293F transfection, the supernatant was combined with CHO-S-GPRC5D cells for verification, and the effective binding clone antibodies were purified and then detected for FACS EC50. The results showed that the candidate antibodies were strongly combined with CHO-S-GPRC5D cells, including C07, F08, B05, A12, and B11. Figure 2 ​

[0084] Table 1. List of candidate antibodies and CDR sequences

[0085] 1) GPRC5D antibody cell binding verification: flow cytometry detection Specific parameters: use 3x10 5 6 cells / well to detect the number of cells. The primary antibody is GPRC5D transfected supernatant (100 μl / well), of 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 .

[0086] 2) GPRC5D purified antibody affinity verification: flow cytometry detection Specific 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.

[0087] Table 2. Antibody affinity analysis table

[0088] 3) GPRC5D candidate purified antibody binding detection with CHO-S-Mouse-GPRC5D: use CHO-S cells overexpressing mouse GPRC5D 3x10 5 6 cells / well, use GPRC5D candidate antibody + positive antibody (10 μg / ml, 100 μl / well) as the primary antibody, and PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution) as the secondary antibody for flow cytometry detection. The results are shown in Figure 5 , showing that B05, F08 candidate antibodies have strong binding to CHO-S-Mouse-GPRC5D cells.

[0089] 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 a BCMA replacement 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 a pCDH-EF1a lentivirus expression plasmid, and the structure is as shown in Figure 6 The lentivirus system plasmid (pCDH-EF1a lentivirus 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 of the cells transfected for 48-72 hours was harvested. After concentration and filtration, the CAR lentivirus was obtained and stored at -80°C for standby.

[0090] Table 3. CAR-T system sequence table

[0091] 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 normal 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 .

[0092] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives examples, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, the present application is intended to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application.

Claims

1. A single domain antibody specifically targeting GPRC5D, characterized in that: The single-domain antibody comprises HCDR1-3 of the heavy chain variable region, wherein HCDR1 consists of SEQ ID NO: 30; HCDR2 consists of SEQ ID NO: 31; and HCDR3 consists of SEQ ID NO:

32.

2. The single domain antibody according to claim 1, characterized in that The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:

33.

3. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the single domain antibody according to any one of claims 1 to 2; 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. An antibody derivative, characterized in that The antibody derivative comprises the single domain antibody according to any one of claims 1 to 2 and a detectable label coupled 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.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the single domain antibody according to any one of claims 1 to 2, the chimeric antigen receptor according to any one of claims 3 to 4, and / or the antibody derivative according to claim 5; 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 any one of claims 1-2 as shown in SEQ ID NO: 55; and / or, the nucleic acid molecule comprises the base sequence of the chimeric antigen receptor as shown in SEQ ID NO: 60; Preferably, the base sequences encoding HCDR1-3 are shown in SEQ ID NO:80-82; 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.

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

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

9. Use of the single domain antibody according to any one of claims 1-2, the chimeric antigen receptor according to any one of claims 3-4, the antibody derivative according to claim 5, the nucleic acid molecule according to claim 6, the vector according to claim 7 and / or the recombinant host cell according to claim 8, 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. Preferably, 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 product includes a kit, a nucleic acid chip, and a nucleic acid membrane strip.

10. A method for preparing the single domain antibody according to any one of claims 1 to 2, the chimeric antigen receptor according to any one of claims 3 to 4, or the antibody derivative according to claim 5, characterized in that: The method includes artificial synthesis or preparation by genetic engineering technology; The preparation by genetic engineering technology refers to culturing the recombinant host cell according to claim 8 under conditions suitable for expressing the antibody.

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