Tumor specific glycoprotein antibody C1 and application thereof

Abstract

The invention discloses a tumor specific glycoprotein antibody C1 and application thereof. The amino acid sequence of the CDR1 of the tumor specific glycoprotein antibody C1 comprises a sequence as shown in SEQ ID NO. 1; the amino acid sequence of the CDR2 of the tumor specific glycoprotein antibody C1 comprises a sequence as shown in SEQ ID NO. 2; the amino acid sequence of the CDR3 of the tumor specific glycoprotein antibody C1 comprises a sequence as shown in SEQ ID NO.3. The tumor specific glycoprotein antibody C1 has affinity to GPC3 and is smaller in molecular weight, and a detection reagent developed on the basis of the tumor specific glycoprotein antibody C1 is higher in sensitivity and lower in false positive.

Description

Technical Field

[0001] The invention belongs to the field of antibody biotechnology and relates to a tumor-specific glycoprotein antibody C1 and an application thereof. Background Art

[0002] Glypican-3 (GPC3) is a membrane heparan sulfate glycoprotein that plays an important regulatory role in cell growth and development. Its expression varies greatly in different malignant tumors and may be one of the important causes of tumorigenesis. GPC3 is highly expressed in liver cancer, moderately expressed in melanoma, ovarian clear cell carcinoma, and lung adenocarcinoma, and expressed in small amounts in tumors such as yolk sac tumor, neuroblastoma, hepatoblastoma, and Wilm's sarcoma cells, while it is silent in breast cancer and mesothelioma.

[0003] Existing design methods have designed antibody-drug conjugates, therapeutic monoclonal antibodies, chimeric antigen receptor T cells, bispecific antibodies, etc. for GPC3, some of which have been put into use, but the GPC3 protein will be divided into N-terminal soluble proteins and C-terminal membrane proteins of different molecular weights. The divided proteins can be released from cancer cells into the microenvironment, so when screening antibodies, specific antibodies that bind to the C-terminal membrane protein are needed. When applied to cell drugs, they are generally expressed in the form of single-chain antibodies (scFv). The long coding sequence is not conducive to the construction and modification of the expression vector. At the same time, it will also cause cross-pairing of VH and VL of adjacent scFv molecules, resulting in non-specific activation of cell drugs.

[0004] Therefore, there is an urgent need to provide a GPC3 antibody with high affinity, high specificity and small molecular weight. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a tumor-specific glycoprotein antibody C1 and its application, which has affinity for GPC3 and a smaller molecular weight. Antibody drugs developed based on it have better effectiveness, stronger tissue penetration, better in vivo stability and better efficacy.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a tumor-specific glycoprotein antibody C1, wherein the amino acid sequence of CDR1 of the tumor-specific glycoprotein antibody C1 includes the sequence shown in SEQ ID NO.1; the amino acid sequence of CDR2 of the tumor-specific glycoprotein antibody C1 includes the sequence shown in SEQ ID NO.2; and the amino acid sequence of CDR3 of the tumor-specific glycoprotein antibody C1 includes the sequence shown in SEQ ID NO.3.

[0008] SEQ ID NO.1: GILFSANS.

[0009] SEQ ID NO. 2: ITSDGYT.

[0010] SEQ ID NO.3: KITNY.

[0011] The tumor-specific glycoprotein antibody C1 of the present invention has affinity for GPC3 and a smaller molecular weight. The antibody drugs developed based on it have better effectiveness, stronger tissue penetration, better in vivo stability, and better efficacy; the engineered immune cells developed based on it have higher exogenous gene expression levels, better effectiveness, better efficacy, and lower difficulty in transformation; the detection reagents developed based on it have higher sensitivity and lower false positives.

[0012] There is a special antibody lacking light chains in the blood of camelids and chondrichthyes, namely heavy chain antibodies. Compared with ordinary antibodies, it only contains one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. By cloning its variable region, a single domain antibody (also known as nanobody) consisting only of the heavy chain variable region can be obtained, which has the advantages of small molecular weight, strong tissue penetration, high stability, and low immunogenicity.

[0013] 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, alpaca peripheral blood mononuclear cells (PBMC) are separated, RNA is extracted and reverse transcribed to obtain cDNA. Alpaca single-domain antibody-specific primers are used to amplify the VHH sequence and clone it into a phage expression plasmid to construct a phage display library. CHO-S cells are used to incubate the phage display library, and antibody clones that only bind to CHO-S cells are eliminated. Then, virus-like particles (VLPs) containing GPC3 are used to perform 3-4 rounds of enrichment and panning on the phage display library. Monoclones are picked from the enriched products, and the specific binding of candidate antibodies to GPC3-VLPs is detected by ELISA. After the positive antibodies are expressed and purified, their affinity to CHO-S cells expressing GPC3 is detected respectively. Antibody clones with strong affinity for GPC3 were selected, and the GPC3 single-domain antibody sequence was cloned into the CAR expression vector to construct CAR-T cells with GPC3 single-domain antibody as the antigen binding domain, and their killing ability against GPC3-positive Huh7 cells in vivo and in vitro was tested. The results showed that compared with the prior art, the GPC3 single-domain antibody obtained in the present invention has a killing effect on the cell line expressing GPC3 and is specific.

[0014] Preferably, the amino acid sequence of the tumor-specific glycoprotein antibody C1 includes the sequence shown in SEQ ID NO.4.

[0015] SEQ ID NO.4:

[0016] QLQLVESGGGLVQAGGSLRLSCAASGILFSANSIRWYRQAPGTQRDMVA SITSDGYTYYADSVKGRFTISRDNTKNTAYLQMNSVKPEDTAVYVCKITNYW GQGTQVTVSS.

[0017] In a second aspect, the present invention provides a single domain antibody, which comprises the variable region sequence of the tumor-specific glycoprotein antibody C1 described in the first aspect and the full-length or partial amino acid sequence of the crystallizable segment of human immunoglobulin Ig Fc.

[0018] Preferably, the Ig Fc comprises the full-length or partial sequence of the Fc fragment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof.

[0019] Preferably, the Fc amino acid sequence of IgG1 includes the sequence shown in SEQ ID NO.5.

[0020] Preferably, the amino acid sequence of the single-domain antibody includes the sequence shown in SEQ ID NO.6.

[0021] SEQ ID NO.5:

[0022] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0023] SEQ ID NO.6:

[0024] QLQLVESGGGLVQAGGSLRLSCAASGILFSANSIRWYRQAPGTQRDMVASITSDGYTYYADSVKGRFTISRDNTKNTAYLQMNSVKPEDTAVYVCKITNYWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0025] In a third aspect, the present invention provides a composition for detecting tumor-specific glycoproteins, wherein the composition comprises the tumor-specific glycoprotein antibody C1 described in the first aspect or the single domain antibody described in the second aspect.

[0026] In a fourth aspect, the present invention provides a chimeric antigen receptor, comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane region and a signal transduction domain; the antigen binding domain comprises the tumor-specific glycoprotein antibody C1 described in the first aspect.

[0027] Preferably, the signal peptide comprises any one or a combination of at least two of CD8, GM-CSF, CD4, CD28, CD137, IgG, IgE, TCRα or TCRβ.

[0028] Preferably, the hinge region comprises any one of CD8, CD28, CD137, IgG1, IgG4, TCRα or TCRβ, or a combination of at least two thereof.

[0029] Preferably, the transmembrane region includes any one of CD28, CD3ε, CD3ζ, CD3γ, CD3δ, TCRα, TCRβ, CD40, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233 or CD314, or a combination of at least two of them.

[0030] Preferably, the signal transduction domain comprises a co-stimulatory domain and a human CD3 intracellular signal transduction domain.

[0031] Preferably, the co-stimulatory domain comprises any one or a combination of at least two of CD3ε, CD3γ, CD3δ, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB, PD1, Dap10, CDS, ICAM-1, ICOS, NKG2D, GITR or OX40L.

[0032] Preferably, the human CD3 intracellular signal transduction domain includes any one of CD3ζ, CD3ε, CD3γ or CD3δ, or a combination of at least two of them.

[0033] Preferably, the signal peptide comprises a human CD8 signal peptide having an amino acid sequence as shown in SEQ ID NO.7.

[0034] Preferably, the hinge region comprises the human IgG4 hinge region as shown in SEQ ID NO.8.

[0035] Preferably, the transmembrane region comprises the human CD8 transmembrane region as shown in SEQ ID NO.9.

[0036] Preferably, the signal transduction domain includes the human 4-1BB intracellular region as shown in SEQ ID NO.10 and / or the human CD3ζ intracellular region as shown in SEQ ID NO.11.

[0037] SEQ ID NO.7: MALPVTALLLPLALLLHAARPS.

[0038] SEQ ID NO.8:

[0039] ESKYGPPCPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH EALHNHYTQKSLSLSLGK.

[0040] SEQ ID NO.9: CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR.

[0041] SEQ ID NO.10:

[0042] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0043] SEQ ID NO.11:

[0044] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR.

[0045] Preferably, the chimeric antigen receptor comprises the human CD8 signal peptide shown in SEQ ID NO.7, the first aspect, the human IgG4 hinge region shown in SEQ ID NO.8, the human CD8 transmembrane region shown in SEQ ID NO.9, the human 4-1BB intracellular region shown in SEQ ID NO.10 and the human CD3ζ intracellular region shown in SEQ ID NO.11.

[0046] Preferably, the amino acid sequence of the chimeric antigen receptor comprises the sequence shown in SEQ ID NO.12.

[0047] SEQ ID NO.12:

[0048] MALPVTALLLPLALLLHAARPSQLQLVESGGGLVQAGGSLRLSCAASGILFSANSIRWYRQAPGTQRDMVASITSDGYTYYADSVKGRFTISRDNTKNTAYLQMNSVKP EDTAVYVCKITNYWGQGTQVTVSSESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0049] In a fifth aspect, the present invention provides a nucleic acid molecule encoding the tumor-specific glycoprotein antibody C1 described in the first aspect, the single domain antibody described in the second aspect, or the chimeric antigen receptor described in the fourth aspect.

[0050] In a sixth aspect, the present invention provides an expression vector, which comprises the nucleic acid molecule described in the fifth aspect.

[0051] 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 fourth aspect, preferably a lentiviral vector.

[0052] In a seventh aspect, the present invention provides a recombinant lentivirus, wherein the recombinant lentivirus contains the expression vector described in the sixth aspect.

[0053] In an eighth aspect, the present invention provides a chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the chimeric antigen receptor described in the fourth aspect.

[0054] Preferably, the chimeric antigen receptor immune cell comprises the expression vector described in the sixth aspect.

[0055] Preferably, the chimeric antigen receptor immune cell comprises any one or a combination of at least two of T cells, TIL cells, B cells, NK cells, mast cells or macrophages.

[0056] In the ninth aspect, the present invention provides the use of the tumor-specific glycoprotein antibody C1 described in the first aspect, the chimeric antigen receptor described in the fourth aspect, the expression vector described in the sixth aspect, or the chimeric antigen receptor immune cell described in the eighth aspect in the preparation of a product that can prevent and / or treat cancer or tumors.

[0057] Preferably, the tumor comprises a GPC3-positive solid tumor, and the cancer comprises any one of melanoma, liver cancer, ovarian cancer or lung cancer.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] (1) The tumor-specific glycoprotein antibody C1 of the present invention has affinity for GPC3 and has a smaller molecular weight;

[0060] (2) Antibody drugs developed based on the tumor-specific glycoprotein antibody C1 of the present invention have better effectiveness, stronger tissue penetration, better in vivo stability, and better efficacy;

[0061] (3) The engineered immune cells developed based on the tumor-specific glycoprotein antibody C1 of the present invention have higher exogenous gene expression levels, better effectiveness, better drug efficacy, and lower modification difficulty;

[0062] (4) The detection reagent developed based on the tumor-specific glycoprotein antibody C1 of the present invention has higher sensitivity and lower false positives. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1A This is the first round PCR amplification diagram of the VHH region of the alpaca antibody;

[0064] Figure 1B This is the second round PCR amplification diagram of the VHH region of the alpaca antibody;

[0065] Figure 2 The specificity diagram of the recombinant single domain antibody is detected by flow cytometry;

[0066] Figure 3 The concentration-effect curve of the antibody tested in CHO-S-GPC3 cells;

[0067] Figure 4A Schematic diagram of the molecular structure of CAR;

[0068] Figure 4B This is a graph showing the expression results of BVHGC3-013 in T cells detected by flow cytometry;

[0069] Figure 4C This is a graph showing the results of flow cytometry detection of the binding efficiency of BN108 and BVHGC3-013 cells to GPC3 protein;

[0070] Figure 5A IncuCyte real-time quantitative live cell imaging analysis of the killing function and specificity of CAR-T cells targeting GPC3 (effect-target ratio = 1:1);

[0071] Figure 5B IncuCyte real-time quantitative live cell imaging analysis of the killing function and specificity of CAR-T cells targeting GPC3 (effect-target ratio = 1:4).

[0072] Reagents used in the following examples:

[0073] SK-Hep-1 cells: ATCC, HTB-52;

[0074] Huh7 cells: Cell Bank of Chinese Academy of Sciences, SCSP-526;

[0075] CHO-S cells: Gibco, R80007;

[0076] FITC-labeled Rabbit anti-Llama IgG (H+L) antibody: Invitrogen, A16155;

[0077] Anti-M13-HRP antibody: SinoBiological, 11973-MM05T-H;

[0078] Anti-VHH antibody: GenScript, A02017;

[0079] PE-labeled anti-human IgG antibody: Biolegend, 410708;

[0080] AF647-labeled Anti-human IgG antibody: Jackson ImmunoResearch, 109-606-170;

[0081] AF647-labeled anti-human GPC3 antibody: R&D system, FAB2119A. DETAILED DESCRIPTION

[0082] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0083] 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.

[0084] Example 1

[0085] (1) Construction of GPC3 overexpressing cells

[0086] According to the amino acid sequence information of human GPC3 (UniProtAccession: P51654), a lentiviral expression vector was constructed and used to infect CHO-S cells to obtain the GPC3-overexpressing cell line CHO-S-GPC3, which was used for alpaca immunization, serum titer detection and antibody affinity verification, respectively.

[0087] (2) Alpaca immunization and serum titer testing

[0088] The CHO-S-GPC3 cells constructed as above were used to immunize three alpacas (73#, 74# and 194#), once every three weeks, for a total of five immunizations.

[0089] 5 mL of peripheral blood was collected before immunization and two weeks after the fourth immunization, and the centrifuge tube containing the blood sample was placed in a centrifuge, centrifuged at 800 × g for 10 min, and the upper serum was collected. Use sterile PBS buffer to dilute the GPC3-His recombinant protein to a final concentration of 1 μg / mL, take a new 96-well ELISA plate, add 1 μg / mL of GPC3-His recombinant protein to 100 μL / well, and coat overnight at 4°C; then shake off the antigen coating liquid, wash 5 times with PBST buffer (containing 0.05% Tween 20); add 200 μL / well of blocking solution and block at 37°C for 2h; after shaking off the blocking buffer, wash the plate 5 times with PBST buffer; use PBS buffer to gradiently dilute the above-collected serum, add 100 μL of gradient diluted serum to the 96-well ELISA plate, incubate at room temperature for 1 hour, and the control well is PBS buffer; shake off the liquid in the well and wash 5 times with PBST buffer; add 100 μL of HRP anti-Llama IgG (H+L) antibody (1:50000 dilution), incubate at room temperature for 60 min; shake off the liquid in the wells, wash the wells 5 times with PBST buffer; add 100 μL / well TMB colorimetric solution; incubate at room temperature in the dark for 10 min; add 50 μL / well stop solution; read the OD in the wells using an ELISA reader 450 value.

[0090] The results are shown in Tables 1, 2 and 3. The binding titers of the serum of the three alpacas before immunization and the GPC3 protein were all lower than 1:2000 (the antibody titer is expressed in the form of dilution multiples, the higher the dilution multiple, the higher the antibody titer), indicating that the content of anti-GPC3 specific antibodies contained therein was extremely low. On the contrary, the binding titers of the serum of the 73# and 74# alpacas after the fourth immunization and the GPC3 protein were both higher than 1:32K, and the binding titer of the serum of the 194# alpacas after the fourth immunization and the GPC3 protein was higher than 1:8K, indicating that the content of anti-GPC3 specific antibodies in the serum of these three alpacas was significantly increased.

[0091] Table 1

[0092]

[0093] Table 2

[0094]

[0095] Table 3

[0096]

[0097] Example 2

[0098] Construction of single domain antibody phage display library.

[0099] (1) VHH antibody fragment cloning

[0100] After confirming that the serum of the two alpacas contained GPC3-specific antibodies, 100 mL of peripheral blood was collected, PBMC was separated using lymphocyte separation solution, RNA was extracted, and reverse transcription was performed to obtain cDNA. Using PBMC cDNA as a template, the alpaca heavy chain antibody sequence was PCR amplified using specific primers (upstream primers bind to the signal peptide of the VHH antibody ORF, and downstream primers bind to the CH2 region). The PCR product was analyzed by electrophoresis, and the target fragment with a molecular weight of about 750 bp was recovered ( Figure 1A ). Then, the first round PCR product was used as a template to amplify the heavy chain antibody VHH fragment using specific primers (the upstream primer binds to the antibody FR1 region, and the 5' end contains the SfiI restriction site GGCCCAGCCGGCC; the downstream primer binds to the antibody Hinge and FR4 regions, and the 5' end contains the SfiI restriction site GGCCACGAAGGCC). The PCR product was analyzed by electrophoresis, and the target fragment with a molecular weight of about 400 bp was recovered ( Figure 1B ). The target fragment was stored at -80℃.

[0101] (2) Electroporation of library vectors and detection of library capacity and diversity

[0102] The phage surface display vector pComf plasmid and the VHH fragment library obtained above were digested with SfiI endonuclease. The linearized pComf vector and the VHH fragment library were connected overnight at 16°C using T4 ligase. 300 μL of the mixture of the above-mentioned connection product and the competent cells of E. coli SS320 was added to the pre-cooled electroporation cup, and the connection product was transformed into the competent cells of E. coli by electroporation, and then 2YT medium was added to resuspend the bacteria for recovery culture. 15 mL of bacterial solution was taken for subsequent phage production enrichment, and 20 μL of bacterial solution was taken and diluted with 2YT medium in a gradient manner and evenly spread on an LB plate containing ampicillin, and cultured at 37°C overnight. The number of clones that can be produced by each connection reaction was calculated to obtain the capacity of the single-domain antibody phage surface display library.

[0103] The results showed that the single domain antibody phage display library obtained from 73# alpaca had a library capacity of 1.60×10 9 The storage capacity obtained from 74# alpaca is 1.32×10 9 The storage capacity obtained from 194# alpaca is 1.15×10 9 . 20 single clones on the plate were picked and Sanger sequencing was performed with M13R primer. The results showed that the phage library sequences were highly different, with no repeated sequences and good diversity.

[0104] (3) Enrichment of phage display libraries

[0105] Take the above electroporation and resuscitation culture solution and adjust it to OD 600 When the OD is about 0.25, add ampicillin at a final concentration of and place in a constant temperature shaker for cultivation. 600 When the pH is 0.6, add M13KO7 helper phage culture. After the helper phage completes the infection of the target strain, centrifuge and discard the supernatant, and culture overnight. Then centrifuge the bacterial solution, transfer the supernatant containing phage particles to a new centrifuge tube, mix well and let stand at 4°C for 2h. Centrifuge to collect the phage precipitate, centrifuge the resuspended phage to remove insoluble impurities, and obtain the original library of single-domain antibody phage surface display.

[0106] Take 10 μL of the precipitate and dilute it 10 times. Add 0.2 mL of OD 600 ER2738 Escherichia coli was added at 0.5, mixed well, placed in a 37°C water bath, left to stand for 10 minutes, and then spread on LB plates. After culturing at 37°C overnight, the plaques were counted to obtain the titer of the phage surface display library.

[0107] Example 3

[0108] Target antibody library screening.

[0109] (1) First round of panning and product amplification

[0110] Seal the centrifuge tube and incubate at 4°C overnight. Dilute the GPC3 antigen to 50 μg / mL with CBS solution, add it to a 96-well solid phase plate, and coat it at 4°C overnight. Resuspend the CHO-S-GPC3 cells and block at 37°C for 60 minutes. At the same time, take the precipitate product of the original library displayed on the surface of the single-domain antibody phage, add PBS buffer A to block for 60 minutes as a premix. Centrifuge the blocked CHO-S cells to discard the supernatant, and incubate with the phage premix for 60 minutes to remove phage clones that non-specifically bind to CHO-S. Discard the GPC3 protein in the 96-well plate, add MPBS buffer PBS buffer and stand at room temperature for 60 minutes to block. Centrifuge the CHO-S-GPC3 cells to discard the supernatant and add them to the GPC3 protein wells for incubation for 60 minutes. The phage supernatant in the GPC3 protein wells was discarded, each well was washed, Gly-HCl elution solution was added, and the wells were incubated at 37°C with shaking. The specifically bound phages were eluted twice, and the eluted products were stored in pre-sealed centrifuge tubes at 4°C.

[0111] Take 2YT medium, add tetracycline at a final concentration of 100 μg / mL and 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, 20 mL of 2YT medium was added, and the culture was continued at 37°C for 30 min.600 When it is 0.5 again, add M13KO7 helper phage, shake well and let stand at 37℃ for 30min. Add ampicillin to the bacterial solution at a final concentration of 100μg / mL, culture for 45min, centrifuge and discard the supernatant and collect the bacteria. Resuspend with 2YT-AK medium and culture overnight. Transfer the phage suspension infected and amplified overnight to a centrifuge tube and centrifuge for 30min, then divide the supernatant into 40mL centrifuge tubes. Add 10mL PEG / NaCl to each tube, mix well and place on ice, let stand for 60min to precipitate the phage, centrifuge and discard the supernatant, resuspend the phage with PBS buffer, centrifuge for 5min, and remove insoluble impurities. Transfer the phage suspension to a new centrifuge tube, add PEG / NaCl, mix well and let stand at 4℃ for 10min to precipitate the phage. Centrifuge at 4℃ for 10min and discard the supernatant. The phage was resuspended in 0.5 mL PBS buffer and centrifuged at 4°C for 5 min to remove insoluble impurities. This was the phage product obtained in the first round of panning amplification.

[0112] (2) Second to fourth rounds of panning and product amplification

[0113] The GPC3 antigen was diluted to 10 μg / mL using CBS solution, added to a 96-well solid phase plate, and coated overnight at 4°C. The phage products amplified by the first round of panning were used for the second to fourth rounds of panning in the same manner as above to obtain a phage library that specifically binds to GPC3.

[0114] (3) ELISA detection and sequencing of monoclonal phage

[0115] The single-domain antibody phage display library after multiple rounds of panning was used to infect ER2738 Escherichia coli, mixed and placed at 37°C for 10 minutes, then spread on LB plates and incubated at 37°C overnight. 2YT-A medium was added to each well of a 96-well deep-well plate at 0.2 mL, and a single clone on the plate was picked and incubated at 37°C overnight. Cultured to OD 600 About 0.5. Add M13KO7 helper phage, mix well, let stand at 37℃ for 15min, and then culture at 37℃ for 45min. After centrifugation and discarding the supernatant, resuspend each well with 0.5mL 2YT-AK medium and culture at 220rpm at 30℃ overnight. Centrifuge at 3900rpm for 10min, and the supernatant obtained is the monoclonal phage particle.

[0116] While amplifying the phage monoclonal clone, the GPC3 antigen protein was coated on the ELISA plate with CBS at pH 9.6. After coating overnight at 4°C, the antigen was discarded, and after washing three times with PBST buffer, 250 μL of 3% MPBS buffer was added to each well and blocked overnight at 4°C. After the blocking solution was mixed, each well was washed 4 times with PBST buffer, and 50 μL of 0.1% PBST buffer was added, and then 50 μL of the above monoclonal phage supernatant was added one by one. After incubation at 4°C for 60 minutes, it was washed 5 times with 0.05% PBST buffer. After diluting anti M13-HRP antibody (1:5000) with 0.05% PBST buffer, 100 μL was added to each well and incubated at 4°C for 45 minutes. After washing 5 times with 0.05% PBST buffer, 100 μL of TMB was added for color development at room temperature for 10 minutes, and then 50 μL of 0.2M hydrochloric acid was added to terminate, and the OD was read on the ELISA plate. 450 The sample / negative control value was calculated, and clones with a ratio significantly greater than that of the positive serum control group were selected for sequencing to obtain antibody 73-G3-1-C1.

[0117] Example 4

[0118] Expression of recombinant single domain antibodies and detection of binding to target proteins.

[0119] The sequence of 73-G3-1-C1 was amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.4, and its C-terminus was fused with the human IgG1 Fc fragment for expression. After the obtained expression plasmid was transiently transfected into HEK293 cells, the harvested cell culture supernatant contained the 73-G3-1-C1 antibody.

[0120] The binding specificity of 73-G3-1-C1 antibody was tested. The culture supernatant containing 73-G3-1-C1 antibody was mixed with 3×10 5 Huh7 (naturally expressing GPC3), SK-Hep-1-GPC3 OE (SK-Hep-1 cells overexpressing GPC3) and 293T (GPC3 negative cell line) cells were incubated at room temperature for 60 minutes. After centrifugation at 800×g for 5 minutes at room temperature, the supernatant was discarded and the cells were washed 3 times with PBS buffer. 100μL PE-labeled anti-human IgG antibody (1:500 dilution) was added and incubated at room temperature for 45 minutes in the dark. After centrifugation at 800×g for 5 minutes at room temperature, the supernatant was discarded and the cells were washed 3 times with PBS buffer. The cells were resuspended in 0.5mL PBS buffer for flow cytometry analysis.

[0121] The results are as follows Figure 2 As shown, the expression supernatant of the negative control group had an effect on Huh7 and SK-Hep-1-GPC3 OE, 293T had no significant binding, while 73-G3-1-C1 recombinant single domain antibody had no significant binding to Huh7 and SK-Hep-1-GPC3 OE The binding of the cells was significantly stronger than that of the negative control antibody, and there was no significant binding to 293T cells, indicating that the 73-G3-1-C1 antibody can effectively and specifically bind to GPC3 on the surface of tumor cell lines.

[0122] Example 5

[0123] Purification and half effective concentration (EC 50 ) determination.

[0124] To determine the EC of 73-G3-1-C1 antibody 50 , transiently transfect the relevant expression plasmid into 293F cells, and shake the flask to culture for antibody expression and purification. 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 in sequence. Add the corresponding volume of Protein A magnetic beads (calculated as 20mg IgG / mL Protein A magnetic beads) to the 293F cell shake flask according to the required amount of the sample. Incubate at room temperature for 4 hours at 120rpm in a shaking incubator. Collect the Protein A magnetic beads with a magnetic separation rack and transfer them to a 50mL centrifuge tube. After washing twice with 30mL PBS buffer and ultrapure water, resuspend them with 0.5mL elution buffer. After incubation at room temperature for 5min, collect the magnetic beads with a magnetic separation rack, and transfer the supernatant containing the target antibody to a 15mL centrifuge tube. After eluting the Protein A magnetic beads twice, the eluates were combined and the neutralization buffer was added to adjust the pH of the solution. The eluted sample was dialyzed with PBS buffer at least 100 times the volume of the sample, first dialyzed at 18°C ​​for 2 hours, and then dialyzed at 8°C for 16 hours after changing the buffer once. Finally, the protein concentration was determined and the sample was filtered with a 0.22μm sterile filter membrane and packaged, and stored in a -80°C refrigerator for use.

[0125] Serial dilutions of the target antibody were added to 3×10 5 CHO-S-GPC3 and CHO-S were incubated at room temperature for 60 minutes. Centrifuge at 800×g for 5 minutes at room temperature, discard the supernatant containing antibodies, and wash the cells three times with PBS buffer. 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 for 5 minutes at room temperature, discard the supernatant containing antibodies, wash the cells three times with PBS buffer, and resuspend the cells with 0.5mL PBS buffer for flow analysis.

[0126] The results are as follows Figure 3 As shown, the EC of 73-G3-1-C1 to CHO-S-GPC3 50 The value is 17.33μg / mL.

[0127] Example 6

[0128] Construction of CAR-T cells based on target antibodies.

[0129] (1) Design of CAR molecules

[0130] The target gene structure of the lentiviral vector involved in this example is as follows Figure 4A shown.

[0131] BVHGC3-013 is composed of the following structures in series: human CD8 signal peptide (SP for short), anti-human GPC3 single-domain antibody 73-G3-1-C1 [VHH(73-G3-1-C1 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).

[0132] BN108 is composed of the following structures in series: SP, scFv (GC33), CD8 hinge, CD8 TM, 4-1BB ICD, and CD3ζ ICD.

[0133] GC33 VL amino acid sequence (SEQ ID NO.13):

[0134] DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSP QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPT FGQGTKLEIKR.

[0135] GC33 VH amino acid sequence (SEQ ID NO.14):

[0136] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLE WMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTR FYSYTYWGQGTLVTVSS.

[0137] (2) Lentivirus preparation

[0138] The above CAR molecule expression sequence was fully synthesized and then connected to the lentiviral vector pCDH-EF1α-MCS plasmid by molecular cloning, so that it was expressed under the regulation of the human EF-1α promoter and Kozak sequence. The transfection reagent Lipofectamine 3000 was used to co-transfect 293T cells with the lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE and pMD2.G according to the instructions. The viral supernatant was collected 2 days after transfection, centrifuged at 3000rpm for 10min at 4°C, filtered through a 0.45μm pore size filter membrane, and finally ultracentrifuged at 25,000rpm for 2h at 4°C. The obtained virus concentrate was stored at -80°C and named BVHGC3-013 and BN108 respectively. Finally, the activity titer of the above lentivirus was detected using Jurkat cells.

[0139] (3) CAR-T cell preparation

[0140] PBMCs from healthy donors were revived in AIM V medium, and 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody and 300 IU / mL recombinant hIL-2 were added, and cultured in a cell culture incubator overnight. The obtained T cells were washed, and lentivirus was added at an MOI of 5TU / cell 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. After overnight culture, the cell density was adjusted to 2×10 6 / mL, and supplemented with 300IU / mL hIL-2. On the 4th day after transduction, the cells were washed to discard the residual lentiviral particles in the supernatant, and then cultured in a cell culture incubator for another 5 days, during which the cell density was maintained at 2×10 6 / mL. The cells were collected on the 10th day after transduction and frozen in liquid nitrogen for future use. The obtained CAR-T cells were named after the corresponding CAR molecules, and the T cells not transduced with lentivirus were named MockT.

[0141] (4) Detection of CAR molecule expression

[0142] The BVHGC3-013 CAR-T cells to be tested were washed twice with PBS buffer and resuspended with FACS buffer (PBS buffer containing 0.1% sodium azide and 0.4% BSA). According to the antibody instructions, the FITC-labeled anti-VHH antibody was incubated with CAR-T cells for 60 minutes. The supernatant was then centrifuged and discarded, and the cells were washed twice with FACS buffer and resuspended. Using Mock T cells as negative controls, the CAR molecule expression rate of BVHGC3-013 cells was detected by flow cytometry.

[0143] The results are as follows Figure 4B The expression rates of BVHGC3-013 CAR were shown to be 85.1%.

[0144] (5) Detection of CAR molecule binding efficiency to GPC3

[0145] The BN108 and BVHGC3-013 CAR-T cells to be tested were washed twice with PBS buffer and resuspended with FACS buffer (PBS buffer containing 0.1% sodium azide and 0.4% BSA). According to the antibody instructions, the GPC-3-His tag protein was incubated with CAR-T cells for 60 minutes, then centrifuged and the supernatant was discarded, and washed twice with FACS buffer and resuspended. Then, the PE-labeled anti-G4S Linker antibody was incubated with CAR-T cells for 60 minutes. Then the supernatant was discarded by centrifugation, and the cells were washed twice with FACS buffer and resuspended. Using Mock T cells as negative controls, the efficiency of BN108 and BVHGC3-013 CAR-T cells binding to GPC3 was detected by flow cytometry.

[0146] The results are as follows Figure 4C The binding efficiencies of BN108 and BVHGC3-013 CAR to GPC3 were shown to be 77.1% and 70.8%, respectively.

[0147] Example 7

[0148] Study on the functions of CAR-T cells in vivo and in vitro.

[0149] In vitro killing experiment of BVHGC3-013 and BN108 CAR-T cells

[0150] Use culture medium at 1×10 5 Huh7 cells expressing mCherry fluorescent protein 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 cell number to target ratio of 1:1 or 1:4, and the killing effect of CAR-T cells on tumor cells was recorded in real time. After the co-culture, the changes in the mCherry fluorescence signal of the target cells in each group were calculated by the IncuCyte software. The lower the signal value, the fewer the number of cells in the group, and the better the killing effect of CAR-T cells.

[0151] The results are as follows Figure 5A and Figure 5BAs shown in the figure, under the condition of 1:1 effect-target ratio, for GPC3-positive Huh7 cells, the killing effect of the BN108 group was significantly improved compared with the Mock group (P<0.05), and the killing effect of the BVHGC3-013 group was also significantly improved (P<0.05), and its effect was close to that of the BN108 group. In addition, under the condition of 1:4 effect-target ratio, there was no significant difference in the killing effect of the BVHGC3-013 group and the BN108 group (P>0.05). The above results show that compared with existing antibodies, CAR-T cells constructed based on 73-G3-1-C1 antibodies have significant specific killing function for GPC3-positive tumor cells.

[0152] In summary, the tumor-specific glycoprotein antibody C1 of the present invention has affinity for GPC3 and a smaller molecular weight. The antibody drugs developed based on it have better effectiveness, stronger tissue penetration, better in vivo stability, and better efficacy; the engineered immune cells developed based on it have higher exogenous gene expression levels, better effectiveness, better efficacy, and lower difficulty in transformation; the detection reagents developed based on it have higher sensitivity and lower false positives.

[0153] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A tumor-specific glycoprotein antibody C1, characterized in that: The amino acid sequence of CDR1 of the tumor-specific glycoprotein antibody C1 includes the sequence shown in SEQ ID NO.1; The amino acid sequence of CDR2 of the tumor-specific glycoprotein antibody C1 includes the sequence shown in SEQ ID NO.2; The amino acid sequence of CDR3 of the tumor-specific glycoprotein antibody C1 includes the sequence shown in SEQ ID NO.

3.

2. The tumor-specific glycoprotein antibody C1 according to claim 1, characterized in that: The amino acid sequence of the tumor-specific glycoprotein antibody C1 includes the sequence shown in SEQ ID NO.

4.

3. A single domain antibody, characterized in that The single-domain antibody comprises the variable region sequence of the tumor-specific glycoprotein antibody C1 according to claim 1 or 2 and the full-length or partial amino acid sequence of the crystallizable segment Ig Fc of human immunoglobulin; The Ig Fc comprises the full length or partial sequence of the Fc segment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof; The Fc amino acid sequence of the IgG1 includes the sequence shown in SEQ ID NO.5; The amino acid sequence of the single-domain antibody includes the sequence shown in SEQ ID NO.

6.

4. A composition for detecting tumor-specific glycoprotein, characterized in that: The composition comprises the tumor-specific glycoprotein antibody C1 according to claim 1 or 2 or the antibody according to claim 3.

5. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane region and a signal transduction domain; The antigen binding domain comprises the tumor-specific glycoprotein antibody C1 according to claim 1 or 2; The signal peptide includes any one or a combination of at least two of CD8, GM-CSF, IgG, IgE, CD4, CD28, CD137, TCRα or TCRβ; The hinge region includes any one or a combination of at least two of IgG1, IgG4, CD8, CD28, CD137, TCRα or TCRβ; The transmembrane region 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 a human CD3 intracellular signal transduction domain and a co-stimulatory domain; The human CD3 intracellular signal transduction domain includes any one or a combination of at least two of CD3ζ, CD3ε, CD3γ or CD3δ; The co-stimulatory domain includes any one or a combination of at least two 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 signal peptide includes a human CD8 signal peptide whose amino acid sequence is shown in SEQ ID NO.7; The hinge region includes the human IgG4 hinge region as shown in SEQ ID NO.8; The transmembrane region includes the human CD8 transmembrane region shown in SEQ ID NO.9; The signal transduction domain includes the human 4-1BB intracellular region as shown in SEQ ID NO.10 and / or the human CD3ζ intracellular region as shown in SEQ ID NO.11; The chimeric antigen receptor comprises the human CD8 signal peptide shown in SEQ ID NO.7, the tumor-specific glycoprotein antibody C1 according to claim 1 or 2, the human IgG4 hinge region shown in SEQ ID NO.8, the human CD8 transmembrane region shown in SEQ ID NO.9, the human 4-1BB intracellular region shown in SEQ ID NO.10, and the human CD3ζ intracellular region shown in SEQ ID NO.11; The amino acid sequence of the chimeric antigen receptor includes the sequence shown in SEQ ID NO.

12.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the tumor-specific glycoprotein antibody C1 according to claim 1 or 2, the single domain antibody according to claim 3, or the chimeric antigen receptor according to claim 5.

7. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 6; 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 5.

8. A chimeric antigen receptor immune cell, characterized in that The chimeric antigen receptor immune cell expresses the chimeric antigen receptor of claim 5; The chimeric antigen receptor immune cell comprises the expression vector of claim 7; The chimeric antigen receptor immune cells include any one or a combination of at least two of T cells, TIL cells, B cells, NK cells, mast cells or macrophages.

9. Use of the tumor-specific glycoprotein antibody C1 according to claim 1 or 2, the chimeric antigen receptor according to claim 5, the expression vector according to claim 7, or the chimeric antigen receptor immune cell according to claim 8 in the preparation of a product for preventing and / or treating cancer or tumors.

10. The use according to claim 9, characterized in that: The tumor includes a GPC3-positive solid tumor, and the cancer includes any one of melanoma, liver cancer, ovarian cancer or lung cancer.

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