Phosphatidylinositol proteoglycan 3 single-domain antibody and application thereof

Abstract

The invention relates to a phosphatidylinositol proteoglycan 3 single-domain antibody and application thereof. The amino acid sequence of the CDR1 of the single-domain antibody comprises a sequence as shown in SEQ ID NO.1, the amino acid sequence of the CDR2 of the single-domain antibody comprises a sequence as shown in SEQ ID NO.2, and the amino acid sequence of the CDR3 of the single-domain antibody comprises a sequence as shown in SEQ ID NO.3. According to the invention, eukaryotically expressed GPC3 fusion protein is utilized to immunize camels, a phage display library is constructed, the glypican 3 single-domain antibody with good specificity and affinity is obtained through screening, the glypican 3 single-domain antibody has the characteristic of small component force, and therapeutic drugs, engineering immune cells, detection reagents and the like can be effectively developed.

Description

Technical Field

[0001] The 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 on the cell membrane surface and a member of the glypican family. GPC3 is hardly expressed in normal adult tissues, but is specifically upregulated in hepatocellular carcinoma (HCC), etc., so GPC3 has become an important biomarker for the diagnosis and treatment of hepatocellular carcinoma. In addition, GPC3 is also expressed in some melanomas, lung cancers, and ovarian cancers. Therefore, GPC3 can also be used as a therapeutic target for such indications.

[0003] Targeted therapy refers to the specific binding of molecular drugs to certain specific action sites of tumor cells, selectively killing tumor cells without killing or only slightly damaging normal tissues and cells, overcoming the drawbacks of traditional treatment. Therefore, targeted therapy of liver cancer has become a hot topic of research in recent years. At present, there are two main types of liver cancer targeted drugs: one is kinase inhibitors; the second is recombinant humanized, human-mouse chimeric monoclonal antibodies, such as bevacizumab. In the development and clinical application of antibody drugs, their large molecular weight is not conducive to improving the drug's penetration rate into solid tumor lesions; in the development and clinical application of cell-related drugs, they are usually expressed in the form of single-chain antibodies (scFv). Their long coding sequences are not only not conducive to the construction and modification of expression vectors, but also not conducive to binding to cell surface antigens with unique spatial structures. At present, the nanoantibodies (single domain antibodies) obtained in the study not only have complete functions, but also have the advantages of good water solubility, small molecular weight, strong tissue penetration, weak immunogenicity, and better recognition of receptor and ligand binding sites compared with conventional antibodies. For example, CN110872351A discloses a nano-antibody GN1 that specifically binds to the GPC3 protein. It uses the GPC3 protein expressed by 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 that highly express 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 view of the deficiencies of the prior art and actual needs, the present invention provides a glypican 3 single-domain antibody and its application, develops a novel 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, constructs a phage display library, mines single-domain antibodies against glypican 3, and obtains single-domain antibodies against glypican 3 with good specificity and affinity, while having the characteristics of small 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 can be understood that based on the single domain antibodies designed by the present invention, the functionally similar Nanobodies obtained by amino acid substitution, deletion or addition by genetic modification means in the art should be within the scope of protection of the present invention, and the number of the above-mentioned substituted, deleted or added amino acids can be any value, such as 1, 5, 10, 15 and above, so that the sequence identity of the changed amino acid sequence and 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 according to 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 Ig Fc includes 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 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 described in the first aspect and / or the second aspect.

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

[0016] In a fifth aspect, the present invention provides a host cell, wherein the host cell contains 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 cell of the present invention expresses the antibody described in the first aspect and / or the second aspect, which may contain a nucleic acid molecule encoding the antibody or a recombinant vector containing the nucleic acid molecule, and the host cell may be a prokaryotic cell, a lower eukaryotic cell or a higher eukaryotic cell, a prokaryotic cell such as a bacterial cell, a lower eukaryotic cell such as a yeast cell, and a higher eukaryotic cell such as a mammalian cell. Representative examples include Escherichia coli, yeast cells, etc.

[0018] In a sixth aspect, the present invention provides use of the antibody described in the first aspect and / or the second aspect in the preparation of an agent 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 in the preparation of 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 described in the first aspect and a conjugated substance conjugated thereto.

[0021] Preferably, the coupling substance includes at least one of a cytotoxin, a radioactive isotope, 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 phosphatidylinositol proteoglycan 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 the co-stimulatory 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 co-stimulatory 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 co-stimulatory 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 described in 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 virus 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 described in 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 a product that enhances the killing of tumor cells by immune cells.

[0040] In the thirteenth 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 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, and after determining the titer of GPC3-specific antibodies in alpaca serum by ELISA and flow cytometry, separates alpaca peripheral blood mononuclear cells, extracts RNA and reverse transcribes to obtain cDNA, constructs a phage display library, and screens to obtain glypican 3 single-domain antibodies with good specificity and affinity, which also have the characteristics of small molecular weight, and can effectively develop therapeutic drugs, engineered immune cells, detection reagents, and the like. 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 This is a graph showing 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 is a graph showing the expression results of BVHGC3-030 in T cells detected by flow cytometry.

[0050] Figure 6 This is a graph showing the results 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 living 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 is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only 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 techniques or conditions described in the literature in the field or the product instructions are used. 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] There is a special antibody lacking light chains in the blood of camelids and chondrichthyes, namely heavy chain antibodies. Compared with ordinary antibodies, they only contain one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. By cloning their heavy chain variable regions, single-domain antibodies (also known as nanobodies) consisting only of heavy chain variable regions can be obtained. In order to solve the problem that existing GPC3 antibodies cannot have both affinity and specificity, the present invention is committed to developing GPC3 single-domain antibodies with smaller molecular weight to improve the drugability of related antibodies and cell 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 testing of serum titer

[0062] CHO-S-GPC3 cells were used to immunize alpacas for a total of 5 immunizations, once every three weeks. Before each immunization, 5 mL of alpaca peripheral blood was collected. Then, the blood sample was placed in a centrifuge tube and centrifuged at 800×g for 10 min. After 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, wash the plate 5 times with PBST (containing 0.05% Tween 20); then add 200 μL of blocking solution to each well, block at 37°C for 2h; after discarding the blocking buffer, wash the plate 5 times with PBST; at the same time, use PBS to gradient dilute the collected serum, add the gradient diluted serum to the 96-well ELISA plate, 100 μL per well (PBS for the control well), and incubate at room temperature for 1h; 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), incubate at room temperature for 1h; discard the liquid in the well, wash the plate with PBST 5 times; then add 100μL / well TMB colorimetric solution; incubate at room temperature for 10min, protect from light; add stop solution, 50μL / well; use an enzyme reader to detect absorbance and read the OD of each well 450 Value. The results are shown in Table 1. The binding titer of serum to GPC3 protein before immunization of alpaca is lower than 1:2000 (antibody titer is defined as 1:X, X is the maximum dilution factor at which the antibody can be detected; the higher the dilution factor, the higher the antibody titer), indicating that the content of anti-GPC3 specific antibodies contained therein is extremely low. On the contrary, the binding titer of serum to GPC3 protein after the fourth immunization is more than 1:32K, indicating that the content of anti-GPC3 specific antibodies in the serum is 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 transcription was performed to obtain cDNA. Then, using cDNA as a template, PCR was performed using specific primers (the upstream primer binds to the signal peptide of the VHH antibody ORF, and the downstream primer binds to the CH2 region) to amplify the alpaca heavy chain antibody sequence. Further, the PCR product was analyzed by electrophoresis, and a fragment with a molecular weight of about 750 bp (the target fragment, Figure 1A ). Then, using the first round PCR product as a template, specific primers (upstream primer, specific primer for antibody FR1 region, with SfiI restriction site GGCCCAGCCGGCC at 5' end; downstream primer, specific primer for antibody Hinge and FR4 region, with SfiI restriction site GGCCACGAAGGCC at 5' end) were used to amplify the VHH fragment of the heavy chain antibody. The PCR product was recovered and separated by electrophoresis, and the target fragment with a molecular weight of about 400bp was ( 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 a single enzyme using SfiI endonuclease. After the enzyme digestion was completed, the VHH fragment library and the linearized pComf vector were connected using T4 ligase at 16°C overnight. Pre-cool the electroporation cup, add 300 μL of the above-mentioned connection product to the E.coli SS320 competent cells, add it to the electroporation cup after mixing, electrotransform, add 20 mL of SOC medium to resuspend the bacteria, resuscitate and culture, shake at 37°C for 1 hour. Take 15 mL of bacterial solution for subsequent phage enrichment production, and the remaining 5 mL of electroporation product is stored at -80°C (storage formula: add an equal volume of 50% glycerol and mix evenly). In addition, take 20 μL of bacterial solution, dilute it with 2YT medium gradient, evenly spread it on an LB plate (containing ampicillin), culture it at 37°C overnight, calculate the number of clones that can be produced by each connection reaction, and obtain 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 with M13R primers. 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 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 value was 0.6, M13KO7 helper phage was added (the volume of M13KO7 helper phage added = 10 × bacterial solution volume × OD 600 ×5×10 8 / M13KO7 titer), shake well, let stand at 37℃ for 30min, and then culture in a shaker at 225rpm for 1h at 37℃. After infection, centrifuge at 6000rpm for 10min, discard the supernatant, resuspend with 2YT-AK medium, and culture 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 volume of bacterial solution PEG / NaCl to the tube), mix well, and place at 4℃. After standing for 2h, centrifuge, collect the phage precipitate, and resuspend 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 stand (4℃, 10min), centrifuge (4℃), and discard the supernatant. The cells were resuspended in 1 mL 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 the precipitate, dilute it 10 times, and add 200 μL of E. coli ER2738 (OD 600 0.5), mix well and place in a 37°C water bath for 10 min, then spread on LB plates. Culture overnight at 37°C, count plaques, and obtain the titer of the phage surface display library.

[0074] Example 3

[0075] In this example, a 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 with CBS solution, add to a 96-well solid phase plate, and coat overnight at 4°C. Take 1×10 7CHO-S-GPC3 cells were washed 3 times with PBS, resuspended with 3% PBSA, blocked at 37°C for 1h. 150μL of the precipitated product of the original library was taken, 350μL of 1% PBSA was added, and blocked at 4°C for 1h, and this solution was used as the premix. The blocked CHO-S cells were centrifuged at 500×g for 10min, the supernatant was removed, and the phage premix was added, incubated for 1h at 4°C to remove the phage clones that non-specifically bound to CHO-S. The GPC3 protein in the 96-well plate was discarded, and 200μL of 3% MPBS buffer was added and allowed to stand at room temperature, blocked for 1h, and 3% MPBS buffer was added. CHO-S-GPC3 cells were centrifuged at 500×g for 10min, the supernatant was taken and added to the GPC3 protein wells respectively, and incubated at room temperature for 1h. Discard the phage supernatant in the GPC3 protein wells, wash the plate: 0.05% PBST × 6 times, PBS × 4 times. Add 100 μL of pH 2.2 Gly-HCl elution solution to each well, incubate at 37°C for 8 minutes, repeat elution twice for the specifically bound phage, and store the eluted product (washed twice with PBS after blocking) in a pre-sealed centrifuge tube at 4°C.

[0078] Take 20 mL of 2YT medium, add tetracycline at a final concentration of 100 μg / mL and 20 μL of 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 it 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 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 infected and amplified overnight to a 50mL centrifuge tube, centrifuge at 8000rpm, 4℃ for 30min, and dispense the supernatant into 40mL centrifuge tubes. Add 10mL PEG / NaCl to each tube, mix well, put on ice, let stand for 1h, centrifuge at 8000rpm, 4℃ for 30min. Discard the supernatant, resuspend the phage in 1mL sterile PBS, centrifuge, and remove insoluble impurities. Transfer 1 mL of phage suspension to a new 1.5 mL centrifuge tube, add 250 μL of PEG / NaCl, mix well, and let stand at 4°C for 10 min to precipitate 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 obtained in 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 at 4°C overnight to ensure sufficient binding of the antigen to the solid phase plate. Next, the phage products amplified in the first round of panning were used for the second to fourth rounds of panning. The purpose of each round of panning is to increase the phages in the phage library that have specific binding ability 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 coating: The single-domain antibody phage display library that has undergone multiple rounds of screening was infected with the E. coli ER2738 strain, mixed and placed in a 37°C water bath for 10 minutes. Subsequently, the cultured bacteria were spread on LB plates and cultured at 37°C overnight.

[0083] 2. Pick a single clone and culture: Pick a single clone of bacteria from the LB plate, transfer it to a 96-well deep-well plate, add 200 μL 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 bacterial solution to a new 96-well deep-well plate, add 150 μL of 2YT-A medium to each well, then add 20 μL of overnight cultured bacterial solution, and continue culturing until the OD600 value is about 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 solution volume × OD600 × 5 × 10^8 / M13KO7 titer), add M13KO7 helper phage, mix and let stand for 15 minutes, and then culture at 37°C, 225rpm for 45 minutes.

[0086] 5. Centrifugation and culture: Centrifuge at 3900 rpm for 10 min, discard the supernatant, resuspend each well with 500 μL 2YT-AK culture medium, and transfer to 30°C, 220 rpm for overnight culture.

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

[0088] 7. Antigen coating: Use CBS buffer at pH 9.6 to coat the GPC3 antigen protein (concentration of 2 μg / mL, 100 μL / well) onto the ELISA plate 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, wash 4 times with 0.05% PBST, add 50 μL of 0.1% PBST after each wash, and then add 50 μL of monoclonal phage supernatant (ie, phage solution).

[0091] 10. Incubation and secondary antibody reaction: After incubation for 1 hour, wash with 0.05% PBST for 5 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 ratio was calculated, and clones with a ratio significantly greater than that of the positive serum control group were selected for further sequencing, and finally obtained and named as 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): EFVAAISRGATYTS.

[0095] The CDR3 amino acid sequence of the single-domain antibody 74-1-C07-1 (SEQ ID NO.3): 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] PCR amplified the sequence of the single domain antibody, and inserted the PCR product into the pcDNA3.4 expression vector to express its C-terminus by fusion with the human IgG1Fc fragment. The obtained 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 way, the GPC3-specific antibody HYP7 was used as a positive control, and cloned and transfected into HEK293 cells according to the same steps. The expression level and function were verified by comparison.

[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 , transfect the plasmid expressing the recombinant antibody into 293F cells, and perform shaking culture to express and purify the antibody. Since the target recombinant antibody contains human IgG fragments, Protein A magnetic beads can be used for affinity purification. Wash the Protein A magnetic beads twice with 30mL PBS buffer, 0.1M sodium hydroxide, and PBS buffer respectively. Add the corresponding volume of Protein A magnetic beads to the 293F cell shaker according to the required amount of the sample (calculated as 1mL Protein A magnetic beads for every 20mg IgG). Incubate in a shaking incubator for 1 to 4 hours or overnight at 4℃, 120rpm, room temperature. Collect the Protein A magnetic beads with a magnetic separation rack and transfer them to a 50mL centrifuge tube. Wash twice with 30mL PBS buffer and deionized water respectively, and resuspend with 1mL elution buffer. Incubate at room temperature for 5min, collect the magnetic beads, and transfer the supernatant containing the target antibody to a 15mL centrifuge tube. Elute Protein A magnetic beads twice, combine the eluates, add neutralization buffer, and adjust the pH of the solution. Use PBS with a volume of at least 100 times the sample volume to dialyze the eluted sample at 18-25°C for 2 hours, and then change the solution at 2-8°C for 14-16 hours. Finally, determine the protein concentration, filter with a 0.22μm sterile filter membrane, and package the samples and store them in a -80°C refrigerator for use.

[0108] Serial dilutions of the target antibody were added to 3×10 5CHO-S-GPC3 and CHO-S were incubated at room temperature for 1 hour. Centrifuge at 800×g for 5 minutes at room temperature, discard the supernatant containing antibodies, and wash the cells 3 times with PBS. Add 100μL PE-labeled Anti-human IgG antibody (1:500 dilution), mix well, and incubate at room temperature in the dark for 45 minutes. Centrifuge at 800×g, room temperature, 5 minutes, discard the supernatant containing antibodies, wash the cells 3 times with PBS, resuspend the cells with 500μL PBS, and perform 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 for CHO-S-GPC3 was 50 The value is 0.6996μg / mL( Figure 2 ), EC of positive antibody 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. Centrifuged at 800×g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed 3 times with PBS. 100μL PE-labeled anti-human IgG antibody (1:500 dilution) was added and incubated at room temperature in the dark for 45 minutes. Centrifuged at 800×g for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed 3 times with PBS. Resuspend the cells with 500μL PBS and analyze by flow cytometry. The negative control group was only added with anti-human IgG antibody (100μL, PE-labeled, 1:500 dilution), incubated at room temperature in the dark for 45 minutes. Centrifuged, 800×g, room temperature, 5 minutes, the supernatant was discarded, and the cells were washed 3 times with PBS. Resuspend the cells with 500μL PBS and analyze 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 example is as follows Figure 4 shown.

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

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

[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 co-stimulatory 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 then connected to the lentiviral vector pCDH-EF1α-MCS plasmid, and its expression was regulated by the human EF-1α promoter and Kozak sequence. The above lentiviral vector expression plasmids were co-transfected with lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE and pMD2.G using the transfection reagent Lipofectamine 3000. 293T cells were collected 48 hours later, centrifuged at 4°C, 3000rpm, 10min, and then filtered through a 0.45μm pore size filter membrane, and finally ultracentrifuged at 25,000rpm for 3h at 4°C. The obtained virus concentrates were stored at -80°C and named BVHGC3-030 and BN108, respectively. Finally, Jurkat cells were used as materials to detect the activity titer of the above lentiviruses.

[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 lentivirus was added at an MOI of 5TU / mL for transduction. 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 the cells were cultured for another 5 days, during which the cell density was maintained at 1.5×10 6 / mL. On the 10th day after transduction, the cells were harvested and frozen in liquid nitrogen for later use. The obtained CAR-T cells were named after the corresponding CAR molecules, and the T cells not transduced with lentivirus were named Mock.

[0142] (4) Detection of CAR molecule expression

[0143] Wash BVHGC3-030 CAR-T cells twice with PBS and resuspend them 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 them. Using Mock cells as negative control, flow cytometry was used to detect the expression rate of CAR molecules in BVHGC3-030 cells. The results are shown in 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 in 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. CAR-T cells were then incubated with PE-labeled anti-G4S Linker antibody for 1 hour. The supernatant was then centrifuged to remove, washed twice with FACS buffer, and resuspended. Using Ctrl T cells as negative controls, flow cytometry was used to detect the efficiency of BN108 and BVHGC3-030 CAR-T cells binding to GPC3. The results are shown in 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 marker) were resuspended at a density of 100 μL / mL and inoculated in 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 effective 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 and a Mock group (i.e., 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 1:1 effect-target ratio, for GPC3-positive Hep3B cells, the killing effect of BN108 group (relative fluorescence intensity at the last time point was 0.51±0.01) was significantly improved compared with Mock group (relative fluorescence intensity at the last time point was 1.19±0.01), while the killing effect of BVHGC3-030 group (relative fluorescence intensity at the last time point was 0.19±0.01) was significantly stronger than that of BN108 group (P<0.05). The above results show that compared with existing antibodies, CAR-T cells constructed based on 74-1-C07-1 antibody have significantly improved killing effects 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 hepatoma 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 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 in the figure, 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, and one week after the CAR T cell infusion, the tumor was completely eliminated, and the mice maintained tumor-free survival for more than 3 weeks. Moreover, compared with the positive control group (BN108), the anti-tumor effect of BVHGC3-030CAR-T was comparable, 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 implementation mode of the present invention, but the protection scope 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 shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A single domain antibody against glypican 3, characterized in that: 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.

2. The glypican 3 single domain antibody according to claim 1, characterized in that: The amino acid sequence of the glypican 3 single domain antibody includes the sequence shown in SEQ ID NO.

4.

3. A heavy chain antibody, characterized in that The heavy chain antibody comprises the variable region of 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; 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; Preferably, the Fc amino acid sequence of IgG1 includes the sequence shown in SEQ ID NO.

5.

4. Use of the antibody according to claims 1-3 in the preparation of an agent targeting glypican 3.

5. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody according to any one of claims 1 to 3 and a conjugated substance conjugated thereto.

6. 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.

7. The chimeric antigen receptor according to claim 6, characterized in that The signal peptide includes at least one of CD8, GM-CSF, IgG, IgE, CD4, CD28, CD137, TCRα or TCRβ; Preferably, the hinge region comprises at least one of IgG1, IgG4, CD8, CD28, CD137, TCRα or TCRβ; Preferably, the transmembrane domain comprises 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 thereof; Preferably, the signal transduction domain includes the intracellular signal transduction domain and the co-stimulatory domain of human CD3; Preferably, the intracellular signal transduction domain of human CD3 includes at least one of CD3ζ, CD3ε, CD3γ or CD3δ; Preferably, the co-stimulatory 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; Preferably, the amino acid sequence of the signal peptide includes the sequence shown in SEQ ID NO.10; Preferably, the amino acid sequence of the hinge region includes the sequence shown in SEQ ID NO.11; Preferably, the amino acid sequence of the transmembrane domain includes the sequence shown in SEQ ID NO.12; Preferably, the amino acid sequence of the co-stimulatory domain includes the sequence shown in SEQ ID NO.13; Preferably, the amino acid sequence of the intracellular signal transduction domain includes the sequence shown in SEQ ID NO.

14.

8. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody according to any one of claims 1 to 3, or the nucleic acid molecule encodes the chimeric antigen receptor according to claim 6 or 7.

9. An expression vector, characterized in that The expression vector comprises the nucleic acid molecule of claim 8; Preferably, the expression vector is any one of a lentiviral vector, a retroviral vector, an adeno-associated virus vector or a liposome vector containing the gene encoding the chimeric antigen receptor according to claim 6 or 7.

10. A host cell, characterized in that The host cell contains at least one copy of the nucleic acid molecule according to claim 4, or at least one copy of the expression vector according to claim 9.

11. A chimeric antigen receptor cell, characterized in that The chimeric antigen receptor cell expresses the chimeric antigen receptor of claim 6 or 7; 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.

12. Use of the antibody according to any one of claims 1 to 3, the antibody conjugate according to claim 5, the chimeric antigen receptor according to claim 6 or 7, the nucleic acid molecule according to claim 8, the expression vector according to claim 9, the host cell according to claim 10, or the chimeric antigen receptor cell according to claim 11 in the preparation of a product for enhancing immune cells to kill tumor cells, or in the preparation of a drug or preparation for preventing and / or treating cancer or tumors; Preferably, the tumor comprises a GPC3 solid tumor, and the cancer comprises at least one of liver cancer, ovarian cancer, melanoma or lung cancer.

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