A single-domain antibody of glypican 3 and its application

Abstract

The present invention relates to a glypican 3 single-domain antibody and its application. The amino acid sequence of CDR1 of the single-domain antibody includes the sequence shown in SEQ ID NO.1, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID NO.2, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID NO.3. The present invention utilizes eukaryotically expressed GPC3 fusion protein to immunize camels, construct a phage display library, and screen to obtain glypican 3 single-domain antibodies with good specificity and affinity. At the same time, they have the characteristics of low molecular weight, which can effectively develop therapeutic drugs, engineered immune cells, and detection reagents.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a glypican 3 single-domain antibody and an application thereof. Background Art

[0002] Glypican-3 (GPC3) is a heparan sulfate proteoglycan found on the cell membrane and belongs to the glypican family. GPC3 is barely expressed in normal adult tissues, but is specifically upregulated in hepatocellular carcinoma (HCC), making it an important biomarker for the diagnosis and treatment of HCC. Furthermore, GPC3 is also expressed in some melanomas, lung cancers, and ovarian cancers. Therefore, GPC3 could also serve as a therapeutic target for these indications.

[0003] Targeted therapy involves directing molecular drugs to specifically bind to specific sites of action on tumor cells, selectively killing them while sparing or minimizing damage to normal tissues and cells. This overcomes the drawbacks of traditional treatments, and as a result, targeted therapy for liver cancer has become a hot topic of research in recent years. Currently, there are two main types of targeted drugs for liver cancer: kinase inhibitors and recombinant humanized or chimeric monoclonal antibodies, such as bevacizumab. In the development and clinical application of antibody-based drugs, their large molecular weight hinders drug penetration into solid tumor lesions. In the development and clinical application of cell-based drugs, they are typically expressed as single-chain Fv (scFv) antibodies. These long coding sequences hinder not only the construction and modification of expression vectors but also hinder binding to the unique spatial structure of cell surface antigens. Currently, nanobodies (single-domain antibodies) have been developed that not only possess full functional properties but also offer advantages over conventional antibodies, such as improved water solubility, lower molecular weight, stronger tissue penetration, weaker immunogenicity, and better recognition of receptor-ligand binding sites. For example, CN110872351A discloses a nano-antibody GN1 that specifically binds to the GPC3 protein. It uses GPC3 protein expressed in eukaryotic cells (HEK293 cells) for immunization to stimulate alpacas to produce high-titer antibodies. Through phage display, the nano-antibody is screened and can specifically bind to liver cancer cells with high expression of the GPC3 protein, thereby inhibiting the proliferation of liver cancer cells.

[0004] In summary, the development of novel GPC3 single-domain antibodies is of great significance for expanding tumor diagnostic and therapeutic tools. Summary of the Invention

[0005] In response to the deficiencies of the existing technology and actual needs, the present invention provides a glypican 3 single-domain antibody and its application, develops a new glypican 3 single-domain antibody, and provides a new tool for the diagnosis and treatment of tumors.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a glypican 3 single-domain antibody, wherein the amino acid sequence of CDR1 of the single-domain antibody includes the sequence shown in SEQ ID NO.1, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID NO.2, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID NO.3.

[0008] The present invention uses CHO-S cells overexpressing GPC3 to immunize alpacas, and constructs a phage display library to mine single-domain antibodies against glypican 3, thereby obtaining single-domain antibodies against glypican 3 with good specificity and affinity. At the same time, they have the characteristics of low molecular weight and can be effectively used in the development of preparations for diagnosing and / or treating GPC3-related diseases.

[0009] Preferably, the amino acid sequence of the glypican 3 single domain antibody includes the sequence shown in SEQ ID NO.4.

[0010] It will be understood that, based on the single domain antibodies designed according to the present invention, functionally similar Nanobodies obtained by amino acid substitution, deletion or addition using genetic modification methods in the art should be within the scope of protection of the present invention, and the number of amino acids substituted, deleted or added can be any value, such as 1, 5, 10, 15 and more, so that the sequence identity of the changed amino acid sequence to its respective corresponding original sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0011] In a second aspect, the present invention provides a heavy chain antibody comprising the variable region of the Glypican 3 single domain antibody described in the first aspect and the full-length or partial sequence of the crystallizable region of human immunoglobulin Ig Fc.

[0012] Preferably, the full-length or partial sequence of the Ig Fc comprises the full-length or partial sequence of the Fc segment of at least one of IgG1, IgG2, IgG3 or IgG4.

[0013] Preferably, the Fc amino acid sequence of the IgG1 includes the sequence shown in SEQ ID NO.5.

[0014] In a third aspect, the present invention provides a nucleic acid molecule encoding the antibody according to the first aspect and / or the second aspect.

[0015] In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the third aspect.

[0016] In a fifth aspect, the present invention provides a host cell containing at least one copy of the nucleic acid molecule described in the third aspect, or at least one copy of the expression vector described in the fourth aspect.

[0017] The host cells of the present invention express the antibodies described in the first and / or second aspects and may contain a nucleic acid molecule encoding the antibody or a recombinant vector containing the nucleic acid molecule. The host cells may be prokaryotic cells, lower eukaryotic cells, or higher eukaryotic cells. Prokaryotic cells include bacterial cells, lower eukaryotic cells include yeast cells, and higher eukaryotic cells include mammalian cells. Representative examples include Escherichia coli and yeast cells.

[0018] In a sixth aspect, the present invention provides use of the antibody described in the first aspect and / or the second aspect in preparing a reagent targeting glypican 3.

[0019] In the present invention, a small molecular weight single-domain antibody with good specificity and affinity is developed, which can be effectively used to prepare a reagent targeting glypican 3. It can be understood that the reagent can be an immunoassay-related reagent or a drug for treating GPC3-related diseases (such as liver cancer, etc.).

[0020] In a seventh aspect, the present invention provides an antibody conjugate, comprising the glypican 3 single-domain antibody according to the first aspect and a coupling substance coupled thereto.

[0021] Preferably, the coupling substance comprises at least one of a cytotoxin, a radioisotope, a luminescent substance, a color-developing substance or an enzyme.

[0022] In an eighth aspect, the present invention provides a chimeric antigen receptor, comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain and a signal transduction domain; the antigen binding domain comprises the glypican 3 single domain antibody described in the first aspect.

[0023] Preferably, the signal peptide comprises at least one of CD8, GM-CSF, IgG, IgE, CD4, CD28, CD137, TCRα or TCRβ.

[0024] Preferably, the hinge region comprises at least one of IgG1, IgG4, CD8, CD28, CD137, TCRα or TCRβ.

[0025] Preferably, the transmembrane domain includes any one of CD28, CD3ε, CD3ζ, CD3γ, CD3δ, TCRα, TCRβ, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233 or CD314, or a combination of at least two of them.

[0026] Preferably, the signal transduction domain includes the intracellular signal transduction domain and costimulatory domain of human CD3.

[0027] Preferably, the intracellular signal transduction domain of human CD3 includes at least one of CD3ζ, CD3ε, CD3γ or CD3δ.

[0028] Preferably, the costimulatory domain comprises at least one of CD3ε, CD3γ, CD3δ, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB, OX40, PD1, Dap10, CDS, ICAM-1, ICOS, NKG2D, GITR or OX40L.

[0029] Preferably, the amino acid sequence of the signal peptide includes the sequence shown in SEQ ID NO.10.

[0030] Preferably, the amino acid sequence of the hinge region includes the sequence shown in SEQ ID NO.11.

[0031] Preferably, the amino acid sequence of the transmembrane domain includes the sequence shown in SEQ ID NO.12.

[0032] Preferably, the amino acid sequence of the costimulatory domain includes the sequence shown in SEQ ID NO.13.

[0033] Preferably, the amino acid sequence of the intracellular signal transduction domain includes the sequence shown in SEQ ID NO.14.

[0034] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the chimeric antigen receptor according to the eighth aspect.

[0035] In a tenth aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the ninth aspect.

[0036] Preferably, the expression vector is any one of a lentiviral vector, a retroviral vector, an adeno-associated viral vector or a liposome vector containing the gene encoding the chimeric antigen receptor described in the ninth aspect.

[0037] In an eleventh aspect, the present invention provides a chimeric antigen receptor cell, wherein the chimeric antigen receptor cell expresses the chimeric antigen receptor according to the eighth aspect.

[0038] Preferably, the starting cells of the chimeric antigen receptor cells include any one of T cells, TIL cells, B cells, NK cells or macrophages, or a combination of at least two of them.

[0039] In the twelfth aspect, the present invention provides the use of the antibody described in the first or second aspect, the nucleic acid molecule described in the third or ninth aspect, the expression vector described in the fourth or tenth aspect, the host cell described in the fifth aspect, the antibody conjugate described in the seventh aspect, the chimeric antigen receptor described in the eighth aspect, and the chimeric antigen receptor cell described in the eleventh aspect in the preparation of products that enhance immune cell killing of tumor cells.

[0040] In the thirteenth aspect, the present invention provides use of the antibody described in the first or second aspect, the nucleic acid molecule described in the third or ninth aspect, the expression vector described in the fourth or tenth aspect, the host cell described in the fifth aspect, the antibody conjugate described in the seventh aspect, the chimeric antigen receptor described in the eighth aspect, and the chimeric antigen receptor cell described in the eleventh aspect in the preparation of a drug or preparation for preventing and / or treating cancer or tumors.

[0041] Preferably, the tumor comprises a GPC3 solid tumor, and the cancer comprises at least one of liver cancer, ovarian cancer, melanoma or lung cancer.

[0042] Compared with the prior art, the present invention has at least the following beneficial effects:

[0043] The present invention uses CHO-S cells overexpressing GPC3 to immunize alpacas. After determining the titer of GPC3-specific antibodies in alpaca serum by ELISA and flow cytometry, the alpaca peripheral blood mononuclear cells are separated, RNA is extracted and reverse transcribed to obtain cDNA, and a phage display library is constructed to screen and obtain glypican 3 single-domain antibodies with good specificity and affinity. At the same time, they have the characteristics of small molecular weight, which can effectively develop therapeutic drugs, engineered immune cells and detection reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1A This is the result of the first round of PCR amplification of the VHH region of the alpaca antibody.

[0045] Figure 1B This is the result of the second round of PCR amplification of the VHH region of the alpaca antibody.

[0046] Figure 2 Detection of EC of recombinant single domain antibodies by flow cytometry50 Result graph.

[0047] Figure 3 The figure shows the specificity results of recombinant single-domain antibodies detected by flow cytometry.

[0048] Figure 4 Schematic diagram of the molecular structure of CAR targeting GPC3.

[0049] Figure 5 This figure shows the results of flow cytometry detection of BVHGC3-030 expression in T cells.

[0050] Figure 6 This is the result of flow cytometry detection of the binding efficiency of BN108 and BVHGC3-030 to GPC3 protein.

[0051] Figure 7 This is a diagram showing the killing effect of Hep3B cells by CAR-T cells targeting GPC3 using IncuCyte real-time quantitative live cell imaging analysis.

[0052] Figure 8 This is a diagram showing the inhibitory effect of CAR-T on tumor growth. DETAILED DESCRIPTION

[0053] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0054] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0055] In the specific embodiments of the present invention, the sources of experimental materials are as follows: CHO-S cells: Gibco, R80007; SK-Hep-1 cells: ATCC, HTB-52; Huh7 cells: Cell Bank of the Chinese Academy of Sciences, SCSP-526; FITC-labeled Rabbit anti-Llama IgG (H+L) antibody: Invitrogen, A16155; anti-M13-HRP antibody: SinoBiological, 11973-MM05T-H; PE-labeled anti-human IgG antibody: Biolegend, 410708; AF647-labeled anti-human IgG antibody: Jackson ImmunoResearch, 109-606-170; anti-VHH antibody: GenScript, A02017; AF647-labeled anti-human GPC3 antibody: R&D system, FAB2119A.

[0056] Heavy-chain antibodies, a special type of antibody lacking light chains, are found in the blood of camelids and elasmobranchs. Compared to ordinary antibodies, they contain only a single heavy-chain variable region (VHH) and two conventional CH2 and CH3 regions. By cloning this heavy-chain variable region, single-domain antibodies (also known as nanobodies) consisting solely of the heavy-chain variable region can be obtained. To address the problem of existing GPC3 antibodies lacking both affinity and specificity, the present invention is dedicated to developing GPC3 single-domain antibodies with smaller molecular weight to improve the drugability of related antibodies and cell-based drugs.

[0057] Example 1

[0058] This example immunizes alpacas and measures antibody titers.

[0059] (1) Construction of GPC3 overexpressing cells

[0060] Based on the amino acid sequence of human GPC3 (UniProt Accession: P51654), a lentiviral expression vector overexpressing human GPC3 was constructed and used to infect CHO-S cells to obtain the GPC3-overexpressing cell line CHO-S-GPC3, which was used for experiments such as alpaca immunization, detection of serum titer, and verification of antibody affinity.

[0061] (2) Immunization of alpacas and detection of serum titer

[0062] Alpacas were immunized with CHO-S-GPC3 cells for a total of five immunizations, one every three weeks. Before each immunization, 5 mL of peripheral blood was collected from the alpacas. The blood samples were then centrifuged at 800 × g for 10 minutes. After overnight centrifugation, the supernatant was transferred to a new sterile centrifuge tube to collect the immune serum. Use sterile PBS to dilute the GPC3-His tag recombinant protein to 1 μg / mL (final concentration), take a new 96-well ELISA plate, add 1 μg / mL of GPC3-His tag recombinant protein to each well at 100 μL, and coat at 4°C overnight; then discard the antigen coating solution and wash the plate 5 times with PBST (containing 0.05% Tween 20); then add 200 μL of blocking solution to each well and block at 37°C for 2 hours; after discarding the blocking buffer, wash the plate 5 times with PBST; at the same time, use PBS to graded dilution of the collected serum, add the graded diluted serum to the 96-well ELISA plate, 100 μL per well (PBS for the control well), and incubate at room temperature for 1 hour; discard the liquid in the well and wash the plate 5 times with PBST; then add 100 μL of HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) was added and incubated at room temperature for 1 hour. After discarding the liquid in the wells, the plate was washed 5 times with PBST. 100 μL / well of TMB colorimetric solution was added. The plate was incubated at room temperature for 10 minutes in the dark. 50 μL / well of stop solution was added. The absorbance was measured using a microplate reader and the OD value of each well was read. 450 The results are shown in Table 1. The binding titer of serum to GPC3 protein before immunization was less than 1:2000 (antibody titer is defined as 1:X, where X is the maximum dilution at which the antibody can be detected; the higher the dilution, the higher the antibody titer), indicating that the anti-GPC3 specific antibody content was extremely low. In contrast, the binding titer of serum to GPC3 protein after the fourth immunization exceeded 1:32K, indicating that the content of anti-GPC3 specific antibodies in the serum was significantly increased.

[0063] Table 1

[0064]

[0065] Example 2

[0066] This example constructs a single domain antibody phage surface display library.

[0067] (1) VHH antibody fragment cloning

[0068] Based on the above results, and on the basis of confirming that alpaca serum contains GPC3-specific antibodies, 100 mL of peripheral blood was collected and peripheral blood mononuclear cells (PBMC) were obtained by enrichment using lymphocyte separation fluid. RNA was extracted and reverse transcribed to obtain cDNA. Then, using cDNA as a template, PCR was performed using specific primers (upstream primers bound to the signal peptide of the VHH antibody ORF, and downstream primers bound to the CH2 region) to amplify the alpaca heavy chain antibody sequence. Further, the PCR products were analyzed by electrophoresis, and fragments with a molecular weight of about 750 bp (target fragments, Figure 1A ). Then, using the first round PCR product as a template, specific primers (upstream primer, specific primer for the antibody FR1 region, with SfiI restriction site GGCCCAGCCGGCC at the 5' end; downstream primer, specific primer for the antibody Hinge and FR4 region, with SfiI restriction site GGCCACGAAGGCC at the 5' end) were used to amplify the VHH fragment of the heavy chain antibody. The PCR products were recovered and separated by electrophoresis, and the target fragment was about 400bp in molecular weight ( Figure 1B ) to obtain a VHH fragment library.

[0069] (2) Electrical stimulation transformation of library vector and library quality detection

[0070] The phage surface display vector pComf and the VHH fragment library obtained above were digested with SfiI endonuclease. After digestion, the VHH fragment library and the linearized pComf vector were ligated using T4 ligase at 16°C overnight. Pre-chill the electroporation cuvette and add 300 μL of the ligation product to E. coli SS320 competent cells. After mixing, add the mixture to the cuvette and electrotransform. Resuspend the cells in 20 mL of SOC medium and resuspend them in a shaker at 37°C for 1 hour. 15 mL of the culture medium was used for subsequent phage enrichment and production, and the remaining 5 mL of the electroporation product was stored at -80°C (storage recipe: add an equal volume of 50% glycerol and mix thoroughly). Separately, 20 μL of the culture medium was serially diluted with 2YT medium, evenly spread on an LB plate (containing ampicillin), and incubated overnight at 37°C. The number of clones generated in each ligation reaction was calculated to determine the capacity of the single-domain antibody phage surface display library. The results showed that the library capacity of the single domain antibody phage display library was 1.60×10 9 Furthermore, 20 single clones on the plate were picked and Sanger sequencing was performed using the M13R primer. The results showed that the sequences of the phage library were highly different, with no repeated sequences and good diversity.

[0071] (3) Enrichment of phage surface display libraries

[0072] Take 15 mL of the bacterial solution after electroporation and recovery culture, and dilute it with 2YT to adjust the OD 600 When OD is about 0.26, add ampicillin (final concentration 100 μg / mL) and culture in a constant temperature shaker at 37°C and 225 rpm. 600 When the OD value is 0.6, add M13KO7 helper phage (the volume of M13KO7 helper phage added = 10 × the volume of bacterial solution × OD 600 ×5×10 8 / M13KO7 titer), shake well, let it stand at 37℃ for 30min, and then culture it in a shaker at 225rpm for 1h at 37℃. After infection, centrifuge at 6000rpm for 10min, discard the supernatant, resuspend it in 2YT-AK medium, and culture it overnight. Then centrifuge the bacterial solution at 10000rpm for 15min, transfer the supernatant containing phage particles to a new centrifuge tube (add 1 / 5 of the bacterial solution volume of PEG / NaCl to the tube), mix well, and let it stand at 4℃. After standing for 2h, centrifuge, collect the phage precipitate, and resuspend it with 1 / 50 of the original volume of PBS. Transfer the resuspended phage to a 1.5mL EP tube and centrifuge to remove insoluble impurities. Transfer the supernatant to a new 1.5mL EP tube, add PEG / NaCl (250μL) to each tube, mix well, let it stand (4℃, 10min), centrifuge (4℃), and discard the supernatant. The cells were resuspended in 1 mL of PBS and centrifuged at 12,000 × g for 5 min at 4°C. The supernatant was transferred to a new 1.5 mL EP tube to obtain the original library of single domain antibody phage surface display.

[0073] Take 10 μL of precipitate, dilute it 10 times, add 200 μL of E. coli ER2738 (OD 600 After mixing, place in a 37°C water bath for 10 minutes, spread on LB plates, culture at 37°C overnight, and count plaques to obtain the titer of the phage surface display library.

[0074] Example 3

[0075] In this example, the target antibody library was screened.

[0076] (1) First round of panning and product amplification

[0077] Seal a 1.5 mL centrifuge tube with 3% MPBS overnight at 4°C. Dilute GPC3 antigen to 50 μg / mL using CBS solution and add to a 96-well solid phase plate. Coat overnight at 4°C. Take 1×10 7CHO-S-GPC3 cells were washed three times with PBS, resuspended in 3% PBSA, and blocked at 37°C for 1 hour. 150 μL of the original library precipitate was added to 350 μL of 1% PBSA and blocked at 4°C for 1 hour. This solution was used as the master mix. The blocked CHO-S cells were centrifuged at 500 × g for 10 minutes, the supernatant removed, and the phage master mix added. The cells were incubated for 1 hour at 4°C to remove phage clones that nonspecifically bound to CHO-S cells. The GPC3 protein in the 96-well plate was discarded, and 200 μL of 3% MPBS buffer was added to the plate. The plate was allowed to stand at room temperature for 1 hour, blocked, and then 3% MPBS buffer was added. The CHO-S-GPC3 cells were centrifuged at 500 × g for 10 minutes. The supernatant was removed and added to the GPC3 protein wells and incubated at room temperature with shaking for 1 hour. Discard the phage supernatant in the GPC3 protein wells and wash the plate with 0.05% PBST six times and PBS four times. Add 100 μL of pH 2.2 Gly-HCl elution buffer to each well and incubate at 37°C with shaking for 8 minutes. Repeat the elution twice to remove specifically bound phage. The eluted product (washed twice with PBS after blocking) was stored in a pre-sealed centrifuge tube at 4°C.

[0078] Take 20mL 2YT medium, add tetracycline at a final concentration of 100μg / mL and 20μL E.coli ER2738, and culture until OD 600 The eluted phage product was added to the ER2738 bacterial solution, mixed, and incubated at 37°C for 30 min. Then 20 mL of 2YT medium was added and cultured at 37°C and 225 rpm for 30 min. 600 When the OD value reaches 0.5, add M13KO7 helper phage (M13KO7 added volume = 10 × bacterial solution volume × OD 600 ×5×10 8 / M13KO7 titer), shake well, and let it stand at 37℃ for 30min. Add ampicillin with a final concentration of 100μg / mL to the bacterial solution, culture at 37℃, 225rpm for 45min, centrifuge at 8000rpm for 20min, discard the supernatant, and collect the bacteria. Resuspend in 40mL 2YT-AK medium and culture overnight at 30℃, 210rpm. Transfer the phage suspension that has been infected and amplified overnight to a 50mL centrifuge tube, centrifuge at 8000rpm, 4℃ for 30min, and divide the supernatant into 40mL centrifuge tubes. Add 10mL PEG / NaCl to each tube, mix well, put on ice, let it stand for 1h, centrifuge at 8000rpm, 4℃ for 30min. Discard the supernatant, resuspend the phage in 1mL sterile PBS, centrifuge to remove insoluble impurities. Transfer 1 mL of phage suspension to a new 1.5 mL centrifuge tube, add 250 μL of PEG / NaCl, mix thoroughly, and incubate at 4°C for 10 minutes to precipitate the phage. Centrifuge and discard the supernatant. Resuspend the phage in PBS and centrifuge to remove insoluble impurities. This product is used as the phage product from the first round of panning amplification.

[0079] (2) Second to fourth rounds of panning and product amplification

[0080] The GPC3 antigen was diluted to 10 μg / mL and added to a 96-well solid-phase plate. The plate was incubated overnight at 4°C to ensure sufficient binding of the antigen to the solid-phase plate. Next, the phage products amplified from the first round of panning were used for the second to fourth rounds of panning. The goal of each round of panning was to increase the number of phages in the phage library that specifically bind to the target antigen (GPC3) to obtain a phage library that specifically binds to GPC3.

[0081] (3) ELISA detection and sequencing of monoclonal phage

[0082] 1. Phage infection and plating: Infect E. coli ER2738 strain with the single-domain antibody phage display library that has undergone multiple rounds of screening. Mix well and place in a 37°C water bath for 10 minutes. Subsequently, spread the cultured bacteria onto LB plates and incubate at 37°C overnight.

[0083] 2. Pick a single colony and culture: Pick a single colony of bacteria from the LB plate, transfer it to a 96-well deep-well plate, add 200 μL of 2YT-A medium to each well, and culture overnight at 37°C and 225 rpm.

[0084] 3. Cultivate to OD 600 =0.5: Transfer the overnight cultured bacteria to a new 96-well deep-well plate, add 150 μL of 2YT-A medium to each well, then add 20 μL of the overnight cultured bacteria, and continue culturing until the OD600 value is approximately 0.5.

[0085] 4. Add M13KO7 helper phage for infection: Calculate the required volume of M13KO7 phage (according to the formula: M13KO7 volume = 10 × bacterial volume × OD600 × 5 × 10^8 / M13KO7 titer). After adding M13KO7 helper phage, mix well and let it stand for 15 minutes. Then, incubate at 37°C and 225 rpm for 45 minutes.

[0086] 5. Centrifugation and incubation: Centrifuge at 3900 rpm for 10 minutes, discard the supernatant, and resuspend each well in 500 μL of 2YT-AK medium. Transfer the cells to a microplate reader at 30°C and 220 rpm for overnight incubation.

[0087] 6. Collect phage particles: After the second centrifugation, the supernatant obtained is the monoclonal phage particles.

[0088] 7. Antigen coating: GPC3 antigen protein (concentration of 2 μg / mL, 100 μL / well) was coated onto the ELISA plate using CBS buffer at pH 9.6 and incubated at 4°C overnight.

[0089] 8. Blocking treatment: discard the antigen solution, wash three times with PBST, then add 250 μL of 3% MPBS blocking solution to each well and incubate at 4°C overnight.

[0090] 9. Washing step: Remove the blocking solution and wash the plate 4 times with 0.05% PBST. After each wash, add 50 μL of 0.1% PBST and then add 50 μL of monoclonal phage supernatant (i.e., phage solution).

[0091] 10. Incubation and secondary antibody reaction: After incubation for 1 hour, wash with 0.05% PBST five times, add diluted anti-M13-HRP antibody (1:5000, 100 μL / well), and incubate at 4°C for 45 minutes.

[0092] 11. Color development reaction: After washing with PBST, add 100 μL TMB color development solution and react at room temperature for 10 minutes. After the reaction is completed, add 50 μL 0.2M hydrochloric acid to terminate the reaction and then read the OD 450 value.

[0093] 12. Screening and Sequencing: Calculate the OD of Samples and Negative Controls 450 The clones with a significantly greater ratio than that of the positive serum control group were selected for further sequencing, and finally obtained and named single-domain antibody 74-1-C07-1. The CDR1 amino acid sequence of the single-domain antibody 74-1-C07-1 (SEQ ID NO. 1) is: GPTFSSYA.

[0094] The CDR2 amino acid sequence of the single-domain antibody 74-1-C07-1 (SEQ ID NO. 2) is: EFVAAISRGATYTS.

[0095] The CDR3 amino acid sequence of the single-domain antibody 74-1-C07-1 (SEQ ID NO. 3) is: AAGPDTVARRTGRGEYEY.

[0096] The amino acid sequence of the single-domain antibody 74-1-C07-1 is (SEQ ID NO. 4):

[0097] QVQLVESGGGWVQPGGSLRLSCAASGPTFSSYAVGWFRQAPGKEREFVAAISRGATYT SYADSVKGRFTISRDNAKNTSYLQMNSLKLEDTAVYYCAAGPDTVARRTGRGEYEYWGQG TQVTVSS.

[0098] Example 4

[0099] In this example, the recombinant antibody was purified and the half effective concentration (EC 50 ) determination.

[0100] The sequence of the single-domain antibody was amplified by PCR, and the PCR product was inserted into the pcDNA3.4 expression vector, fused to the human IgG1 Fc fragment at its C-terminus. The resulting recombinant plasmid was transfected into HEK293 cells. After a period of culture, the cell culture supernatant was harvested, which contained the recombinant single-domain antibody. In the same manner, the GPC3-specific antibody HYP7 was used as a positive control, cloned and transfected into HEK293 cells according to the same steps. Expression levels and functional verification were compared.

[0101] Human IgG1 Fc amino acid sequence (SEQ ID NO.5):

[0102] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0103] HYP7 light chain variable region amino acid sequence (SEQ ID NO.6):

[0104] DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASS RESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYPLTFGAGTKLELK.

[0105] HYP7 heavy chain variable region amino acid sequence (SEQ ID NO.7):

[0106] EVQLVETGGGMVQPEGSLKLSCAASGFTFNKNAMNWVRQAPGKGLEWVARIRNKTN NYATYYADSVKARFTISRDDSQSMLYLQMNNLKIEDTAMYYCVAGNSFAYWGQGTLVTVS A.

[0107] The recombinant antibody was affinity purified by Protein A magnetic beads and further purified for EC50 determination: To determine the EC50 of the 74-1-C07-1 antibody 50 293F cells were transfected with a plasmid expressing the recombinant antibody and cultured in a shaker flask for antibody expression and purification. Since the target recombinant antibody contains a human IgG fragment, affinity purification using Protein A magnetic beads was performed. The Protein A magnetic beads were washed twice with 30 mL of PBS buffer, 0.1 M sodium hydroxide, and PBS buffer, respectively. Based on the sample volume, the appropriate volume of Protein A magnetic beads was added to the 293F cell shaker flask (calculated as 1 mL of Protein A magnetic beads per 20 mg of IgG). The cells were incubated in a shaking incubator at 120 rpm for 1–4 hours or at room temperature at 4°C overnight. The Protein A magnetic beads were collected using a magnetic separation rack and transferred to a 50 mL centrifuge tube. The cells were washed twice with 30 mL of PBS buffer and twice with deionized water, and resuspended in 1 mL of elution buffer. After incubation at room temperature for 5 minutes, the beads were collected and the supernatant containing the target antibody was transferred to a 15 mL centrifuge tube. Elute the Protein A beads twice, combine the eluates, add neutralization buffer, and adjust the pH. Dialyze the eluted sample against at least 100 times the volume of PBS as the sample at 18-25°C for 2 hours. After changing the buffer, dialyze for 14-16 hours at 2-8°C. Finally, determine the protein concentration, filter through a 0.22 μm sterile filter, and aliquot the sample and store at -80°C until use.

[0108] Target antibodies were serially diluted and mixed with 3×10 5Incubate CHO-S-GPC3 and CHO-S cells at room temperature for 1 hour. Centrifuge at 800 × g for 5 minutes at room temperature, discard the supernatant containing the antibody, and wash the cells three times with PBS. Add 100 μL of PE-conjugated anti-human IgG antibody (1:500 dilution), mix thoroughly, and incubate at room temperature in the dark for 45 minutes. Centrifuge at 800 × g for 5 minutes at room temperature, discard the supernatant containing the antibody, wash the cells three times with PBS, and resuspend the cells in 500 μL of PBS for flow cytometry analysis.

[0109] Flow cytometry detection of EC of recombinant single domain antibodies 50 The results showed that the positive control antibody HYP7 had an EC 50 The value was 12.93 μg / mL, and the EC value of 74-1-C07-1 on CHO-S-GPC3 was 50 The value was 0.6996 μg / mL ( Figure 2 ), EC of positive antibodies 50 The above results show that, compared with the prior art, 74-1-C07-1 has a stronger binding ability to GPC3.

[0110] Example 5

[0111] This example detects the binding of recombinant single-domain antibodies to target proteins and tumor cells.

[0112] The culture supernatant containing 74-1-C07-1 antibody or positive control antibody was mixed with 3×10 5 Huh7 cells (naturally expressing GPC3), CHO-S cells (GPC3-negative), and CHO-S-GPC3 cells (exogenously overexpressing GPC3) were incubated at room temperature for 1 hour. The cells were centrifuged at 800 × g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed three times with PBS. 100 μL of PE-conjugated anti-human IgG antibody (1:500 dilution) was added and incubated at room temperature in the dark for 45 minutes. The cells were centrifuged at 800 × g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed three times with PBS. The cells were resuspended in 500 μL of PBS and analyzed by flow cytometry. For the negative control, only 100 μL of PE-conjugated anti-human IgG antibody (1:500 dilution) was added and incubated at room temperature in the dark for 45 minutes. The cells were centrifuged at 800 × g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed three times with PBS. The cells were resuspended in 500 μL of PBS and analyzed by flow cytometry.

[0113] The negative control group had no significant binding to any cells; the positive control antibodies HYP7 and 74-1-C07-1 had significant binding to Huh7 and CHO-S-GPC3 cell lines, but had no significant binding to CHO-S cells, indicating that the 74-1-C07-1 antibody had good specificity for binding to GPC3.

[0114] Example 6

[0115] This example constructs CAR-T cells based on the target antibody.

[0116] (1) Design of CAR molecules

[0117] The target gene structure of the lentiviral vector involved in this embodiment is as follows Figure 4 shown.

[0118] BVHGC3-030 is composed of the following structures in series: human CD8 signal peptide (abbreviated as SP), anti-human GPC3 single-domain antibody 74-1-C07-1 [abbreviated as VHH(74-1-C07-1)], human IgG4 hinge region (abbreviated as IgG4 hinge), human CD8 transmembrane domain (abbreviated as CD8 TM), human 4-1BB intracellular co-stimulatory domain (abbreviated as 4-1BB ICD), and human CD3ζ intracellular signal transduction domain (abbreviated as CD3ζICD).

[0119] BN108 is composed of the following structures in series: human CD8 signal peptide (SP), anti-human GPC3 single-chain antibody [scFv (GC33)], human CD8 hinge region (CD8 hinge), human CD8 transmembrane domain (CD8 TM), human 4-1BB intracellular co-stimulatory domain (4-1BB ICD), and human CD3ζ intracellular signal transduction domain (CD3ζICD).

[0120] Anti-human GPC3 single-chain antibody light chain variable region amino acid sequence (SEQ ID NO.8):

[0121] DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKR.

[0122] Anti-human GPC3 single-chain antibody heavy chain variable region amino acid sequence (SEQ ID NO.9):

[0123] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKT GDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS.

[0124] Human CD8 signal peptide amino acid sequence (SEQ ID NO.10):

[0125] MALPVTALLLPLALLLHAARPS.

[0126] Human IgG4 hinge amino acid sequence (SEQ ID NO.11):

[0127] ESKYGPPCPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0128] Human CD8 transmembrane domain amino acid sequence (SEQ ID NO.12):

[0129] CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR.

[0130] Human 4-1BB intracellular costimulatory domain amino acid sequence (SEQ ID NO.13):

[0131] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0132] Human CD3ζ intracellular signal transduction domain amino acid sequence (SEQ ID NO.14):

[0133] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0134] The amino acid sequence of the anti-human GPC3 chimeric antigen receptor (BVHGC3-030) (SEQ ID NO.15):

[0135] MALPVTALLLPLALLLHAARPSQVQLVESGGGWVQPGGSLRLSCAASGPTFSSYAVGWFRQAPGKEREFVAAISRGATYTSYADSVKGRFTISRDNAKNTSYLQMNSLKLEDTAVYYCAAGPDTVARRTGRGEYEYWGQGTQVTVSSESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0136] The amino acid sequence (SEQ ID NO.16) of the anti-human GPC3 chimeric antigen receptor (BN108):

[0137] MALPVTALLLPLALLLHAARPSDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPP TFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCT RFYSYTYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0138] (2) Lentivirus preparation

[0139] The CAR molecule expression sequence was fully synthesized and ligated into the lentiviral vector pCDH-EF1α-MCS, where its expression was controlled by the human EF-1α promoter and Kozak sequence. Using Lipofectamine 3000, the lentiviral expression plasmids were co-transfected into 293T cells with the lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE, and pMD2.G, respectively. After 48 hours, the viral supernatant was collected and centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was then filtered through a 0.45 μm pore size filter and ultracentrifuged at 25,000 rpm for 3 hours at 4°C. The resulting viral concentrates were stored at -80°C and designated BVHGC3-030 and BN108, respectively. Finally, the activity titers of the lentiviruses were assayed using Jurkat cells.

[0140] (3) CAR-T cell preparation

[0141] PBMCs from healthy donors were revived in AIM V medium (containing 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody, 300 IU / mL recombinant hIL-2) and cultured in a cell culture incubator for 24 h (culture conditions: 37°C, 5% carbon dioxide). The obtained T cells were washed and transduced with lentivirus at an MOI of 5 TU / mL. At the same time, 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody, and 300 IU / mL recombinant hIL-2 were supplemented and cultured in a cell culture incubator for 24 h (culture conditions: 37°C, 5% carbon dioxide). After 24 h of culture, the cell density was adjusted to 1.8×10 6 / mL, and add 300IU / mL of hIL-2. On the 4th day after transduction, the cells were washed by centrifugation to remove the residual lentivirus in the supernatant, and cultured for another 5 days, during which the cell density was maintained at 1.5×10 6 / mL. Ten days after transduction, cells were harvested and frozen in liquid nitrogen for future use. The resulting CAR-T cells were named after the corresponding CAR molecules, while T cells not transduced with lentivirus were designated Mock.

[0142] (4) Detection of CAR molecule expression

[0143] Wash BVHGC3-030 CAR-T cells twice with PBS and resuspend in FACS buffer. Add FITC-labeled anti-VHH antibody to the CAR-T cell suspension according to the antibody instructions and incubate for 1 hour. After incubation, centrifuge to remove the supernatant, wash the cells twice with FACS buffer and resuspend. Using Mock cells as a negative control, flow cytometry was used to detect the CAR molecule expression rate of BVHGC3-030 cells. The results are shown in Figure 2. Figure 5 The expression rates of BVHGC3-030 CAR were shown to be 65.3%.

[0144] (4) Detection of CAR molecule binding efficiency to GPC3

[0145] BN108 and BVHGC3-030 CAR-T cells were washed twice with PBS and resuspended with FACS buffer (PBS containing 0.1% sodium azide and 0.4% BSA). According to the antibody instructions, GPC-3-His tag protein was incubated with CAR-T cells for 1 hour, then centrifuged to remove the supernatant, washed twice with FACS buffer, and resuspended. PE-labeled anti-G4S Linker antibody was then incubated with CAR-T cells for 1 hour. The supernatant was then centrifuged to remove the supernatant, washed twice with FACS buffer, and resuspended. Ctrl T cells were used as negative controls, and the efficiency of BN108 and BVHGC3-030 CAR-T cells binding to GPC3 was detected by flow cytometry. The results are shown in Figure 2. Figure 6The binding efficiencies of BN108 and BVHGC3-030 CAR to GPC3 were shown to be 58.2% and 64.5%, respectively.

[0146] Example 7

[0147] This example studies the function of CAR-T cells.

[0148] In vitro killing experiments were performed using BVHGC3-030 and BN108 CAR-T cells. 5 The target cells Hep3B (with mCherry fluorescent labeling) were resuspended at a density of 100 μL / mL and inoculated into a 96-well plate at a volume of 100 μL per well. After being cultured overnight in the IncuCyte SX5 live cell imaging analysis system, CAR-T cells were added for co-culture at an effect-target ratio of 1:4 to the effective number of cells, and the killing effect of CAR-T cells on tumor cells was recorded in real time. In addition, a target cell group only and a mock group (ie, T cells without lentiviral transduction) were set as controls. After the co-culture, the changes in the mCherry fluorescence signals of the target cells in each group were calculated using the IncuCyte SX5 software. The lower the signal value, the fewer cells in the group and the better the killing effect of CAR-T cells.

[0149] The results are as follows Figure 7 As shown, under the condition of an effector-target ratio of 1:1, the killing effect of the BN108 group (relative fluorescence intensity at the last time point was 0.51±0.01) on GPC3-positive Hep3B cells was significantly improved compared with the mock group (relative fluorescence intensity at the last time point was 1.19±0.01), while the killing effect of the BVHGC3-030 group (relative fluorescence intensity at the last time point was 0.19±0.01) was significantly stronger than that of the BN108 group (P<0.05). These results show that compared with existing antibodies, CAR-T cells constructed based on the 74-1-C07-1 antibody have significantly improved the killing effect on GPC3-positive tumor cells and have the potential to be further developed into highly effective anti-tumor drugs.

[0150] Example 8

[0151] This example studies the in vivo efficacy of CAR-T cells.

[0152] A subcutaneous tumor-bearing model of the human hepatocellular carcinoma cell line Hep3B was established in severely immunodeficient mice. 14 days after tumor formation, BVHGC3-030 and BN108 CAR-T cells were intravenously infused for in vitro efficacy experiments. The tumor volume of the mice was measured twice a week to reflect the inhibitory effect of CAR-T on tumor growth. The solvent and mock cells were intravenously infused under the same culture conditions as controls.

[0153] The results are as follows Figure 8 As shown, compared with the vehicle group, the T cells in the mock group had no significant inhibitory effect on the growth of Hep3B tumors, while the BN108 and BVHGC3-030 groups significantly inhibited the growth of Hep3B tumors. One week after the CAR T cell infusion, the tumors were completely eliminated, and the mice maintained tumor-free survival for more than 3 weeks. Moreover, the anti-tumor effect of BVHGC3-030CAR-T was comparable to that of the positive control group (BN108), indicating that CAR-T cells constructed based on the 74-1-C07-1 antibody have the potential to be further developed into highly effective anti-tumor drugs.

[0154] In summary, the present invention develops a new GPC3 single-domain antibody with good specificity and affinity, and has broad application prospects. It can be effectively used to prepare reagents targeting GPC3, including detection reagents and drugs, such as constructing chimeric antigen receptors and corresponding immune cells expressing chimeric antigen receptors, which can effectively and specifically kill tumor cells.

[0155] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A glypican 3 single domain antibody, characterized in that: The amino acid sequence of CDR1 of the single-domain antibody is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

3.

2. The glypican 3 single domain antibody according to claim 1, wherein The amino acid sequence of the glypican 3 single domain antibody is shown in SEQ ID NO.

4.

3. A glypican 3 heavy chain antibody, characterized in that: The heavy chain antibody comprises the glypican 3 single domain antibody according to claim 1 or 2 and the full-length or partial sequence of the crystallizable segment Ig Fc of human immunoglobulin.

4. The glypican 3 heavy chain antibody according to claim 3, wherein The full-length or partial sequence of the Ig Fc comprises the full-length or partial sequence of the Fc segment of at least one of IgG1, IgG2, IgG3 or IgG4; The Fc amino acid sequence of the IgG1 includes the sequence shown in SEQ ID NO.

5.

5. Use of the antibody according to any one of claims 1 to 4 in the preparation of a medicament for preventing and / or treating tumors; The tumor is a GPC3-positive solid tumor; The GPC3-positive solid tumor is liver cancer.

6. An antibody conjugate, characterized in that The antibody conjugate comprises the antibody according to any one of claims 1 to 4 and a conjugated substance conjugated thereto.

7. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain and a signal transduction domain; The antigen binding domain comprises the glypican 3 single domain antibody according to claim 1 or 2.

8. The chimeric antigen receptor according to claim 7, characterized in that The signal peptide includes at least one of CD8, GM-CSF, IgG, IgE, CD4, CD28, CD137, TCRα or TCRβ; The hinge region comprises at least one of IgG1, IgG4, CD8, CD28, CD137, TCRα or TCRβ; The transmembrane domain includes any one or a combination of at least two of CD28, CD3ε, CD3ζ, CD3γ, CD3δ, TCRα, TCRβ, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233 or CD314; The signal transduction domain includes the intracellular signal transduction domain and the costimulatory domain of human CD3; The intracellular signal transduction domain of human CD3 includes at least one of CD3ζ, CD3ε, CD3γ or CD3δ; The costimulatory domain includes at least one of CD3ε, CD3γ, CD3δ, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB, OX40, PD1, Dap10, CDS, ICAM-1, ICOS, NKG2D, GITR or OX40L; The amino acid sequence of the signal peptide includes the sequence shown in SEQ ID NO.10; The amino acid sequence of the hinge region includes the sequence shown in SEQ ID NO.11; The amino acid sequence of the transmembrane domain includes the sequence shown in SEQ ID NO.12; The amino acid sequence of the costimulatory domain includes the sequence shown in SEQ ID NO.13; The amino acid sequence of the intracellular signal transduction domain includes the sequence shown in SEQ ID NO.

14.

9. The chimeric antigen receptor according to claim 7, wherein The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.

15.

10. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody according to any one of claims 1 to 4, or the nucleic acid molecule encodes the chimeric antigen receptor according to any one of claims 7 to 9.

11. An expression vector, characterized in that The expression vector comprises the nucleic acid molecule of claim 10.

12. The expression vector according to claim 11, characterized in that The expression vector is any one of a lentiviral vector, a retroviral vector, an adeno-associated viral vector or a liposome vector containing the gene encoding the chimeric antigen receptor according to any one of claims 7 to 9.

13. A host cell, characterized in that The host cell contains at least one copy of the nucleic acid molecule according to claim 10, or at least one copy of the expression vector according to claim 11 or 12.

14. A chimeric antigen receptor cell, characterized in that The chimeric antigen receptor cell expresses the chimeric antigen receptor according to any one of claims 7 to 9.

15. The chimeric antigen receptor cell according to claim 14, characterized in that The starting cells of the chimeric antigen receptor cells include any one of T cells, TIL cells, B cells, NK cells or macrophages, or a combination of at least two of them.

16. Use of the antibody according to any one of claims 1 to 4, the antibody conjugate according to claim 6, the chimeric antigen receptor according to any one of claims 7 to 9, the nucleic acid molecule according to claim 10, the expression vector according to claim 11 or 12, the host cell according to claim 13, or the chimeric antigen receptor cell according to claim 14 or 15 in the preparation of a product that enhances immune cell killing of tumor cells, or in the preparation of a drug or preparation for preventing and / or treating tumors; The tumor includes a GPC3-positive solid tumor; The GPC3-positive solid tumor is liver cancer.

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