Single-domain antibody capable of specifically recognizing GPRC5D

Abstract

The invention provides a single-domain antibody capable of specifically recognizing GPRC5D, which has relatively strong binding activity and reaction specificity, also has good species cross reactivity, is of great significance to research and development in different species models, and is beneficial to acceleration of a transformation process from a laboratory to clinic. The antibody sequences A12 and C07 provided by the invention can be used for constructing chimeric antigen receptor T cells, and the chimeric antigen receptor T cells can be used for treating multiple myeloma.

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 a single domain antibody that specifically recognizes GPRC5D, and relates to the fields of genetic engineering and antibody technology. 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 that B lymphocytes develop to the final functional stage. 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 suppressed, various bacterial infections are prone to occur.

[0004] At present, the main treatment method for MM is through the 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 crucial to find a better specific MM target point (such as GPRC5D) or to 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 antibody sequences targeting GPCR5D and their applications in CAR-T and immune cell construction, thereby providing new treatment ideas 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 A12 and C07 that specifically recognizes GPRC5D, and its application in CAR-T construction.

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

[0008] The present application provides a single-domain antibody specifically recognizing GPRC5D, which comprises a heavy chain complementarity determining region HCDR1, and the sequence of the HCDR1 is shown as SEQ ID NO: 9.

[0009] Further, the single-domain antibody further comprises HCDR2-HCDR3.

[0010] In some embodiments, the sequence of the HCDR2 is shown as SEQ ID NO: 10, and the HCDR3 is shown as SEQ ID NO: 11.

[0011] In some embodiments, the sequence of the HCDR2 is shown as SEQ ID NO: 20, and the HCDR3 is shown as SEQ ID NO: 21.

[0012] Further, the single-domain antibody comprises a heavy chain variable region, and the sequence of the heavy chain variable region is shown as any one of the following groups: 1) the heavy chain variable region comprises or consists of an amino acid sequence shown as SEQ ID NO: 12; 2) the heavy chain variable region comprises or consists of an amino acid sequence shown as SEQ ID NO: 22.

[0013] The present application provides an antibody derivative, which comprises any one of the following: 1) a single-domain antibody according to the first aspect of the present application and a detectable label coupled thereto; 2) a chimeric antigen receptor comprising a heavy chain variable region of the single-domain antibody according to the first aspect of the present application; and 3) a pharmaceutical composition comprising the single-domain antibody according to the first aspect of the present application and a pharmaceutically acceptable carrier.

[0014] 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.

[0015] Further, the chimeric antigen receptor further comprises an Fc sequence, an SP membrane-out signal, a hinge region, a transmembrane region, a costimulatory signal domain, and an intracellular signal transduction region.

[0016] Further, the Fc sequence is shown as SEQ ID NO: 35.

[0017] Further, the sequence of the SP membrane-out signal is shown as SEQ ID NO: 34.

[0018] In some embodiments, 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 R1 gamma, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and variants thereof.

[0019] Further, the transmembrane region is CD8 TM.

[0020] Further, the amino acid sequence of the CD8 TM is set forth in SEQ ID NO: 38.

[0021] In some embodiments, 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 R1 gamma, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and variants thereof.

[0022] Further, the transmembrane region is CD8 TM.

[0023] Further, the amino acid sequence of the CD8 TM is set forth in SEQ ID NO: 38.

[0024] In some embodiments, the co-stimulatory signaling domain is selected from the co-stimulatory signaling domain of 4-1BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8 alpha, CD8 beta, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278, and variants thereof.

[0025] Further, the co-stimulatory signaling domain is 4-1BB.

[0026] Further, the amino acid sequence of the 4-1BB is set forth in SEQ ID NO: 39.

[0027] In some embodiments, the intracellular signaling region is selected from the intracellular signaling region of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD278, CD21, CD22, Fc epsilon R1, FcR gamma, FcR beta, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d, and variants thereof.

[0028] Further, the intracellular signaling region is CD3 zeta.

[0029] Further, the amino acid sequence of the CD3 zeta is shown as SEQ ID NO: 40.

[0030] In some embodiments, the chimeric antigen receptor further comprises a suicide gene, a 2A peptide.

[0031] Further, the suicide gene comprises EGFRt.

[0032] Further, the amino acid sequence of the EGFRt is shown as SEQ ID NO: 43.

[0033] Further, the suicide gene further comprises a signal peptide of the EGFRt.

[0034] Further, the amino acid sequence of the signal peptide of the EGFRt is shown as SEQ ID NO: 42.

[0035] In some embodiments, the 2A peptide is T2A.

[0036] Further, the amino acid sequence of the T2A is shown as SEQ ID NO: 41.

[0037] In some embodiments, the chimeric antigen receptor further comprises a signal peptide.

[0038] Further, the amino acid sequence of the signal peptide is shown as SEQ ID NO: 36.

[0039] The third aspect of the present application provides a biological material, which comprises any one of the following: 1) a nucleic acid molecule encoding the single-domain antibody of the first aspect of the present application and / or the antibody derivative of the second aspect of the present application; 2) a vector comprising the nucleic acid molecule of 1); 3) a recombinant host cell comprising the nucleic acid molecule of 1) or the vector of 2).

[0040] Further, the nucleic acid molecule comprises the base sequence encoding HCDR1, HCDR2-3 in the heavy chain variable region of the single-domain antibody of the first aspect of the present application shown as SEQ ID NO: 47, 52; and / or, the nucleic acid molecule comprises the base sequence of the antibody derivative shown as SEQ ID NO: 57, 59.

[0041] Further, the base sequence encoding HCDR1 is shown as SEQ ID NO: 67.

[0042] Further, the base sequence encoding HCDR2-3 is shown as SEQ ID NO: 68-69.

[0043] Further, the base sequence encoding HCDR2-3 is shown as SEQ ID NO: 73-74.

[0044] Further, the nucleic acid molecule further comprises a base sequence encoding Fc sequence as shown in SEQ ID NO: 84.

[0045] Further, the nucleic acid molecule further comprises a base sequence encoding SP membrane egress signal as shown in SEQ ID NO: 83.

[0046] Further, the nucleic acid molecule further comprises a base sequence encoding CD8 Hinge as shown in SEQ ID NO: 86.

[0047] Further, the nucleic acid molecule further comprises a base sequence encoding CD8 TM as shown in SEQ ID NO: 87.

[0048] Further, the nucleic acid molecule further comprises a base sequence encoding CD3 zeta as shown in SEQ ID NO: 89.

[0049] Further, the nucleic acid molecule further comprises a base sequence encoding 4-1BB as shown in SEQ ID NO: 88.

[0050] Further, the nucleic acid molecule further comprises a base sequence encoding EGFRt as shown in SEQ ID NO: 92.

[0051] Further, the nucleic acid molecule further comprises a base sequence encoding signal peptide of EGFRt as shown in SEQ ID NO: 91.

[0052] Further, the nucleic acid molecule further comprises a base sequence encoding T2A as shown in SEQ ID NO: 90.

[0053] Further, the nucleic acid molecule further comprises a base sequence encoding signal peptide as shown in SEQ ID NO: 85.

[0054] The fourth 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 and / or the biological material of the third aspect of the present application, which use comprises any one of the following: 1) use in constructing CAR-T cells targeting GPRC5D; 2) use in constructing bi-specific antibodies comprising targeting GPRC5D; 3) use in preparing products for diagnosing, treating the progression, regression of GPRC5D positive cancer, and / or monitoring the stability of GPRC5D positive cancer; 4) use in preparing products for detecting GPRC5D protein.

[0055] Further, the bi-specific antibody targeting GPRC5D can also target a T cell marker.

[0056] Further, the T cell marker comprises CD3, CD19, CD38.

[0057] Further, the T cell marker is CD3.

[0058] Further, the GPRC5D positive cancer comprises multiple myeloma, plasma cell leukemia.

[0059] Further, the GPRC5D positive cancer is multiple myeloma.

[0060] Further, the product comprises a pharmaceutical composition, a kit, a nucleic acid chip, a nucleic acid strip.

[0061] The fifth aspect of the present application provides a method for preparing the single domain antibody of the first aspect of the present application or the antibody derivative of the second aspect of the present application, which comprises artificial synthesis or genetic engineering technology.

[0062] The genetic engineering technology refers to culturing the recombinant host cell of the third aspect of the present application under conditions suitable for expression of the antibody.

[0063] Advantages and beneficial effects of the present application: the GPRC5D single domain antibodies A12 and C07 provided by the present application have specific amino acid sequences, strong binding activity and reaction specificity to GPRC5D. The anti-GPRC5D antibody sequence can be used to construct chimeric antigen receptor (CAR) and bi-specific antibody (double specificity 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 bi-specific antibody designed based on the antibody sequence can simultaneously target GPRC5D and T cells, thereby activating T cells and guiding them to attack tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 It is a mixed library sequence case diagram.

[0065] Figure 2 It is a schematic diagram of pcDNA3.4-IgG1Fc vector construction structure.

[0066] Figure 3 It is a GPRC5D antibody cell binding verification diagram.

[0067] Figure 4 It is a GPRC5D purified antibody affinity verification diagram.

[0068] Figure 5Figure 1 shows the binding ability of GPRC5D candidate purified antibodies to CHO-S-Mouse-GPRC5D.

[0069] Figure 6 Figure 2 shows the structure of CAR-T vector.

[0070] Figure 7 Figure 3 shows the killing rate of GPRC5D candidate purified antibodies CAR-T. DETAILED DESCRIPTION

[0071] The application will be further described in conjunction with the preferred embodiments thereof, given only by way of illustration of the present application, and not intended to limit the scope of the present 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 present application.

[0072] In the context of the present application, the term "antibody" is used in the broadest sense and specifically covers single domain antibodies, monoclonal antibodies, polyclonal antibodies, humanized 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.

[0073] In the present application, the term "single-domain antibodies" (sdAbs) is also known as VHH antibodies or camelid antibodies, which is an artificially designed antibody molecule, a kind of naturally light chain missing heavy chain antibody (HCAbs) found in camelids, such as llamas, dromedaries, and sharks, skates and other cartilaginous fish, 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. The crystal structure of VHH antibody is an ellipse of 4 nm x 2.5 nm x 3 nm, with a molecular weight of only 1 / 10 of ordinary antibody, about 12-14 kDa, which is the smallest complete antigen binding fragment, so it is also called nanobody.

[0074] In the present application, the term "CDR" refers to the "hypervariable region" or "complementarity determining region" of an antibody, both the heavy chain variable region and the light chain variable region have 3 CDRs, 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.

[0075] 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.

[0076] In the present application, the antibody sequence modified from the antibody sequence of the first aspect of the present application also falls within the protection 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 replacement of one or more amino acids in the original amino acid sequence with different amino acids. The term "deletion" refers to 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 addition of one or more amino acids compared to the original amino acid sequence.

[0077] In the present application, the antibody sequence modified from the antibody sequence of the first aspect of the present application also falls within the protection 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 replacement of one or more amino acids in the original amino acid sequence with different amino acids. The term "deletion" refers to 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 addition of one or more amino acids compared to the original amino acid sequence.

[0078] In certain embodiments, the skilled person can change the combination 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 of the heavy chain variable region or the heavy chain variable region sequence of the single domain antibody of the present application, which all fall within the protection scope of the present application.

[0079] In the present application, the term "nucleic acid molecule" refers to any polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. 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 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 signal at the 3' (carboxy) terminus. Coding sequences can include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (for example, mammalian) DNA, and even recombinant DNA sequences. A transcription termination sequence will typically be located 3' to the coding sequence.

[0080] 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 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 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). In the present application, the term "recombinant host cell" refers to a host cell into which a vector of the present application has been introduced, enabling the host cell after recombination to transcribe the nucleic acid sequence of interest and / or to translate the protein of interest.

[0081] In the present application, the term "pharmaceutical composition" refers to a composition comprising at least one biologically active compound. The pharmaceutical compositions described herein 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 instances, a pharmaceutically-acceptable acid, base or buffer 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 herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions described herein can be administered to a subject by any route of administration which results in the target tissue, provided that the target tissue is reached. The pharmaceutical compositions of the present application can also be used in combination 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, concurrently or sequentially with 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.

[0082] In the present application, the term "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful 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 can also be present in such media, such as wetting or emulsifying agents, pH buffering substances, and the like.

[0083] In the present application, the term "progression" refers to a malignant change in the disease, including the appearance of growth, spread or metastasis of a lesion (e.g., a tumor). In some embodiments, the relative increase in the sum of the diameters of all measured target lesions is at least 20% (with reference to the smallest sum of diameters of all measured target lesions during the entire study) or the absolute increase in the sum of the diameters is at least 5 mm (with reference to the baseline measurement if the baseline measurement is the smallest) and in addition, the appearance of one or more new lesions is considered disease progression.

[0084] In the present application, the term "regression" refers to a decrease in tumor volume or complete disappearance of the tumor, 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.

[0085] In the present invention, the term "stability" means that the degree of reduction of target lesions has not reached the level of disease remission, and the degree of increase has not reached the level of disease progression, but is somewhere in between. The minimum value of the sum of diameters can be used as a reference for research.

[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 2 After expression in 293F cells, the supernatant was tested for binding to CHO-S-GPRC5D cells. Effectively binding cloned antibodies were purified and FACS EC50 values ​​were determined. The results showed that the candidate antibodies bound to CHO-S-GPRC5D cells, with C07, F08, B05, A12, and B11 showing the strongest binding.

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

[0090] 1) GPRC5D antibody cell binding verification: flow cytometry detection specific parameters: using 3 x 10 5 cells / well to detect the number of cells. The primary antibody is GPRC5D transfected supernatant (100 μl / well), and 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 specific parameters: using CHO-S-GPRC5D cells (3 x 105 / 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: using CHO-S cells overexpressing mouse GPRC5D 3 x 10 5 cells / well, using 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.

[0094] Example 2 Construction of CAR-T cells and verification of killing ability of candidate antibodies Sequence 1, CAR lentivirus preparation: positive control CAR-T is 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 pCDH-EF1a lentiviral expression plasmid, and the structure is as 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 of the cells transfected for 48-72 hours was harvested. After concentration and filtration, the CAR lentivirus was stored at -80°C for standby.

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

[0096] 2, CAR-T cell construction: 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 cell mixing (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 .

[0097] 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 that specifically recognizes GPRC5D, characterized in that: The single-domain antibody comprises a heavy chain complementarity determining region HCDR1, and the sequence of the HCDR1 is shown in SEQ ID NO:

9.

2. The single domain antibody according to claim 1, characterized in that The single domain antibody further comprises HCDR2-HCDR3; Preferably, the sequence of HCDR2 is shown as SEQ ID NO: 10, and the sequence of HCDR3 is shown as SEQ ID NO: 11; Preferably, the sequence of HCDR2 is shown as SEQ ID NO: 20, and the sequence of HCDR3 is shown as SEQ ID NO:

21.

3. The single domain antibody according to claim 1, characterized in that The single-domain antibody comprises a heavy chain variable region, the sequence of which is shown in any one of the following groups: 1) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 12; 2) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:

22.

4. An antibody derivative, characterized in that The antibody derivative comprises any one of the following: 1) The single domain antibody according to any one of claims 1 to 3 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 chimeric antigen receptor comprising the heavy chain variable region of the single domain antibody according to any one of claims 1 to 3; 3) A pharmaceutical composition comprising the single domain antibody according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

5. The antibody derivative according to claim 4, characterized in that 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.

6. The antibody derivative according to claim 4, 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.

7. A biomaterial, characterized in that The biological material includes any one of the following: 1) A nucleic acid molecule encoding the single domain antibody according to any one of claims 1 to 3 and / or the antibody derivative according to any one of claims 5 to 6; 2) a vector comprising the nucleic acid molecule described in 1); 3) a recombinant host cell, comprising the nucleic acid molecule described in 1) or the vector described in 2); Preferably, the nucleic acid molecule comprises the base sequence encoding HCDR1 and HCDR2-3 in the heavy chain variable region of the single-domain antibody according to any one of claims 1 to 3 as shown in SEQ ID NOs: 47 and 52; and / or, the nucleic acid molecule comprises the base sequence of an antibody derivative as shown in SEQ ID NOs: 57 and 59; Preferably, the base sequence encoding HCDR1 is shown in SEQ ID NO: 67; Preferably, the base sequences encoding HCDR2-3 are shown in SEQ ID NO:68-69; Preferably, the base sequences encoding HCDR2-3 are shown in SEQ ID NO:73-74; 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.

8. Use of the single domain antibody according to any one of claims 1 to 3, the antibody derivative according to any one of claims 5 to 6, and / or the biomaterial 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 according to any one of claims 1 to 3 or the antibody derivative according to any one of claims 5 to 6, said method comprising artificial synthesis or preparation using genetic engineering technology; The preparation by genetic engineering technology refers to culturing the recombinant host cell described in claim 7 under conditions suitable for expressing the antibody.

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